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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Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor
Yousefi, O.S. ; Günther, Matthias ; Hörner, Maximilian ; Chalupsky, Julia ; Wess, Maximilian ; Brandl, Simon M. ; Smith, Robert W. ; Fleck, Christian ; Kunkel, Tim ; Zurbriggen, Matias D. ; Höfer, Thomas ; Weber, Wilfried ; Schamel, Wolfgang W.A. - \ 2019
eLife 8 (2019). - ISSN 2050-084X
A. thaliana - dynamics - human - immunology - inflammation - ligand-receptor - optogenetics - signaling - T cells

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis
Pomeroy, Brianna ; Sipka, Anja ; Hussen, Jamal ; Eger, Melanie ; Schukken, Ynte - \ 2017
medicine - cell biology - genetics - ecology - immunology - mathematical sciences - developmental biology - infectious diseases - computational biology
The heightened susceptibility to infectious diseases in postpartum dairy cows is often attributed to immune dysfunction associated with the transition period. However, the cell populations involved in this immune dysfunction and the dynamics between those populations are not well defined. Monocytes play a crucial role in governing initial immune response in bacterial infections. Bovine monocytes are subdivided in classical (CD14+/CD16−), intermediate (CD14+/CD16+) and non-classical monocytes (CD14−/CD16+) with distinct phenotypic and functional differences. This study investigated the relationship of monocyte subsets counts in blood at 42 and 14 days prior to expected calving date to occurrence of metritis and mastitis within 2 weeks postpartum. In the enrolled prospective cohort of 27 German Holstein cows, housed at the Institute of Animal Nutrition of the Friedrich-Loeffler-Institute Braunschweig, Germany, n = 13 developed metritis and/or mastitis postpartum. A multivariable logistic regression was used to analyze the relationship between prepartum cell counts of monocyte subsets and neutrophils with postpartum disease. Our model revealed that higher counts of the two CD14+ monocyte subsets were predictive of disease. In contrast, higher numbers of the CD14− monocyte subset were negatively associated with disease. Interestingly, the neutrophil count, a common hallmark for inflammatory response, was not associated with the outcome variable at either time point. The results indicate that the number and composition of monocyte subsets before calving are related to the susceptibility to infectious disease within 2 weeks postpartum. Furthermore the oppositional effect of CD14+ and CD14− subsets strengthens the hypothesis that these subsets have different functional roles in the inflammatory response in dairy cows.
Consequences of dry period length and dietary energy source on physiological health variables in dairy cows and calves
Mayasari, Nova - \ 2017
Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Ariette van Knegsel; Henk Parmentier. - Wageningen : Wageningen University - ISBN 9789463431408 - 221
dairy cows - calves - dry period - feed rations - feeds - energy balance - animal health - inflammation - antibodies - adaptation physiology - immunology - melkkoeien - kalveren - gustperiode - voedingsrantsoenen - voer - energiebalans - diergezondheid - ontsteking - antilichamen - adaptatiefysiologie - immunologie

During the transition period, dairy cows experience a negative energy balance (NEB) caused by the high energy requirement for milk yield, while feed intake is limited. Severity of the NEB has been associated with an increased incidence of metabolic disorders and infectious diseases, inflammation, immunosuppression and oxidative stress. It is known that shortening or omitting the dry period or feeding a glucogenic ration improves the energy balance (EB) in dairy cows in early lactation. It can be expected that an improvement of the EB due to shortening or omitting the dry period results in reduced inflammation, immunosuppression and less oxidative stress in dairy cows in early lactation. The first objective of this thesis was to study the effects of dry period length and dietary energy source on immune competence, inflammatory biomarkers and oxidative stress in dairy cows over 2 subsequent lactations. The second objective was to study the consequences of maternal dry period length on colostrum immunoglobulin content and immune competence of calves in the first 12 weeks of life. In the current study, 167 cows were assigned to 3 dry period lengths (0, 30, or 60 d) and 2 early lactation rations (glucogenic or lipogenic). Cows were planned to have the same dry period length and ration over 2 subsequent lactations. Omitting the dry period reduced plasma bilirubin levels compared with a conventional dry period, which is line with the better EB in cows with a 0-d dry period. Effects of dry period length on inflammatory biomarkers, oxidative stress variables and natural antibodies (NAb) titers were, however, less consistent. Omitting the dry period increased not only negative acute phase proteins (APP) in plasma, but also positive APP, oxidative stress variables in plasma, and NAb in milk. Shortening the dry period to 30-d did not influence inflammatory biomarkers and oxidative stress compared with a conventional dry period of 60-d. Occurrence of clinical health problems did not differ between cows with different dry period lengths. In the current study, changes in positive APP and oxidative stress variables in plasma and NAb in milk could be explained by the occurrence of clinical health problems related to inflammation (clinical mastitis, fever, metritis and retained placenta), rather than a better EB due to a shorter or no dry period. Moreover, a higher titer of IgG binding lipopolysaccharide in plasma was associated with decreased odds of high somatic cell count and occurrence of clinical mastitis. In the first lactation after implementation of dry period length and dietary treatments, feeding a glucogenic ration in early lactation increased NAb titers in milk compared with a lipogenic ration, which could be explained partly by a better EB. In the second lactation after implementation of dry period length and dietary treatments, feeding a lipogenic ration in early lactation increased cholesterol levels in plasma compared with a glucogenic ration, which could be related to the high fat content in this ration. Cows with a 0-d dry period had a lower colostrum production and less immunoglobulins in colostrum compared with cows with a 30-d or 60-d dry period. After colostrum uptake, NAb titers in plasma of calves from cows with a 0-d dry period were lower during the first week of life compared with calves from cows with a 30-d or 60-d dry period. Levels of specific antibodies in calves, after immunization in week 6 and 10, in calves were not affected by the maternal dry period length. Birth weight of calves from cows with a 0-d dry period was lower compared with calves from cows with a 30-d dry period, but not compared with calves from cows with a 60-d dry period. Growth of calves until 12 weeks of life was not affected by dry period length. In conclusion, although shortening and omitting the dry period improved the EB in early lactation, this did not result in clear consistent effects of dry period length on inflammation or oxidative stress. Changes in inflammation biomarkers, oxidative stress variables and NAb in milk were a reflection of the occurrence of health problems related to inflammation in particular clinical mastitis and compromised uterine health. Furthermore, albeit omitting the dry period compared with shortening or conventional dry period cows resulted in a reduced immunoglobulin content in colostrum and reduced NAb titers in plasma of their calves in the first week of life, but did not affect specific immune response of the calves in the first 12 weeks of life.

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 - 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.

LPS challenge in jonge biggen : VDI-12: effect voerinterventie op biggen
Greeff, Astrid de; Allaart, Janneke ; Bruijn, Carlijn de; Schokker, Dirkjan ; Roubos, Petra ; Winkelman-Goedhart, Hélène ; Vastenhouw, Stéphanie ; Ruuls, Lisette ; Rebel, Johanna ; Smits, Mari - \ 2016
Wageningen : Wageningen Livestock Research (Wageningen Livestock Research rapport 1009) - 21
biggen - maatregel op voedingsgebied - adequate immuniteit - diergezondheid - lipopolysacchariden - varkenshouderij - dierhouderij - immunologie - piglets - nutritional intervention - immune competence - animal health - lipopolysaccharides - pig farming - animal husbandry - immunology
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 p.
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - 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 p.
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.

Effect of fructooligosaccharides on gut health in neonatal piglets : VDI-3 Piglet experimen
Schokker, D. ; Jansen, R. ; Jansman, A.J.M. ; Vastenbouw, S. ; Bree, F.M. de; Bossers, A. ; Rebel, J.M.J. ; Smits, M.A. - \ 2015
Wageningen : Wageningen UR Livestock Research (Livestock Research report 913) - 39
piglets - intestines - animal health - oligosaccharides - intestinal microorganisms - immunology - biggen - darmen - diergezondheid - oligosacchariden - darmmicro-organismen - immunologie
Gut microbial colonization and immune competence development are affected by early-life environmental and dietary interventions. The interplay between microbiota in the intestinal tract and the gut mucosal surfaces of the host is critical for the development of an accurate immune competence. In the present study we intervened during early life of suckling piglets by a daily oral administration of fructooligosaccharides (FOS solution) from day 2 – 14 and investigated the effects on intestinal microbiota composition (by 16S rDNA sequencing) and biological processes of the intestinal mucosal tissue (by genome-wide intestinal gene expression analysis) during the suckling phase.
Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins

The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.

In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.

In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.

Biological processes induced by ZnO, Amoxicillin, Rye and Fructooligosaccharides in cultured Intestinal Porcine Epithelial Cells : VDI-4; In-vitro tests 2013-2014
Hulst, M.M. ; Hoekman, A.J.W. ; Wijers, I. ; Schokker, D. ; Smits, M.A. - \ 2015
Wageningen : Wageningen UR Livestock Research (Livestock Research report 882) - 42
in vitro - bioassays - epithelium - livestock - feed additives - genes - immunology - biotesten - epitheel - vee - voedertoevoegingen - genen - immunologie
The objective of this study was to develop an in-vitro bioassay using cultured Intestinal Porcine Epithelial Cells (IPEC-J2) and evaluate the capability of this assay to predict enterocyte-specific physiological and immunological processes induced by nutrients/additives in the intestines of farm animals. Responses to five nutrients/feed-additives, similar to those studied in animal trials, performed in the Feed4Foodure framework, were measured by gene expression analysis of IPEC-J2 cells either under stressed (Salmonella) or non-stressed conditions. Response genes were analysed using bioinformatics web-tools in order to identify dominant biological processes induced by these nutrients/feed-additives and to predict key-genes/proteins important for regulation of these biological proc
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
Good memories for details improve fish health
Wiegertjes, G.F. - \ 2014
Wageningen : Wageningen University, Wageningen UR - ISBN 9789461739759 - 20
vissen - diergezondheid - immunologie - aquacultuur - celbiologie - vaccinatie - fishes - animal health - immunology - aquaculture - cellular biology - vaccination
Samen een practicum immunologie ontwerpen
Versluis, K. ; Savelkoul, H.F.J. - \ 2014
Bionieuws 24 (2014)9. - ISSN 0924-7734 - p. 14 - 14.
onderwijsmethoden - lesmaterialen - laboratoriummethoden - proeven - immunologie - elisa - docenten - voortgezet onderwijs - teaching methods - teaching materials - laboratory methods - trials - immunology - teachers - secondary education
Een ontwikkelteam van docenten werkt samen met immunologen van Wageningen Universiteit aan een lessenserie. Hoogleraar Savelkoul: "Ik kan in een dag een Elisa-expert van je maken". Het project wordt begeleid door Arjen Nawijn, leraar STOAS.
1Health4Food : focus op gezondheid mens-dier
Kimman, T.G. ; Mevius, D.J. ; Antonis, A.F.G. ; Parée, P. - \ 2014
V-focus 2014 (2014)1. - ISSN 1574-1575 - p. 22 - 24.
veehouderij - dierlijke productie - diergezondheid - volksgezondheid - wetenschappelijk onderzoek - onderzoeksprojecten - diagnostiek - antibioticaresistentie - volksgezondheidsbevordering - verbreed spectrum bèta-lactamases - immuniteit - immuunsysteem - immunologie - voedselveiligheid - voeding en gezondheid - mens-dier relaties - livestock farming - animal production - animal health - public health - scientific research - research projects - diagnostics - antibiotic resistance - sanitation - extended spectrum beta-lactamases - immunity - immune system - immunology - food safety - nutrition and health - human-animal relationships
1Health4Food is een ambitieus onderzoeksprogramma op het gebied van dier- en volksgezondheid. Het landbouwbedrijfsleven heeft in sterke mate bepaald waar de prioriteiten moeten liggen: bij de ESBL’s en de snelle diagnostiek. Binnen 1Health4Food wordt kennis ontwikkeld voor meerdere sectoren, kennis die veehouders en hun adviseurs, zoals dierenartsen, in staat stellen om rendabel te produceren op een wijze die ook veilig is voor de mens. De ambitie is om in de toekomst gezamenlijk een gezonde en veilige veehouderij te realiseren.
Hoe - naar modern inzicht - een garnalenkwekerij op te zetten? : Gesprek met viroloog prof.dr. Just M. Vlak, over WSSV en de immunologische weerstand van een garnaal
Vlak, Just - \ 2013
shrimps - shrimp culture - white spot syndrome virus - animal viruses - vaccination - immunology - immune system
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 - 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.

Immunogenetics in dairy cattle : somatic cell count and natural antibody levels
Wijga, S. - \ 2013
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Henk Bovenhuis; John Bastiaansen. - S.l. : s.n. - ISBN 9789461737274 - 178
melkvee - melkkoeien - immunogenetica - celgetal - natuurlijke antilichamen - genomica - immunologie - genetica - dairy cattle - dairy cows - immunogenetics - somatic cell count - natural antibodies - genomics - immunology - genetics

There remains is a lot to be learned about the interpretation of genetic parameters and the biology of disease resistance and SCS. This PhD thesis aimed to obtain additional insight in disease resistance and SCS by: 1) quantifying the impact of genetics on innate immunity, represented by natural antibodies (NAb), through estimation of heritabilities and genetic correlations; 2) identifying the genomic regions involved in SCS and NAb levels; 3) quantifying the impact of genetics on environmental sensitivity for SCS.

Natural antibody levels are heritable with heritabilities ranging from 0.06 to 0.55 and in general, heritabilities for NAb isotypes were higher than heritabilities for total NAb levels, the latter making no distinction between isotypes. Genetic correlations suggest that isotypes IgA and IgM have a common genetic basis, but that the genetic basis for IgG1 differs from that for IgA or IgM. An additional genome-wide association study for NAb levels showed that information can be gained when total NAb levels are further subdivided into isotype levels. A region on chromosome 23 was significantly associated with genetic variation in isotype IgM levels. The bovine major histocompatibility complex (MHC) is located near this region, making this a region of candidate gene(s) involved in NAb expression in dairy cows both from a functional and positional perspective. Results from the study on genetic parameters and the genome-wide association study suggest that NAb isotypes may provide a better characterization of different elements of the immune response or immune competence and enable more effective decisions when breeding programs start to include innate immune parameters. A genome-wide association study was not only performed for NAb levels, but also for SCS. Relatively few associations, however, were found, which suggests that SCS is controlled by multiple loci, each with a relatively small effect, distributed across the genome.

Somatic cell score is partly under genetic control, but is also affected by the environment. Sensitivity to respond to environmental factors, however, can have a genetic origin.

Environmental factors can be divided into known and unknown factors, referred to as macro- and micro environment, respectively. Macro-environmental sensitivity can be expressed as genetic variation in the slope of a reaction norm, whereas micro-environmental sensitivity can be expressed as differences in residual variance that have a genetic origin. Both macro- and micro-environmental sensitivity were found for SCS and these sensitivities were positively correlated. Knowledge on both forms of sensitivity can aid in optimization of selection as correlations between the additive genetic variance in intercept, slope and environmental variance were all away from unity. Selection for reduced environmental sensitivity has the potential to reduce variability in animal performance due to environmental factors and herewith increase predictability of performance across and within environments.

Knowledge on disease biology is important to fully understand the processes involved when selecting for increased disease resistance, as a better understanding enables a better prediction of the consequences. In this context, the general discussion involved the phenotype definition and statistical modeling, influence of maternal effects and genetic variation in the MHC region. The discussion contained three conclusions: 1) analyses of cell types (detailed phenotypes) rather than SCS can provide further insight in the genetic control of SCS and mastitis; 2) no evidence was found for maternal genetic effects on NAb levels in milk. Maternal environmental effects, however, could play a role in NAb levels; 3) genetic diversity in the MHC region is maintained by natural selection. Selective breeding and farm management practices may affect this genetic diversity, which could bring about negative effects on animal fitness, such as fertility problems. Selective breeding for specific MHC haplotypes may therefore impose a risk for negative effects on animal health.

Culicoides obsoletus allergens for diagnosis of insect bite hypersensitivity in horses
Meide, N.M.A. van der - \ 2013
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - S.l. : s.n. - ISBN 9789461736697 - 228
paarden - equus - culicoides obsoletus - insectenbeten - allergenen - overgevoeligheid - allergieën - cytokinen - diagnose - elisa - vectoren, ziekten - immunologie - horses - insect bites - allergens - hypersensitivity - allergies - cytokines - diagnosis - disease vectors - immunology

AInsect Bite Hypersensitivity (IBH) is the most common skin allergy in horses and involves a Type I (IgE mediated) hypersensitivity reaction against bites of insects, mainly of the Culicoides species. Welfare of affected horses is seriously reduced and no fully curative treatment is yet available. Furthermore, current diagnostic tests are unreliable because of their low sensitivity and specificity. Aim of our research was to increase the understanding of immunological aspects of IBH, with special attention to improving diagnosis by the characterization and production of recombinant allergens.

Whole body extracts (WBE) of three Culicoides species: C. obsoletus C. nubeculosus and C. sonorensis were evaluated for their applicability for diagnosis of IBH in horses in The Netherlands. They were tested for IgE binding by ELISA and Western blotting and for their capacity to degranulate basophils in a histamine release test. For all tests, best results were obtained with C. obsoletus. The ELISA was further evaluated using C. obsoletus extract on approximately 200 IBH affected and healthy horses, which demonstrated high test sensitivity and specificity. C. obsoletus-specific IgE serum levels were found to be the same in the IBH season and off season, suggesting that the test can be used to diagnose horses in winter when clincial symptoms are absent.

Since C. obsoletus was found to be the most important species for diagnosis of IBH in The Netherlands, mRNA of this Culicoides species was sequenced and assembled to create a transcriptome. Using the sequences from in literature described allergens from C. nubeculosus and C. sonorensis, similarity searches were performed on this transcriptome,. This resulted in the identification of seven allergens from C. obsoletus. These allergens were cloned and expressed as recombinant proteins in E. coli and named Cul o 1 – Cul o 7. The frequency of positive test results by ELISA within IBH affected horses ranged from 38 % to 67 %. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test.

The applicability of the 7 C. obsoletus derived recombinant allergens was further evaluated and compared with C. obsoletus WBE in an IgE ELISA using a large number of horses.The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISA’s with a combination of recombinant allergens, combi-1 (Cul o 3, 5 and 7) and combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Both combi-1 and combi-2 resulted in a lower test sensitivity with samples collected in winter compared to samples collected in IBH season, but most IBH affected horses could still also be correctly diagnosed in winter.

The association between several factors and IgE levels against C. obsoletus whole body extract and the 7 recombinant allergens was quantified. Furthermore, the relation between IgE levels and severity of symptoms was examined. Severity of symptoms and IgE levels against several C. obsoletus allergens were found to be related. Factors that were found to be associated with IgE levels were: breed, age, month of scoring, interaction between IBH status and month of scoring, degree of itchiness and number of seasons horses were affected with IBH.

The general discussion discussed the prospects to use the produced recombinant allergens for immunotherapy treatment of IBH affected horses. The panel of all 7 recombinant allergens allows to determine for which exact components of C. obsoletus the IBH horses are allergic (“component resolved diagnosis”). This will enable a tailor made composition of (recombinant) allergens for use in immunotherapy.

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 - 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.

Carp mucus and its role in mucosal defense
Marel, M.C. van der - \ 2012
Wageningen University. Promotor(en): Huub Savelkoul; D. Steinhagen, co-promotor(en): Jan Rombout. - S.l. : s.n. - ISBN 9789461734273 - 183
karper - cyprinus - slijm - verdedigingsmechanismen - vissen - immunologie - glycoproteïnen - carp - mucus - defence mechanisms - fishes - immunology - glycoproteins
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