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Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Fur

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In early life and around weaning piglets are susceptible to infection due to abrupt dietary, social and environmental changes that may directly or indirectly impact gut health and animal performance. In the past, antibiotic growth promoters (AGPs) were intensively used in the livestock industry to prevent the development of gastro-intestinal disorders and to optimise animal performance. However, because of the worldwide concern on the emergence of multi-drug resistant bacteria, the use of in-feed AGPs has been banned in the European Union since 2006. As a result, sustainable alternatives to in-feed AGPs that promote animal health and performance received great interest. In the last few decades, researchers have been studying the effects of several promising microbial and non-microbial nutritional factors for pigs. Pre- and probiotics, for example, have been shown to influence the porcine gut microbiota, improve gut epithelial barrier function and modulate the immune system. It is evident that these complex systems are inherently linked to each other and are important determinants of health and disease. However, dietary interventions are mostly administered post-weaning. During this period, feed additives are less likely to exert strong effects because of the with age increasing resilience of the gut microbiota to (dietary) perturbations. In order to induce transient and/or long-lasting changes, we hypothesized that early life serves as a ‘window of opportunity’ to modulate the gut microbiota, the gut epithelial barrier, and the immune system with the aim to make pigs more resilient to infections during the weaning period and later in life.Dendritic cells (DCs) are important immune cells that link the innate and the adaptive immune system and are the first cells to encounter (dietary) antigens that pass the gut epithelial barrier. In this thesis we assessed the immunostimulating potential of feed additives in vitro, using fresh and cryopreserved bone marrow-derived d

The basophil activation test (BAT) is an ex vivo functional assay that measures by flow cytometry the degree of basophil degranulation after stimulation with an allergen. In recent years, there has been an increased interest in the diagnostic value of the BAT as it has the potential to mimic the clinical phenotype of sIgE sensitized patients, in contrast to allergen-specific IgE levels. This diagnostic potential would be of particular interest for food allergies present early in life such as peanut, cow’s milk and eggs, which require an expensive, time-consuming and patient unfriendly oral food challenge (OFC) for diagnosis. However, routine applications of the BAT for clinical use are not yet feasible due to the lack of standardized protocols and large clinical validation studies. This review will summarize the current data regarding the application of the BAT in food allergy (FA) for cow’s milk, egg and peanut, being the most common causes of FA in children. Additionally, it will discuss the hurdles for widespread clinical use of the BAT and possible future directions for this diagnostic procedure.

Tree nut-allergic individuals are often sensitised towards multiple nuts and seeds. The underlying cause behind a multi-sensitisation for cashew nut, hazelnut, peanut and birch pollen is not always clear. We investigated whether immunoglobulin E antibody (IgE) cross-reactivity between cashew nut, hazelnut and peanut proteins exists in children who are multi-allergic to these foods using a novel IMMULITE®-based inhibition methodology, and investigated which allergens might be responsible. In addition, we explored if an allergy to birch pollen might play a role in this co-sensitisation for cashew nut, hazelnut and peanut. Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to i

Cashew nut allergy has been recognized as a severe tree nut allergy amongst (Dutch) children and young adults and its prevalence seems to be increasing. For clinical diagnosis of an allergy, it is essential to know the causative agents in the food product causing the allergic symptoms.In this thesis entitled ‘Cracking the cashew nut: strategies to identify and characterize novel allergens’, we aimed to apply innovative strategies and technologies to identify and characterize putative allergenic proteins in cashew nut, to broaden the current knowledge on cashew nut allergens beyond those already known (Ana o 1, Ana o 2 and Ana o 3). Our knowledge of cashew nut proteins that can trigger an allergic reaction is currently very limited, especially compared to other nuts or seeds in which the allergen repertoire has been researched much more widely. Using several different strategies, we evidenced that additional allergens must be present in cashew nuts, which presumably contribute to the elicitation of allergic symptoms in cashew nut allergic patients. Knowledge of newly identified cashew nut proteins provides a basis for further research to extend clinical diagnostic tests and treatments currently available for cashew nut allergy.Chapter 2 includes an opinion on the use of current in vivo and ex vivo endpoints in murine food allergy models and their suitability for evaluating the sensitizing capacity of protein concentrates and/or food products. An overview is given of the best predictive risk assessment methods and endpoint parameters currently relied on in in vivo food allergy models with a focus on milk, egg and peanut allergens, addressing their strengths and limitations for assessing sensitization risks. Findings indicated that, although the current available models are suitable for studying the pathophysiology of food allergy, they still couldn’t predict the magnitude of the allergic potential of a particular allergen. Thus, there is still a strong need to better

Young piglets are very susceptible to infections and therefore vaccination early after birth can help to decrease the disease incidence on swine farms. However, vaccination during the neonatal period is a challenge, because vaccination of newborn pigs is in general less successful compared to adult pigs. The innate and adaptive immune system are still developing during early-life and this results in qualitative and quantitative differences between the immune responses of newborn or neonatal pigs (< 1-week-old) and adult or immunocompetent (> 8-week-old) pigs after vaccination. Research has indicated that effective immuno-stimulators (adjuvants) in adult vaccines may not fulfil the requirements to stimulate the neonatal immune system effectively. Therefore, age-group-adapted vaccine formulations may be necessary to enhance the success of vaccination at an early age. The aim of this thesis was to investigate immune responses in neonatal and adult pigs after Toll-like receptor (TLR) stimulation to increase our understanding of the porcine (neonatal) immune system and the immune responses after vaccination in pigs of different age-groups. In addition to conventional intramuscular (i.m.) vaccination, skin vaccination was used as a delivery route, because skin has the potential to increase vaccine efficacy due to its abundance of antigen presenting cells (APCs). Porcine reproductive and respiratory syndrome virus (PRRSV) was selected as vaccine-antigen, because this is an endemic and important disease in the swine farming. In addition, to date no effective inactivated PRRSV-vaccine has been developed yet.In chapter 2, we studied dendritic cells (DCs) and showed that neonatal porcine DCs derived from blood were composed of the same DC subsets and DC proportions as their adult counterparts, namely: plasmacytoid DCs (pDC), and two types of conventional DCs (cDC1 and cDC2). After TLR1/2 or TLR9 stimulation peripheral blood mononuclear cells (PBMCs) and DCs were activated in b

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ECF caused by the hemoprotozoan pathogen Theileria parva is the leading tick-borne disease of cattle in sub-Saharan Africa, causing death of cattle and significant economic losses. Currently control of the disease is dependent on vaccination and drug treatment. The infection and treatment method of vaccination involves deliberate infection with live sporozoites from three strains namely T. parva Muguga, Kiambu5 and Serengeti (known as Muguga cocktail) and simultaneous treatment with a long-acting oxytetracycline. Although effective, this method has serious limitations; the immunity generated by the vaccine is strain specific and immunized cattle can become life-long asymptomatic carriers of the parasite, posing risk for the spread of the disease through introduction of ‘foreign’ T. parva strains in areas where the strains in the vaccine are not present. The production and distribution of the vaccine involves complex procedures requiring infrastructure and specialized expertise which are not always available. These concerns undermine the widespread uptake of this vaccine. There have been efforts to identify sporozoite antigens that are targets of neutralizing antibodies and one protein, p67 is considered the lead candidate for development of a subunit vaccine. However, this protein only protects only a proportion of immunized cattle. Although research is ongoing on different formulations and immunization regimen to improve the efficacy of this antigen, protection against ECF might be improved by additional sporozoite antigens that induce sporozoite neutralizing antibodies. In an attempt to identify such antigens and to support research on the biology of T. parva we employed two strategies. Firstly, we characterized the sporozoite proteome by LC-MS/MS analysis. In total, 4,780 proteins were identified in an enriched preparation of sporozoites. Of these, 2,007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot

Contains fulltext : 196406.pdf (publisher's version ) (Open Access), Innate immune memory, also termed "trained immunity" in vertebrates, has been recently described in a large variety of plants and animals. In most cases, trained innate immunity is induced by pathogens or pathogen-associated molecular patterns (PAMPs), and is associated with long-term epigenetic, metabolic, and functional reprogramming. Interestingly, recent findings indicate that food components can mimic PAMPs effects and induce trained immunity. The aim of this study was to investigate whether bovine milk or its components can induce trained immunity in human monocytes. To this aim, monocytes were exposed for 24 h to beta-glucan, Toll-like receptor (TLR)-ligands, bovine milk, milk fractions, bovine lactoferrin (bLF), and bovine Immunoglobulin G (bIgG). After washing away the stimulus and a resting period of five days, the cells were re-stimulated with TLR ligands and Tumor necrosis factor (TNF-) and interleukin (IL)-6 production was measured. Training with beta-glucan resulted in higher cytokine production after TLR1/2, TLR4, and TLR7/8 stimulation. When monocytes trained with raw milk were re-stimulated with TLR1/2 ligand Pam3CSK4, trained cells produced more IL-6 compared to non-trained cells. Training with bIgG resulted in higher cytokine production after TLR4 and TLR7/8 stimulation. These results show that bovine milk and bIgG can induce trained immunity in human monocytes. This confirms the hypothesis that diet components can influence the long-term responsiveness of the innate immune system.

Both infants and elderly people have compromised immune systems. For infants human milk provides support for the immune system. Infants need immunological support, as their immune system needs time to develop. Furthermore, infants are faced with microbial colonisation and changed nutrition from breast milk towards solid food. In addition to commensal bacteria, infants are increasingly exposed to food- and airborne pathogens as well, which can cause infections in the intestine and airways. As especially, RSV and Influenza infections in the airways can be quite severe, it would be beneficial to improve airway immunity by dietary modulation. Next to this, the epithelial barrier of infants is not fully closed, especially just after birth. As a result intact food an microbial components can cross the epithelium into the mucosa. The infant should not develop allergies or anti-inflammatory immune responses towards these macronutrients and is supposed to induce oral tolerance against both food components as well as to commensal bacteria. Elderly people often suffer from non-communicable inflammation-related inflammatory diseases as a result of immunosenescence and inflammaging. Next to this, elderly respond less to vaccination and are more vulnerable to infections. The ageing population is growing worldwide and therefore the burden of age-related disease on the global health care system is increasing as well. Hence, it would be beneficial when people become older without developing these diseases, which is termed healthy ageing. The main objective of this project was to study the immunomodulatory effect of raw bovine milk and its ingredients in nutritional intervention, oral cholera vaccination and trained immunity and its possible relevance for different stages in life. Consumption of raw bovine milk is associated in epidemiological studies with a reduced incidence of asthma, allergies, rhinitis and respiratory tract infections, which are all related to immunity in the air

The non-specific adsorption of biomolecules, often referred to as fouling, is a recurring issue in many biomedical and bioanalytical applications. For example, the non-specific adsorption of proteins diminishes the signal-to-noise ratio of biosensing systems, while protein coronas formed on drug delivery vehicles hamper the drug to reach its target site due to fast removal by phagocytic cells. Zwitterionic polymer brushes have been brought forward as excellent candidates to prevent fouling. This thesis provides tools and insights to improve antifouling performance in general, to efficiently introduce functionality to zwitterionic polymer brushes and to transfer existing knowledge of antifouling coatings on flat surfaces onto microbeads. In addition, two applications for the antifouling zwitterionic polymer-coated microbeads are also presented. Chapter 1 provides an introduction to antifouling coatings in general and to zwitterionic polymer brushes in particular. This chapter also introduces the concept of a romantic surface: a surface that repels all biomolecules except that special one it is interested in. Chapter 2 describes a novel approach on how to create such romantic surfaces. An azide-functionalized sulfobetaine is introduced that enables the 3D-functionalization of fully zwitterionic polymer brushes. The number of built-in functional groups could be tuned by the copolymerization of this novel azido-SB monomer with a standard sulfobetaine. The incorporated azide moieties can then be used to couple recognition units of choice using “click” chemistry. Functionalization of the brushes with biotin units enabled the specific binding of avidin without the interference of the non-specific binding of fibrinogen. Particles of various sizes are being used for a wide range of biomedical application, including drug delivery, imaging, cells sorting and as biosensing platform. As on flat surfaces, fouling compromises the performance of these particles. Chapter 3 describes

In a growing world population in which chronic inflammatory diseases are on the rise and allergies and asthma affect 30-40% of the population, there is a pressing need to understand underlying mechanisms to develop successful preventive strategies for these diseases. Raw cow’s milk consumption in early life has been associated in epidemiological studies with a significant decrease in the prevalence of allergies and asthma. In this thesis we investigated the effect of components present in raw cow’s milk on maintenance of mucosal immune functioning in the gut and discussed their implications for respiratory health. We thereby give novel insights into the underlying mechanisms by which raw cow’s milk components display their immunomodulatory capacity and may contribute to regulation of mucosal immune responses. Future studies should further investigate the effects of these components in in vivo allergy and respiratory infection models, to better understand the link between diet and respiratory health.

This review aims to provide an in depth overview of the current knowledge of the effects of bovine immunoglobulins on the human immune system. The stability and functional effects of orally ingested bovine immunoglobulins in milk products are described and potential mechanisms of action are discussed. Orally ingested bovine IgG (bovine IgG) can be recovered from feces, ranging from very low levels up to 50% of the ingested IgG that has passed through the gastrointestinal tract. In infants the recovered levels are higher than in adults most likely due to differences in stomach and intestinal conditions such as pH. This indicates that bovine IgG can be functionally active throughout the gastrointestinal tract. Indeed, a large number of studies in infants and adults have shown that bovine IgG (or colostrum as a rich source thereof) can prevent gastrointestinal tract infections, upper respiratory tract infections, and LPS-induced inflammation. These studies vary considerably in target group, design, source of bovine IgG, dosage, and endpoints measured making it hard to draw general conclusions on effectiveness of bovine immunoglobulin rich preparations. Typical sources of bovine IgG used in human studies are serum-derived IgG, colostrum, colostrum-derived IgG, or milk-derived immunoglobulins. In addition, many studies have used IgG from vaccinated cows, but studies using IgG from nonimmunized animals have also been reported to be effective. Mechanistically, bovine IgG binds to many human pathogens and allergens, can neutralize experimental infection of human cells, and limits gastrointestinal inflammation. Furthermore, bovine IgG binds to human Fc receptors which, enhances phagocytosis, killing of bacteria and antigen presentation and bovine IgG supports gastrointestinal barrier function in in vitro models. These mechanisms are becoming more and more established and explain why bovine IgG can have immunological effects in vivo. The inclusion of oral bovine immunoglobuli

Class-switching of B cells to IgA can be induced via both T-cell-dependent and T-cell-independent mechanisms. IgA is most predominantly produced mucosally and is important for combating infections and allergies. In contrast to mice, humans have two forms of IgA; IgA1 and IgA2 with diverse tissue distribution. In early life, IgA levels might be sub-optimal especially during the fall season when bacterial and viral infections are more common. Therefore, we investigated using human B cells whether T-cell-independent factors -promoting cell survival, class switching and immunoglobulin secretion- BAFF, APRIL, IL-10 and retinoic acid can boost IgA production in the context of viral or bacterial infection. To this end total and naive peripheral blood B cells were stimulated with these factors for 6 days in the presence or absence of TLR7/8 agonist R848 (mimicking viral infection) or TLR9 agonist CpG-ODN (mimicking bacterial infection). We show that BAFF significantly augments IgA2 production in TLR7/8 stimulated mature, but not naïve B cells. In addition, BAFF augments IL-10 production and viability in TLR7/8 and TLR9 stimulated mature B cells. These data warrant further investigation of its role in immune regulation both in the periphery and mucosal tissues in early life or during disease.

Advanced age is associated with chronic low-grade inflammation, which is usually referred to as inflammaging. Elderly are also known to have an altered gut microbiota composition. However, whether inflammaging is a cause or consequence of an altered gut microbiota composition is not clear. In this study gut microbiota from young or old conventional mice was transferred to young germ-free mice. Four weeks after gut microbiota transfer immune cell populations in spleen, Peyer’s patches, and mesenteric lymph nodes from conventionalized germ-free mice were analyzed by flow cytometry. In addition, whole-genome gene expression in the ileum was analyzed by microarray. Gut microbiota composition of donor and recipient mice was analyzed with 16S rDNA sequencing. Here we show by transferring aged microbiota to young germ-free mice that certain bacterial species within the aged microbiota promote inflammaging. This effect was associated with lower levels of Akkermansia and higher levels of TM7 bacteria and Proteobacteria in the aged microbiota after transfer. The aged microbiota promoted inflammation in the small intestine in the germ-free mice and enhanced leakage of inflammatory bacterial components into the circulation was observed. Moreover, the aged microbiota promoted increased T cell activation in the systemic compartment. In conclusion, these data indicate that the gut microbiota from old mice contributes to inflammaging after transfer to young germ-free mice.

A dysbiosis in the intestinal microbiome plays a role in the pathogenesis of several immunological diseases. These diseases often show a gender bias, suggesting gender differences in immune responses and in the intestinal microbiome. We hypothesized that gender differences in immune responses are associated with gender differences in microbiota. We demonstrated mouse strain dependent gender differences in the intestinal microbiome. Interestingly, a cluster of colonic genes (related to humoral and cell-mediated immune responses) correlated oppositely with microbiota species abundant in B6 females and in BALB/c males. This suggests that with different genetic backgrounds, gender associated immune responses are differentially regulated by microbiota. The net result was the same, since both mouse strains showed similar gender induced differences in immune cell populations in the mesenteric lymph nodes. Therefore, host-microbe interactions might be more complicated than assumed, as bacterial-species adaptations might be highly dependent on the genetic make-up of the individual.

A dysbiosis in the intestinal microbiome plays a role in the pathogenesis of several immunological diseases. These diseases often show a gender bias, suggesting gender differences in immune responses and in the intestinal microbiome. We hypothesized that gender differences in immune responses are associated with gender differences in microbiota. We demonstrated mouse strain dependent gender differences in the intestinal microbiome. Interestingly, a cluster of colonic genes (related to humoral and cell-mediated immune responses) correlated oppositely with microbiota species abundant in B6 females and in BALB/c males. This suggests that with different genetic backgrounds, gender associated immune responses are differentially regulated by microbiota. The net result was the same, since both mouse strains showed similar gender induced differences in immune cell populations in the mesenteric lymph nodes. Therefore, host-microbe interactions might be more complicated than assumed, as bacterial-species adaptations might be highly dependent on the genetic make-up of the individual.

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Contains fulltext : 177266.pdf (publisher's version ) (Open Access), Males and females are known to have gender-specific differences in their immune system and gut microbiota composition. Whether these differences in gut microbiota composition are a cause or consequence of differences in the immune system is not known. To investigate this issue, gut microbiota from conventional males or females was transferred to germ-free (GF) animals of the same or opposing gender. We demonstrate that microbiota-independent gender differences in immunity are already present in GF mice. In particular, type I interferon signaling was enhanced in the intestine of GF females. Presumably, due to these immune differences bacterial groups, such as Alistipes, Rikenella, and Porphyromonadaceae, known to expand in the absence of innate immune defense mechanism were overrepresented in the male microbiota. The presence of these bacterial groups was associated with induction of weight loss, inflammation, and DNA damage upon transfer of the male microbiota to female GF recipients. In summary, our data suggest that microbiota-independent gender differences in the immune system select a gender-specific gut microbiota composition, which in turn further contributes to gender differences in the immune system.

The prevalence of autism spectrum disorders (ASDs) is causally dependent on genetic and environmental influences. We investigated whether autism spectrum disorders are associated with month of birth compared to the general population using a retrospective study, comparing ASD cases (n = 3478) with the general population (n = 2,716,876) born between 1995 and 2008. Associations were examined using χ2 tests and Walter and Elwood’s seasonality χ2 tests for the total ASD group, and separately for autistic disorder and Asperger syndrome. For the total ASD group, the distribution of month of birth was different compared to the general population (p < 0.0001), with July as the highest contributor, and a season-of-birth effect was found for this group (p = 0.02). For the autistic disorder group, the months of birth distribution were different (p = 0.01), with July as the highest contributor. No season-of-birth effect over the year was found (p = 0.09). No association was found for the months of birth of individuals with Asperger syndrome (p = 0.06), with no seasonal trend over the year (p = 0.60). In conclusion, a drop in sun exposure during the first trimester of pregnancy might explain the peak in July births and the associated risk for ASD development.

Background: The double-blind, placebo-controlled food challenge test (DBPCFC) is the gold standard in cashew nut allergy. This test is costly, time consuming and not without side effects. Analysis of IgE reactivity to cashew nut components may reduce the need for food challenge tests. Methods: In a prospective and multicentre study, children with suspected cashew nut allergy underwent a DBPCFC with cashew nut. Specific IgE to cashew nut and to the components Ana o 1, 2 and 3 were determined. A skin prick test (SPT) with cashew nut extract was performed. The association between the outcome of the food challenge test and specific IgE to Ana o 1, 2 and 3 was assessed with logistic regression analyses, unadjusted and adjusted for other diagnostic variables. Discriminative ability was quantified with a concordance index (c). Results: A total of 173 children (103 boys, 60%) with a median age of 9 years were included. About 79% had a positive challenge test outcome. A steep rise in the risk of a positive challenge was observed for specific IgE to each individual component Ana o 1, 2 and 3 with estimated risks up to approximately 100%. Median values of Ana o 1, 2, 3 were 1.29 kU/l (range 0–100 kU/l), 4.77 kU/l (range 0–100 kU/l) and 8.33 kU/l (range 0–100 kU/l) respectively and varied significantly (p < 0.001). Specific IgE to Ana o 1, 2 and 3 was better distinguished between cashew-allergic and tolerant children (c = 0.87, 0.85 and 0.89, respectively) than specific IgE to cashew nut or SPT (c = 0.76 and 0.83, respectively). Conclusion: The major cashew nut allergens Ana o 1, 2 and 3 are each individually predictive for the outcome of food challenge tests in cashew-allergic children.

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