Perfluorooctanesulfonic acid (PFOS) disrupts cadherin-16 in the developing rat thyroid gland.

Perfluorooctanesulfonic acid (PFOS) can disrupt the thyroid hormone (TH) system in rodents, potentially affecting perinatal growth and neurodevelopment. Some studies also suggest that gestational exposure to PFOS can lead to lower TH levels throughout life, indicating that PFOS may compromise thyroid gland development. To address this question, we utilized a rat thyroid gland ex vivo culture system to study direct effects of PFOS on the developing thyroid. No significant changes to follicular structure or size were observed with 1 µM or 10 µM PFOS exposure. However, the transcription factor Foxe1, together with Tpo and Lrp2, were upregulated, whereas the key transcription factor Pax8 and its downstream target gene Cdh16 were significantly downregulated at the transcript level, observed with both RT-qPCR and RNAscope. Notably, Cdh16 expression was not uniformly downregulated across Cdh16-postive cells, but instead displayed a patchy expression pattern across the thyroid gland. This is a significant change in expression pattern compared to control thyroids where Cdh16 is expressed relatively uniformly. The disrupted expression pattern was also seen at the protein level. This suggests that PFOS exposure can impact follicular growth and structure. Compromised follicle integrity, if irreversible, could help explain reduced TH synthesis postnatally. This view is supported by observed changes to Tpo and Lrp2 expression, two factors that play a role in TH synthesis.

Allergenicity evaluation of quinoa proteins - A study in Brown Norway rats.

The popularity of quinoa seeds has increased in the last decade due to their high nutritional value and natural gluten-free composition. Consumption of new proteins may pose a risk of introducing new allergies. In the present study the immunogenicity and sensitising capacity of quinoa proteins were assessed in a dose-response experiment in Brown Norway rats in comparison to proteins from spinach and peanut. Cross-reactivity between quinoa proteins and known allergens was evaluated by in silico analyses followed by analyses with 11 selected protein extracts and their anti-sera by means of ELISAs and immunoblotting. Further, an in vitro simulated gastro-duodenal digestion was performed. Quinoa proteins were found to have an inherent medium to high immunogenicity and sensitising capacity, being able to induce specific IgG1 and IgE levels higher than spinach but lower than peanut and elicit reactions of clinical relevance similar to peanut. Quinoa proteins were generally shown to resist digestion and retain capacity to bind quinoa-specific antibodies. Quinoa proteins were shown to be cross-reactive with peanut and tree nut allergens as high sequence homology and antibody cross-binding were demonstrated. Present study suggests that quinoa pose a medium to high level of allergenicity that should be further investigated in human studies.

Intestinal protein uptake and IgE-mediated food allergy.

Food allergy is affecting 5-8% of young children and 2-4% of adults and seems to be increasing in prevalence. The cause of the increase in food allergy is largely unknown but proposed to be influenced by both environmental and lifestyle factors. Changes in intestinal barrier functions and increased uptake of dietary proteins have been suggested to have a great impact on food allergy. In this review, we aim to give an overview of the gastrointestinal digestion and intestinal barrier function and provide a more detailed description of intestinal protein uptake, including the various routes of epithelial transport, how it may be affected by both intrinsic and extrinsic factors, and the relation to food allergy. Further, we give an overview of in vitro, ex vivo and in vivo techniques available for evaluation of intestinal protein uptake and gut permeability in general. Proteins are digested by gastric, pancreatic and integral brush border enzymes in order to allow for sufficient nutritional uptake. Absorption and transport of dietary proteins across the epithelial layer is known to be dependent on the physicochemical properties of the proteins and their digestion fragments themselves, such as size, solubility and aggregation status. It is believed, that the greater an amount of intact protein or larger peptide fragments that is transported through the epithelial layer, and thus encountered by the mucosal immune system in the gut, the greater is the risk of inducing an adverse allergic response. Proteins may be absorbed across the epithelial barrier by means of various mechanisms, and studies have shown that a transcellular facilitated transport route unique for food allergic individuals are at play for transport of allergens, and that upon mediator release from mast cells an enhanced allergen transport via the paracellular route occurs. This is in contrast to healthy individuals where transcytosis through the enterocytes is the main route of protein uptake. Thus, knowledge on factors affecting intestinal barrier functions and methods for the determination of their impact on protein uptake may be useful in future allergenicity assessments and for development of future preventive and treatment strategies.

Camel Milk Cannot Prevent the Development of Cow's Milk Allergy-A Study in Brown Norway Rats.

SCOPE: Currently there are no specific recommendations for the use of any particular infant formula in the prevention of cow's milk allergy (CMA). Recently, there has been an increasing interest in alternative infant formulas based on milk proteins from other sources than the cow, including milk from other mammalians such as goat, sheep, donkey, horse, and camel. Whereas these have been studied for their usability in CMA management, there are no studies of their CMA preventive capacity. Thus, the aim of this study is to evaluate whether camel milk can prevent CMA and vice versa. METHODS AND RESULTS: The capacity of camel milk in preventing CMA and vice versa is evaluated in a well-established prophylactic Brown Norway rat model. IgG1, IgE, and IgA responses, allergy elicitation, intestinal and mLN gene expression, and protein uptake are analyzed. The study demonstrates that camel and cow's milk in general has an insignificant cross-preventive capacity. Yet, whereas cow's milk is shown to have a low transient capacity to prevent sensitization and clinically active camel milk allergy, camel milk does not show this effect for CMA. CONCLUSIONS: This study suggests that due to lack of cross-tolerance camel milk cannot be used for CMA prevention.

The role of skin inflammation, barrier dysfunction, and oral tolerance in skin sensitization to gluten-derived hydrolysates in a rat model.

BACKGROUND: Adverse reactions to wheat-containing skin care products have been linked to food allergy development. OBJECTIVES: To determine the role of skin barrier dysfunction and inflammation in sensitization to gluten-derived hydrolysates via the skin in Brown Norway rats with and without oral tolerance to wheat. METHODS: Skin barrier defect was induced by mechanical disruption, and skin inflammation was induced by topical application of SLS or MC903. Unmodified, enzyme hydrolyzed, or acid hydrolyzed gluten products were applied to the skin three times per week for 5 weeks. Subsequently, rats were orally gavaged with unmodified gluten. RESULTS: Wheat-naïve rats were readily sensitized to gluten hydrolysates via the skin. Skin barrier defect and skin inflammation had little effect on the skin sensitization and hydrolysate-specific IgE levels. Oral administration of unmodified gluten promoted the production of unmodified gluten-specific IgE in rats sensitized via the skin. Sensitization through intact skin, disrupted skin barrier, or inflamed skin was unable to break tolerance to unmodified gluten in rats on a wheat-containing diet. CONCLUSIONS: Mechanical skin barrier disruption and skin inflammation play a limited role in experimental skin sensitization to gluten-derived hydrolysates.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

Endocrine disrupting chemicals (EDCs) are a matter of great concern. They are ubiquitous in the environment, are considered harmful to humans and wildlife, yet remain challenging to identify based on current international test guidelines and regulatory frameworks. For a compound to be identified as an EDC within the EU regulatory system, a plausible link between an endocrine mode-of-action and an adverse effect outcome in an intact organism must be established. This requires in-depth knowledge about molecular pathways regulating normal development and function in animals and humans in order to elucidate causes for disease. Although our knowledge about the role of the endocrine system in animal development and function is substantial, it remains challenging to predict endocrine-related disease outcomes in intact animals based on non-animal test data. A main reason for this is that our knowledge about mechanism-of-action are still lacking for essential causal components, coupled with the sizeable challenge of mimicking the complex multi-organ endocrine system by methodological reductionism. Herein, we highlight this challenge by drawing examples from male reproductive toxicity, which is an area that has been at the forefront of EDC research since its inception. We discuss the importance of increased focus on characterizing mechanism-of-action for EDC-induced adverse health effects. This is so we can design more robust and reliable testing strategies using non-animal test methods for predictive toxicology; both to improve chemical risk assessment in general, but also to allow for considerable reduction and replacement of animal experiments in chemicals testing of the 21st Century.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Quinoa (Chenopodium quinoa Willd.) Seeds Increase Intestinal Protein Uptake.

SCOPE: Within the last decade, quinoa seeds have gained much popularity as a new food and have recently been proposed as an appropriate food for early introduction in infants. Quinoa contains high levels of saponins, which are known for their adjuvant activity and effect on the intestinal barrier function. The aim of this study is to investigate the impact of quinoa on intestinal permeability and inflammation in comparison with the positive controls; cholera toxin (CT), and capsaicin. METHODS AND RESULTS: The effect of quinoa on intestinal barrier function and inflammation is investigated in vitro using a Caco-2 cell line and in vivo using a Brown Norway rat model. Effects in vivo are analyzed by protein uptake, histology, gene expression, antibody levels, and flow cytometry. Quinoa and the positive controls all increased the intestinal permeability, but distinct patterns of absorbed protein are observed in the epithelium, Peyer's patches, lamina propria, and serum. The quinoa-mediated effect on intestinal barrier function is found to be distinct from the effect of the two positive controls. CONCLUSION: The findings demonstrate the ability of quinoa to increase intestinal permeability and to promote compartment-specific protein uptake via mechanisms that may differ from CT and capsaicin.

Short-Term Amoxicillin-Induced Perturbation of the Gut Microbiota Promotes Acute Intestinal Immune Regulation in Brown Norway Rats.

The intestinal gut microbiota is essential for maintaining host health. Concerns have been raised about the possible connection between antibiotic use, causing microbiota disturbances, and the increase in allergic and autoimmune diseases observed during the last decades. To elucidate the putative connection between antibiotic use and immune regulation, we have assessed the effects of the antibiotic amoxicillin on immune regulation, protein uptake, and bacterial community structure in a Brown Norway rat model. Daily intra-gastric administration of amoxicillin resulted in an immediate and dramatic shift in fecal microbiota, characterized by a reduction of within sample (α) diversity, reduced variation between animals (ß diversity), increased relative abundance of Bacteroidetes and Gammaproteobacteria, with concurrent reduction of Firmicutes, compared to a water control group. In the small intestine, amoxicillin also affected microbiota composition significantly, but in a different way than observed in feces. The small intestine of control animals was vastly dominated by Lactobacillus, but this genus was much less abundant in the amoxicillin group. Instead, multiple different genera expanded after amoxicillin administration, with high variation between individual animals, thus the small intestinal α and ß diversity were higher in the amoxicillin group compared to controls. After 1 week of daily amoxicillin administration, total fecal IgA level, relative abundance of small intestinal regulatory T cells and goblet cell numbers were higher in the amoxicillin group compared to controls. Several bacterial genera, including Escherichia/Shigella, Klebsiella (Gammaproteobacteria), and Bifidobacterium, for which the relative abundance was higher in the small intestine in the amoxicillin group than in controls, were positively correlated with the fraction of small intestinal regulatory T cells. Despite of epidemiologic studies showing an association between early life antibiotic consumption and later prevalence of inflammatory bowel diseases and food allergies, our findings surprisingly indicated that amoxicillin-induced perturbation of the gut microbiota promotes acute immune regulation. We speculate that the observed increase in relative abundance of small intestinal regulatory T cells is partly mediated by immunomodulatory lipopolysaccharides derived from outgrowth of Gammaproteobacteria.

An Animal Model for Wheat Allergy Skin Sensitisation: A Comparative Study in Naive versus Tolerant Brown Norway Rats.

BACKGROUND: Allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food, leading to the assumption that food allergy sensitisation may occur through the skin. Concerns have been raised regarding the safety of use of personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy through the skin, and even cause an abrogation of an already established oral tolerance. OBJECTIVE: To establish an animal model for food allergy skin sensitisation and compare the sensitising capacity of an unmodified and an acid-hydrolysed gluten product via slightly damaged skin in naïve versus tolerant rats. METHODS: Gluten products were applied on the slightly damaged skin of naïve or tolerant Brown Norway (BN) rats without adjuvant 3 times per week for 3 or 5 consecutive weeks. The effect of the skin applications was evaluated by means of different ELISAs and immunoblotting. RESULTS: A robust animal model was developed for food allergy skin sensitisation. In naïve rats, both gluten products were able to induce a statistically significant level of specific antibodies and sensitise through the skin, but in the wheat-tolerant rats, only the acid-hydrolysed gluten was able to sensitise through the skin, albeit at a level much lower than in the naïve rats. Results showed that new epitopes had been developed as a result of acid hydrolysis but original epitopes were maintained. This may explain why only the acid-hydrolysed gluten could induce specific antibody responses in the tolerant animals. CONCLUSIONS: This study showed that it is possible to sensitise BN rats through slightly damaged skin, and that the sensitising capacity is heavily influenced by the tolerance status of their immune system and the degree of modification of the wheat products.

Comparison of the Allergenicity and Immunogenicity of Camel and Cow's Milk-A Study in Brown Norway Rats.

BACKGROUND: When breastfeeding is impossible or insufficient, the use of cow's milk-based hypoallergenic infant formulas is an option for infants suffering from or at risk of developing cow's milk allergy. As the Camelidae family has a large evolutionary distance to the Bovidae family and as camel milk differs from cow's milk protein composition, there is a growing interest in investigating the suitability of camel milk as an alternative to cow's milk-based hypoallergenic infant formulas. METHODS: The aim of the study was to compare the allergenicity and immunogenicity of camel and cow's milk as well as investigating their cross-reactivity using a Brown Norway rat model. Rats were immunised intraperitoneally with one of four products: camel milk, cow's milk, cow's milk casein or cow's milk whey fraction. Immunogenicity, sensitising capacity, antibody avidity and cross-reactivity were evaluated by means of different ELISAs. The eliciting capacity was evaluated by an ear swelling test. RESULTS: Camel and cow's milk showed similarity in their inherent immunogenicity, sensitising and eliciting capacity. Results show that there was a lower cross-reactivity between caseins than between whey proteins from camel and cow's milk. CONCLUSIONS: The study showed that camel and cow's milk have a low cross-reactivity, indicating a low protein similarity. Results demonstrate that camel milk could be a promising alternative to cow's milk-based hypoallergenic infant formulas.