Search

Background: We examined the feasibility, efficacy and safety of slow low-dose oral immunotherapy (SLOIT) for egg, milk, wheat allergies, with accepted severity-stratified initial and maintenance doses., Methods: Children with food allergies defined by low-dose oral food challenges (LD-OFCs) to hen's egg (cumulative protein dose up to 983 mg, n = 133), cow's milk (287 mg, n = 50), and wheat (226 mg, n = 45) were recruited. Participants were divided into two groups [SLOIT and control (complete avoidance]) based on their preferences. Participants who selected SLOIT were instructed to take the safe dose daily, with monthly increases, aiming to increase the dose by 10 times in one year. The primary outcome was the proportion of participants who passed the LD-OFCs following 1 year of therapy., Results: The participants in SLOIT group ingested their antigen 92.9% of the therapy's day on average. The proportion of participants who passed LD-OFCs was 35.9% (61/170) in the SLOIT group and 8.7% (4/46) in the control group (P < .001); no large differences were observed among allergens. Among the subjects who failed LD-OFCs, the median change in the total dose in the LD-OFC was 235% (interquartile range: 100%-512%) in the SLOIT group and 100% (42%-235%) in the control group (P < .001). Provoked allergic symptoms were observed in only 0.58% (280/48,486) per programmed intake and approximately 50% of the SLOIT group did not experience any obvious allergic symptoms throughout therapy., Conclusions: SLOIT showed significant feasibility, efficacy and safety, providing a promising option to manage patients with severe food allergies., (Copyright © 2020 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Exosomes are extracellular vesicles involved in intercellular communication. The objectives were to characterize bovine milk exosomes (BME) and determine its effect on RAW 264.7 macrophages., Methods: BME were isolated using differential centrifugation and characterized by particle size and the presence of exosomal markers Alix, TSG101, and CD81. The effect of in vitro digestion and different pH on the stability of BME was investigated. The biological activity of BME in RAW 264.7 macrophages was conducted by assessing proliferation and cell cycle. Moreover, the protective effect of exosomes on cisplatin-induced cytotoxicity was evaluated., Results: BME have an average particle size of 106.8 ± 3.4 nm and expressed Alix, TSG101, and CD81. TSG101 was detected after digestion and exposure to different pH values. Cell-cycle analysis showed that BME reduced the percentage of apoptotic cells while arresting the cells in G2/M phase accompanied by differential expression of proliferation markers p53, p21, cyclin D1, and β-catenin. Exosomes protected macrophages against cisplatin-induced cytotoxicity., Conclusion: Our results showed for the first time the effect of BME on the proliferation of RAW 264.7 macrophages and its protective effect against chemotherapeutic drug-induced cytotoxicity. Potential effect of BME on immune system must be studied.

Background: The role of breast-feeding in the development of oral tolerance and allergic diseases is controversial, which could be related to variability in milk components. Toll-like receptor 2 (TLR2) is an innate immune receptor implicated in regulating allergic disease development., Objectives: We examined whether deficiency of maternal TLR2 affects the normal development of oral tolerance and related immune parameters during lactation in a mouse model., Methods: Heterozygous TLR2 +/- pups from wild-type (WT) or TLR2 -/- dams were fed either by their biologic dam or a dam of the alternate genotype. Development of oral tolerance to ovalbumin, levels of tolerogenic CD103 + dendritic cells, and regulatory T (Treg) cells, as well as intestinal permeability, were evaluated in these pups. The levels of key immune mediators in milk from TLR2 -/- and WT mothers were also examined., Results: Heterozygous TLR2 +/- pups that were born to and nursed by TLR2 -/- dams exhibited impaired oral tolerance. This was prevented by cross-fostering onto WT (TLR2 +/+ ) dams. Impairments included selective elevation in anti-ovalbumin IgE in plasma following immunization, reduced numbers of tolerogenic dendritic cells and Treg cells in the intestinal tract, and increased intestinal permeability. TLR2 deficiency also affected milk content of insulin-like growth factor-1, IFN-γ, IL-6, and IL-13., Conclusion: Our results underline a critical role for TLR2 in regulating milk components that are essential for development of oral tolerance in early life and demonstrate the importance of considering the immune status of nursing mothers in studies of immune development and responses., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Formulas adapted to infant feeding, although most of the time made from cow's milk proteins, can be made from hydrolyzed rice protein but they must be classified as "formulas for specific medical needs", according to European regulations. The nutritional quality of rice proteins is thus suitable to be used in infant formulas giving that it is supplemented by certain amino acids which can be lacking. Besides, hydrolysis is required to facilitate their water solubility and digestibility. Owing to a low allergenicity of rice and to the absence of the cross-allergy between milk proteins and rice proteins, these formulas are adapted to the diet of children with cow's milk protein allergy (CMPA), which explains their growing use in some countries. However, CMPA, an expanding disorder, has consequences for growth, bone mineralization, and often has an association with allergy to other foods, including cow's milk extensive hydrolysate, so that a surveillance of the adaption of hydrolyzed rice protein formulas (HRPF) to CMPA, the absence of unexpected side effects, and the appropriate response to its various health hazards seems mandatory. This paper analyses the health problem deriving from CMPA, the industrial development of hydrolyzed rice protein formulas, and the limited number of clinical studies, which confirms, at the moment, a good allergic tolerance and safety. The goal is to better advise heath care professionals on their use of HRPFs during CMPA.

Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells ( p < 2.3 × 10 -9 ). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2 s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7 , was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.

Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.

A national control program against bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) was launched in Norway in 2016. A key strategy in the program is to test for presence of antibodies and protect test-negative herds from infection. Because these viruses are endemic, the rate of re-introduction can be high, and a disease-free status will become more uncertain as time from testing elapses. The aim of this study was to estimate the probability of freedom (PostPFree) from BRSV and BCV antibodies over time by use of bulk tank milk (BTM) antibody-testing, geographic information and animal movement data, and to validate the herd-level estimates against subsequent BTM testing. BTM samples were collected from 1148 study herds in West Norway in 2013 and 2016, and these were analyzed for BRSV and BCV antibodies. PostPFree was calculated for herds that were negative in 2013/2014, and updated periodically with new probabilities every three months. Input variables were test sensitivity, the probability of introduction through animal purchase and local transmission. Probability of introduction through animal purchase was calculated by using real animal movement data and herd prevalence in the region of the source herd. The PostPFree from the final three months in 2015 was compared to BTM test results from March 2016 using a Wilcoxon Rank Sum Test. The probability of freedom was generally high for test-negative herds immediately after testing, reflecting the high sensitivity of the tests. It did however, decrease with time since testing, and was greatly affected by purchase of livestock. When comparing the median PostPFree for the final three months to the test results in 2016, it was significantly lower (p < 0.01) for test positive herds. Furthermore, there was a large difference in the proportion of test positive herds between the first and fourth quartile of PostPFree. The results show that PostPFree provides a better estimate of herd-level BTM status for both BRSV and BCV than what can be achieved by relying solely on the previous test-result., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hypoallergenic formulas are recommended for infants who are not breastfed and cannot tolerate cow milk formulas due to allergy. These formulas are hydrolyzed to break down larger protein chains into shorter, easy-to-digest, and potentially less allergenic proteins. Hydrolysis, however, possibly occurs at the expense of the transforming growth factor beta (TGF-β) and anti-inflammatory activity that is inherent in regular formula. Our objective was to determine the TGF-β and the anti-inflammatory activity of commercially available hypoallergenic and regular formulas. Human gingival fibroblasts were incubated with reconstituted formulas followed by detection of TGF-β target genes and activation of Smad2/3 signaling. Gingival fibroblasts and the oral squamous cell carcinoma cell line HSC-2 were also exposed to formulas before adding interleukin (IL)1β and tumor necrosis factor (TNF)α to provoke expression of pro-inflammatory cytokines. For murine bone marrow-derived macrophages, pro-inflammatory cytokine expression was stimulated with saliva. Changes in p65 nuclear translocation and phosphorylation of smad3 and p38 were analyzed by immunostaining. Our study demonstrated that regular formula, but not hypoallergenic formula, enhanced the expression of TGF-β target genes IL11, PRG4, and NOX4 in gingival fibroblasts. Hypoallergenic formulas also failed to initiate nuclear translocation of Smad2/3 and phosphorylation of Smad3. Moreover, regular formulas were more potent than hypoallergenic formulas in reducing the expression of pro-inflammatory cytokines in gingival fibroblasts, HSC-2 epithelial cells, and murine bone marrow macrophages. Hypoallergenic and regular formulas had a similar capacity to reduce p65 nuclear translocation and phosphorylation of p38 in fibroblasts. These findings suggest that hypoallergenic formulas lack in vitro TGF-β activity and have a lower anti-inflammatory activity compared with regular formulas., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In this review, we discuss an immunobiology model of farm exposure towards the protective effect of asthma. Unraveling the protective effect of farming exposure could help develop novel strategies to prevent asthma. Asthma is a chronic airway inflammation that causes coughing, wheezing, chest tightness or shortness of breath. The reasons for the increase in the prevalence of asthma worldwide is still unclear but has been hypothesized to be attributable to westernization/urbanization of rural regions thus resulting in the loss of rural farming environmental. In this review we discuss the effect of the environmental factors, specifically farming, on the risk of asthma in children. Here, we will summarize the main findings of 27 studies related to 11 different cohorts. Several studies have shown preventive effect of traditional farming on the prevalence and incidence of asthma in childhood. Furthermore, consumption of unpasteurized cow's milk, exposure to farm animals as well as fodder have been shown to have a protective effect on asthma. The precise mechanism of the protective effect is still unclear. There are assumptions, that maternal/childhood exposures to farm animals result in higher microbial exposures through which the protective effect might be mediated. Also, consumption of unpasteurized milk (when consumed during pregnancy by mother or early childhood by children) can modulate cytokine production patterns which could be responsible for the observed protective effect. CONCLUSION: This review provides evidence of the protective effect of farming environment i.e., exposure to farm animals, their fodder as well as consumption of unpasteurized cow's milk suggesting that novel strategies could be developed to prevent asthma., (Copyright © 2020 by Academy of Sciences and Arts of Bosnia and Herzegovina.)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes severe respiratory tract infections in humans (COVID-19), has become a global health concern. Currently, several vaccine candidates against SARS-CoV-2 are in clinical trials but approval of these vaccines is likely to take a long time before they are available for public use. In a previous report, the importance of passive immunity and how immunoglobulin (Ig)G collected from recovered coronavirus patients could help in the protection against COVID-19 and boost the immune system of new patients was reported. Passive immunity by immunoglobulin transfer is a concept employed by most mammals and bovine IgG has a role to play in human therapy. IgG is one of the major components of the immunological activity found in cow's milk and colostrum. Heterologous transfer of passive immunity associated with the consumption of bovine immune milk by humans has been investigated for decades for its immunological activity against infections. This short review focuses on passive immunity and how microfiltered raw immune milk or colostrum collected from cows vaccinated against SARS-CoV-2 could provide short-term protection against SARS-CoV-2 infection in humans and could be used as an option until a vaccine becomes commercially available., (Copyright © 2020 Jawhara.)

Dairy cattle health, wellbeing and productivity are deeply affected by stress. Its influence on metabolism and immune response is well known, but the underlying epigenetic mechanisms require further investigation. In this study, we compared DNA methylation and gene expression signatures between two dairy cattle populations falling in the high- and low-variant tails of the distribution of milk cortisol concentration (MC), a neuroendocrine marker of stress in dairy cows. Reduced Representation Bisulfite Sequencing was used to obtain a methylation map from blood samples of these animals. The high and low groups exhibited similar amounts of methylated CpGs, while we found differences among non-CpG sites. Significant methylation changes were detected in 248 genes. We also identified significant fold differences in the expression of 324 genes. KEGG and Gene Ontology (GO) analysis showed that genes of both groups act together in several pathways, such as nervous system activity, immune regulatory functions and glucocorticoid metabolism. These preliminary results suggest that, in livestock, cortisol secretion could act as a trigger for epigenetic regulation and that peripheral changes in methylation can provide an insight into central nervous system functions.

There is a need for efficient methods to treat food allergy; however, no immunotherapeutic method has yet been satisfactory due to the high rate of unpredictable severe reactions and the limited efficacy. Therefore, modified versions of food allergens have been suggested as alternatives to the parent proteins for immunotherapy. The aim of the study was to compare the inherent allergenicity of the native and denatured version of the cow's milk proteins β-lactoglobulin and α-lactalbumin, and to study the impact of the use of Al(OH) 3 as an adjuvant. Brown Norway rats were immunized intraperitoneally with either native or denatured β-lactoglobulin or α-lactalbumin, with or without the use of Al(OH) 3 as adjuvant. Antibody responses were analysed in various ways by means of different ELISAs. Both the immunogenicity and the sensitizing capacity of the cow's milk allergens were influenced by their globular folding, with the native version being more allergenic than the denatured counterpart. The native folded proteins mainly raised antibodies against conformational epitope, whereas the denatured versions predominantly raised antibodies against linear epitopes. Most interestingly, the study showed that the use of Al(OH) 3 , besides increasing immunogenicity and sensitizing capacity of the cow's milk allergens, caused a modification of the specificity of the antibodies raised against the native version of the proteins. Adsorption of the native forms of the allergens to Al(OH) 3 caused a significant greater proportion of antibodies raised against linear epitopes, stressing that the adsorption induced a partly unfolding of the proteins. This may have implications for IT safety and efficacy., (© 2020 The Scandinavian Foundation for Immunology.)

Summary: Cut-off values for both skin prick tests (SPT) and specific IgE (sIgE) levels for predicting cow´s milk allergy (CMA) diagnosis are not universally defined. This study is a retrospective analysis of consecutive children (0-18 years-old) with suspected CMA tested with SPT and sIgE for cow's milk (CM) and its fractions between 2016-2017. CMA diagnosis was defined by a positive oral food challenge or a highly suggestive clinical history of CMA and SPT and/or sIgE positive to CM and/or its fractions. One hundred and five patients were included, 58% males with a median age of 2.5 (P25-P75:1-6) years and the diagnosis was confirmed in 83 patients (79%). The variables associated with CMA diagnosis were SPT with CM (p minor 0,05) and casein (p minor 0,05) and all sIgE to CM and its fractions (alfa Lactalbumin, beta Lactoglobulin and casein; p minor 0,05 for all). Optimal cut39 off points (Youden's index) for CMA diagnosis were, for the mean wheal diameter, to CM milk of 4.5mm and to casein of 3mm. For sIgE levels the optimal cut-off points were: for CM of 4.36 kUA/L, alfa-lactalbumin of 1.6 kUA/L, beta-lactoglobulin of 1.7 kUA/L and for casein of 2.6 kUA/L. The role of SPT and sIgE levels to cow´s mlik and its fractions is unequivocal in CMA follow-up. Moreover, sIgE levels seem to be more discriminatory than SPT.

Background: Hidradenitis suppurativa is a complex disorder, the pathogenesis of which is still unsolved. The known association between hidradenitis suppurativa and Crohn's disease, an autoimmune disease diagnosed with the presence of Anti-Saccharomyces cerevisiae antibodies of the IgG family, suggests that a much more complex mechanism than a simple infectious disorder is involved. The goal of this study is to report patients' characteristics and the outcome of 6 years of a yeast (Saccharomyces cerevisiae)-exclusion diet and surgery in the treatment of hidradenitis suppurativa., Method: We analyzed 185 patients with hidradenitis suppurativa with a self-evaluative questionnaire. Thirty-seven patients were treated in our center following our protocol. The other 148 were members of a support group for patients with hidradenitis suppurativa treated by other centers., Results: In 80% of patients who had the onset of hidradenitis suppurativa before the age of 30, the female to male ratio was 3.34:1, 74% were active smokers, and 5% also had Crohn's disease. In the diet group, 70% had an improvement of hidradenitis suppurativa symptomatology, 81% of whom in less than 6 months. Also, 87% of patients demonstrated an immediate recurrence of skin lesions less than a week after consuming a food containing the yeast. Immunologic testing showed intolerance to yeast, wheat, and cow's milk in 20%, 29%, and 23% of patients, respectively., Conclusion: The analysis confirmed the stabilization and regression of hidradenitis suppurativa with our diet, presumably by decreasing the local and systemic inflammation, leading to a less invasive operative treatment. These new findings seem to link hidradenitis suppurativa to food intolerance and gut dysbiosis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Immune response to cow's milk allergen (CMA) has been analyzed mostly using crude milk antigen or a mixture of various caseins. This study aimed to assess the changes in the immunological response against αS1-casein during oral immunotherapy (OIT) and to investigate the mechanism of tolerance., Methods: We have performed rush OIT to 39 patients with CMA and obtained the serum samples up to 3 years after OIT. Immunoglobulin E (IgE) and IgG4 antibodies specific to highly purified αS1-casein as well as passively-sensitized basophil activation were evaluated using the serial samples. Furthermore, we examined whether basophil activation led by the pre-OIT serum was suppressed by the post-OIT serum, or by the tolerant serum obtained from naturally outgrown patients., Results: Specific IgE to αS1-casein was significantly reduced after OIT. Specific IgG4 (sIgG4) to αS1-casein was also detected in most of the pre-OIT sera, which was not significantly increased after OIT. Activation of passively-sensitized basophils to αS1-casein was significantly reduced after 2 years (14% ± 19%) and 3 years (19% ± 18%) post-OIT compared with pre-OIT (%CD63 high basophils; 51% ± 27%). Furthermore, the addition of post-OIT or tolerant serum to pre-OIT serum significantly suppressed the basophil activation. This suppression was abrogated by washing the supernatant after passive sensitization, but not by depleting IgG antibodies from post-OIT or tolerant sera, nor by blocking FcγRIIb using an anti-FcγR antibody., Conclusions: αS1-casein-sIgG4 plays a minor role in tolerance mechanisms in cases of CMA; humoral factors other than antigen-sIgG4 may be involved., (© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Importance: Sales of specialized formula for managing cow's milk allergy (CMA) have increased, triggering concern that attribution of common infant symptoms, such as crying, vomiting, and rashes, to CMA may be leading to overdiagnosis, which could undermine breastfeeding., Objective: To understand whether CMA guideline recommendations might promote CMA overdiagnosis or undermine breastfeeding., Evidence Review: We reviewed recommendations made in CMA guidelines and critically appraised 2 key recommendations. First, we reviewed relevant literature summarizing whether maternal or infant dietary exclusion of cow's milk is effective for managing common infant symptoms. Second, we reviewed published data on breastmilk composition and thresholds of reactivity in CMA to estimate the probability that cow's milk protein in human breastmilk can trigger symptoms in infants with CMA. We also documented the level of commercial involvement in CMA guidelines., Findings: We reviewed 9 CMA guidelines published from 2012 to 2019. Seven suggest considering CMA as a cause of common infant symptoms. Seven recommend strict maternal cow's milk exclusion for managing common symptoms in breastfed infants. We found CMA proven by food challenge affects approximately 1% of infants, while troublesome crying, vomiting, or rashes are each reported in 15% to 20% of infants. We found clinical trials do not provide consistent support for using maternal or infant cow's milk exclusion to manage common symptoms in infants without proven CMA. We estimated that for more than 99% infants with proven CMA, the breastmilk of a cow's milk-consuming woman contains insufficient milk allergen to trigger an allergic reaction. Three CMA guidelines were directly supported by formula manufacturers or marketing consultants, and 81% of all guideline authors reported a conflict of interest with formula manufacturers., Conclusions and Relevance: Recommendations to manage common infant symptoms as CMA are not evidence based, especially in breastfed infants who are not directly consuming cow's milk. Such recommendations may cause harm by undermining confidence in breastfeeding.

In the present review, we highlight the possible "extra-immunological" effects of maternal immunoglobulins (Ig) transferred to the blood circulation of offspring, either via the placenta before birth or via the colostrum/milk across the gut after birth in different mammalian species. Using the newborn pig as a model, since they are naturally born agammaglobulinemic, intravenously (i.v.) infused purified serum Ig rapidly improved the vitality, suckling behavior, and ensured the survival of both preterm and term piglets. In further studies, we found that proper brain development requires i.v. Ig supplementation. Studies have reported on the positive effects of i.v. Ig treatment in children with epilepsy. Moreover, feeding newborn pigs an elementary diet supplemented with Ig improved the gut structure, and recently a positive impact of enteral or parenteral Ig supplementation on the absorption of polyunsaturated fatty acids (PUFAs) was observed in the newborn pig. Summarized, our own results and those found in the literature, indicate the existence of important extra-immune effects of maternal Ig, in addition to the classical protective effects of transferred maternal passive immunity, including effects on the development of the brain, gut, and possibly other organ systems in the neonate. These additional properties of circulating Ig could have an impact on care guidelines for human neonates, especially those born prematurely with low plasma Ig levels., (Copyright © 2020 Pierzynowska, Woliński, Weström and Pierzynowski.)

Food allergy and allergen management are important global public health issues. In 2011, the first iteration of our allergen threshold database (ATDB) was established based on individual NOAELs and LOAELs from oral food challenge in roughly 1750 allergic individuals. Population minimal eliciting dose (EDp) distributions based on this dataset were published for 11 allergenic foods in 2014. Systematic data collection has continued (2011-2018) and the dataset now contains over 3400 data points. The current study provides new and updated EDp values for 14 allergenic foods and incorporates a newly developed Stacked Model Averaging statistical method for interval-censored data. ED01 and ED05 values, the doses at which 1%, and respectively 5%, of the respective allergic population would be predicted to experience any objective allergic reaction were determined. The 14 allergenic foods were cashew, celery, egg, fish, hazelnut, lupine, milk, mustard, peanut, sesame, shrimp (for crustacean shellfish), soy, walnut, and wheat. Updated ED01 estimates ranged between 0.03 mg for walnut protein and 26.2 mg for shrimp protein. ED05 estimates ranged between 0.4 mg for mustard protein and 280 mg for shrimp protein. The ED01 and ED05 values presented here are valuable in the risk assessment and subsequent risk management of allergenic foods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Introduction and gradual incremental escalation of a low dose of baked milk may accelerate the resolution of severe cow's milk (CM) allergy for some children. The purpose of our study was to evaluate the efficacy and safety of baked milk oral immunotherapy (OIT) in children with CM allergy after a low-dose baked milk oral food challenge (OFC)., Methods: In a retrospective analysis of OFC performed between 2013 and 2018 at the Children's Hospital of Toulouse (France), we identified 64 children with CM allergy and high milk and casein-specific IgE levels, who underwent a total of 171 milk OFC. Mean age at 1st OFC was 4.8 years. Mean CM-specific IgE was 47.9 kUA/L, and mean casein-specific IgE was 42.3 kUA/L., Results: Most children were treated with baked milk OIT. Our study shows that 67.2% of the children did not react to 1st low-dose baked milk OFC (168.6 mg of CM protein). Eighteen percent of children stopped the OIT at home. Finally, desensitization to fresh milk was achieved in 27 children (42.2% of children allergic to CM). Children with lower CM-specific IgE levels have a significantly higher probability of becoming desensitized to unbaked CM., Conclusion: Most children with CM allergy and high milk and casein-specific IgE levels tolerate the introduction of baked milk. However, the occurrence of anaphylactic reactions during OIT remains possible., (© 2020 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Successful prevention of food allergy requires the identification of the factors adversely affecting the capacity to develop oral tolerance to food antigen in early life., Objectives: This study sought to determine whether oral exposure to Dermatophagoides pteronyssinus through breast milk affects gut mucosal immunity with long-term effects on IgE-mediated food allergy susceptibility., Methods: Gut immunity was explored in 2-week-old mice breast-fed by mothers exposed to D pteronyssinus, protease-inactivated D pteronyssinus, or to PBS during lactation. We further analyzed oral tolerance to a bystander food allergen, ovalbumin (OVA). In a proof-of-concept study, Der p 1 and OVA levels were determined in 100 human breast milk samples and the association with prevalence of IgE-mediated egg allergy at 1 year was assessed., Results: Increased permeability, IL-33 levels, type 2 innate lymphoid cell activation, and T h 2 cell differentiation were found in gut mucosa of mice nursed by mothers exposed to D pteronyssinus compared with PBS. This pro-T h 2 gut mucosal environment inhibited the induction of antigen-specific FoxP3 regulatory T cells and the prevention of food allergy by OVA exposure through breast milk. In contrast, protease-inactivated D pteronyssinus had no effect on offspring gut mucosal immunity. Based on the presence of Der p 1 and/or OVA in human breast milk, we identified groups of lactating mothers, which mirror the ones found in mice to be responsible for different egg allergy risk., Conclusions: This study highlights an unpredicted potential risk factor for the development of food allergy, that is, D pteronyssinus allergens in breast milk, which disrupt gut immune homeostasis and prevents oral tolerance induction to bystander food antigen through their protease activity., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Milk allergenic proteins are the main reason of cow's milk allergy. The most common method for removing milk allergenic proteins is enzymatic hydrolysis. However, the direct application of protease to hydrolyze allergens will lead to the introduction of new allergenic proteins. An ideal strategy for removing milk allergenic proteins without introducing new allergenic proteins is immobilization of protease. Herein, we established a simple method to synthesize a novel papain-Cu 3 (PO 4 ) 2 ·3H 2 O-magnetic nanoflowers (PCMNs). The PCMNs demonstrated 1556% higher activity than the free alkaline papain. Moreover, the PCMNs could hydrolyze most of allergenic proteins in cow's milk, generating low-sensitive milk. In cycle analysis, the PCMNs also exhibited good reusability and were easily separated from the product., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano-therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use., (© 2019 John Wiley & Sons, Ltd.)

Background: Buffalo milk is rich in various nutritional components and bioactive substances that provide more essential health benefits to human body. Recently, exosome identified in the breast milk has been reported as a neotype nutrient and can mediate intercellular communication with exosomal miRNAs. In the present study, we therefore hypothesized that exosome-derived miRNAs from buffalo milk would play the potential physiological importance of consumption of buffalo milk., Results: We isolated exosomes from buffalo and cow milk samples that were obtained at mid-lactation period, and the exosomal miRNA profiles were then generated using miRNA-seq. In addition, miRNAomes of pig, human and panda milk exosomes were downloaded from GEO database. Finally, a total of 27 milk exosomal miRNA profiles that included 4 buffalo, 4 cow, 8 pig, 4 human and 7 panda were analyzed using the miRDeep2 program. A total of 558 unique miRNA candidates existed across all species, and the top 10 highly expressed miRNA were evolutionarily conserved across multiple species. Functional analysis revealed that these milk enriched miRNAs targeted 400 putative sites to modulate disease resistance, immune responsiveness and basic metabolism events. In addition, a total of 32 miRNAs in buffalo milk were significantly up-regulated compared with non-buffalo milks, while 16 were significantly down-regulated. Of interest, functional analysis showed that up-regulated miRNAs were mainly related to host metabolism processes, while the predicted functions of down-regulated miRNAs were enriched in immune response., Conclusion: In this study, we explored the exosomal miRNAome differences between milks of different animals, expanding the theoretical basis for potential applications of the miRNA-containing vesicles.

Cow's milk protein allergy, which occurs in approximately 5-10% of the population of infants and children, has become an important public food safety problem. As a major allergen in cow's milk, the most abundant protein casein (CN) is considered to be potent in inducing food allergy. In recent years, ultrasound treatment has played a significant role in the field of colloidal particulate system. In this study, we found that ultrasound treatment dramatically decreased the diameter of a CN particle to less than 100 nm in the presence of Tween 80, producing colloidal casein (c-CN) with high transparency. The electrophoretic and transmission electron microscopy analysis showed that the advanced protein structure of CN changed significantly. In addition, the enzyme-linked immunosorbent assay with allergic sera showed that the immunoglobulin-E-binding capacity of c-CN was significantly decreased. In the meantime, the LAD2 mast cell line degranulation assay demonstrated that ultrasound treatment made CN hypoallergenic. The colloidal and hypoallergenic properties of c-CN were stably maintained for more than 30 days. Likewise, the allergenicity of fresh whole milk also decreased after ultrasound treatment. This work provided an effective way to reduce the allergenicity of milk allergen, which could be beneficial to the production of hypoallergenic cow's milk.

Background: Tick distribution in Sweden has increased in recent years, with the prevalence of ticks predicted to spread towards the northern parts of the country, thus increasing the risk of tick-borne zoonoses in new regions. Tick-borne encephalitis (TBE) is the most significant viral tick-borne zoonotic disease in Europe. The disease is caused by TBE virus (TBEV) infection which often leads to severe encephalitis and myelitis in humans. TBEV is usually transmitted to humans via tick bites; however, the virus can also be excreted in the milk of goats, sheep and cattle and infection may then occur via consumption of unpasteurised dairy products. Virus prevalence in questing ticks is an unreliable indicator of TBE infection risk as viral RNA is rarely detected even in large sample sizes collected at TBE-endemic areas. Hence, there is a need for robust surveillance techniques to identify emerging TBEV risk areas at early stages., Methods: Milk and colostrum samples were collected from sheep and goats in Örebro County, Sweden. The milk samples were analysed for the presence of TBEV antibodies by ELISA and validated by western blot in which milk samples were used to detect over-expressed TBEV E-protein in crude cell extracts. Neutralising titers were determined by focus reduction neutralisation test (FRNT). The stability of TBEV in milk and colostrum was studied at different temperatures., Results: In this study we have developed a novel strategy to identify new TBEV foci. By monitoring TBEV antibodies in milk, we have identified three previously unknown foci in Örebro County which also overlap with areas of TBE infection reported during 2009-2018. In addition, our data indicates that keeping unpasteurised milk at 4 °C will preserve the infectivity of TBEV for several days., Conclusions: Altogether, we report a non-invasive surveillance technique for revealing risk areas for TBE in Sweden, by detecting TBEV antibodies in sheep milk. This approach is robust and reliable and can accordingly be used to map TBEV "hotspots". TBEV infectivity in refrigerated milk was preserved, emphasising the importance of pasteurisation (i.e. 72 °C for 15 s) prior to consumption.

Background: The detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling., Methods: Samples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity., Results: From 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p < 0.05) was observed between two incubation protocols. The detection of LSD-specific antibodies from bulk milk samples (pools of 2-10 individuals) came along with a reduced sensitivity over the sample of individual animals., Conclusions: Results show that the detection of capripoxvirus specific antibodies in milk samples using the commercially available ELISA from IDvet is feasible and can represent a helpful tool for LSDV monitoring programs.

In this study, the pathogenicity of porcine deltacoronavirus (PDCoV) strain NH (passage 10, P10) was evaluated. We found that PDCoV strain NH is enteropathogenic in 5-day-old pigs. Pathogenicity experiments provided a challenge model for studying the protection efficiency of passive immunity. In order to investigate the protective efficacy of passive immunity in newborn piglets, pregnant sows were vaccinated with either a PDCoV-inactivated vaccine at the Houhai acupoint (n = 5) or DMEM as a negative control (n = 2) using a prime/boost strategy 20 and 40 days before delivery. PDCoV spike (S)-specific IgG and neutralizing antibody (NA) responses were detected in immunized sows and piglets born to immunized sows. PDCoV spike (S)-specific sIgA was also detected in the colostrum and milk of immunized sows. Five days post-farrowing, piglets were orally challenged with PDCoV strain NH (10 5 TCID 50 /piglet). Severe diarrhoea, high levels of viral RNA copies and substantial intestinal villus atrophy were detected in piglets born to unimmunized sows. Only 4 of 31 piglets (12.9%) born to immunized sows in the challenge group displayed mild to moderate diarrhoea, lower viral RNA copies and minor intestinal villi damage compared to piglets born to unimmunized sows post-challenge. Mock piglets exhibited no typical clinical symptoms. The challenge experiment results indicated that the inactivated PDCoV vaccine exhibited 87.1% protective efficacy in the piglets. These findings suggest that the inactivated PDCoV vaccine has the potential to be an effective vaccine, providing protection against virulent PDCoV., (© 2019 Blackwell Verlag GmbH.)

Goat milk (GM), as compared to cow milk (CM), is easier for humans to digest. It also has antioxidant and anti-inflammatory effects and can improve minor digestive disorders and prevent allergic diseases in infants. It is unclear whether GM consumed in pregnant mothers has any protective effects on allergic diseases in infants. In this experimental study with mice, we found GM feeding enhanced immunoglobulin production, antigen-specific (ovalbumin, OVA) immune responses, and phagocytosis activity. The GM-fed mice had an increasing proportion of CD3 + T lymphocytes in the spleen. Splenocytes isolated from these animals also showed significantly increased production of cytokines IFN-γ and IL-10. More importantly, GM feeding during pregnancy and lactation periods can confer protective activity onto offspring by alleviating the airway inflammation of allergic asthma induced by mite allergens. There was a remarkably different composition of gut microbiota between offspring of pregnant mice fed with water or with milk (GM or CM). There was a greater proportion of beneficial bacterial species, such as Akkermansia muciniphila, Bacteroides eggerthii , and Parabacteroides goldsteinii in the gut microbiota of offspring from GM- or CM-fed pregnant mice compared to the offspring of water-fed pregnant mice. These results suggested that improving the nutrition of pregnant mice can promote immunological maturation and colonization of gut microbiota in offspring. This mother-to-child biological action may provide a protective effect on atopy development and alleviate allergen-induced airway inflammation in offspring., (Copyright © 2020 Kao, Wang, Tseng, Wu, Tsai, Hsieh, Chen, Kuo, Liu, Liu and Wang.)

Caprine tuberculosis (TB) is a zoonosis with sanitary and economic repercussions. Caprine TB control programs are based on a test and cull strategy using the intradermal tuberculin tests and slaughterhouse surveillance. However, this approach is not always feasible and may have a limited sensitivity under specific circumstances. In this study, performance of a new experimental test based on the P22 protein complex (P22 ELISA) was evaluated in two TB-infected herds using milk and serum samples and compared with cell-based diagnostic tests. Samples from a low (n = 62, herd 1) and a high (n = 52, herd 2) TB prevalence herd were selected. Moreover, bulk tank milk samples from both herds were analysed using the P22 ELISA. At the end of the study, a group of animals (n = 21) was euthanized and subjected to post-mortem analysis and bacteriological culture. Significant differences (p < .001) on the qualitative and quantitative (ODs) results were observed between herds using both serum and milk samples in the P22 ELISA. The correlation observed in the quantitative results obtained in serum and milk samples was very strong in animals from flock 2 (r s  = 0.91) and moderate in animals from flock 1 (r s  = 0.46). Among the slaughtered animals, the P22 ELISA detected a higher proportion of lesion-culture positive animals than cell-based diagnostic tests (61.9 and 66.7% using milk and serum samples, respectively). The P22 ELISA using milk samples demonstrated a similar sensitivity compared with serum samples, suggesting it might be a valuable test for TB control in dairy goats., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: A large proportion of cow's milk (CM)-allergic children are able to tolerate extensively heated forms of CM such as baked goods. Little is known about whether ultra-heat-treated (UHT) forms of cow's milk are immunologically similar to extensively heated cow's milk and therefore may be tolerated by these children., Objective: To determine whether skin test wheal size using UHT CM was significantly different from other forms of CM and CM extracts., Methods: Children presenting for oral food challenges with either extensively heated or unheated cow's milk underwent skin prick test (SPT) to commercial CM, UHT CM, evaporated CM, and fresh whole CM. The results were compared between groups of children., Results: At study exit, only 14% of children were avoiding all forms of CM, compared with 70% at study entry. No difference was seen in the mean SPT results for UHT CM between those children that could tolerate heated CM compared with those that could not. The mean SPT result for casein was significantly lower in those that could tolerate heated CM. However, within the group of heated milk-tolerant children, the mean SPT for UHT CM was significantly lower than the SPT for fresh whole CM., Conclusion: Ultra-heat-treated CM does not behave significantly differently from other forms of CM when evaluated by SPT in heated milk-allergic vs heated milk-tolerant children. This suggests that UHT CM is not sufficiently immunologically different from unheated CM to be tolerated by heated CM-tolerant children., (Copyright © 2019 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The present study aimed to analyze the rat breast milk profile of fatty acids (FA), immunoglobulins (Ig), microbiota, and their relationship, and to further assess their associations in the mother-offspring pair. Dams were monitored during the three weeks of gestation, allowed to deliver at term, and followed during two weeks of lactation. At the end of the study, milk was obtained from the dams for the analysis of fatty acids, microbiota composition, immunoglobulins, and cytokines. Moreover, the cecal content and plasma were obtained from both the dams and pups to study the cecal microbiota composition and the plasmatic levels of fatty acids, immunoglobulins, and cytokines. Rat breast milk lipid composition was ~65% saturated FA, ~15% monounsaturated FA, and ~20% polyunsaturated FA. Moreover, the proportions of IgM, IgG, and IgA were ~2%, ~88%, and ~10%, respectively. Breast milk was dominated by members of Proteobacteria , Firmicutes , and Bacteroidetes phyla. In addition, forty genera were shared between the milk and cecal content of dams and pups. The correlations performed between variables showed, for example, that all IgGs subtypes correlated between the three compartments, evidencing their association in the mother-milk-pup line. We established the profile of FA, Ig, and the microbiota composition of rat breast milk. Several correlations in these variables evidenced their association through the mother-milk-pup line. Therefore, it would be interesting to perform dietary interventions during pregnancy and/or lactation that influence the quality of breast milk and have an impact on the offspring., Competing Interests: The authors declare that they have no conflicts of interest.

Background: Most of children with cow's milk (CM) and hen's egg allergies are known to outgrow their diseases with time, but recent studies have demonstrated that children tend to continue to have allergic symptoms in adolescence and adulthood. The aim of this study was to investigate the natural course of CM and egg allergies in Korean children and analyze prognostic factors., Methods: In this retrospective study, we reviewed data of children with CM allergy and with egg allergy using medical records and parental telephone interviews. Diagnosis of CM or egg allergy was based on positive oral food challenge test or convincing history of allergic symptoms in combination with positive allergen-specific IgE. Acquisition of tolerance was defined by the absence of allergic symptoms after reintroduction of the offending foods., Results: Half of the children outgrew CM allergy at a median age of 8.7 years. CM-specific IgE level at the first reaction was a significant prognostic factor for oral tolerance in CM allergy (p < 0.05). The median age to acquire oral tolerance in 50% of patients with egg allergy was 5.6 years. Egg-specific IgE level at the first reaction and family history of allergic diseases significantly affected the prognosis in children with egg allergy (p < 0.05)., Conclusions: Half of Korean children with CM and egg allergies had symptom resolution at 8.7 and 5.6 years of age, respectively. Our results also suggest that CM- and egg-specific IgE levels at the first reaction are the most significant prognostic factors in predicting acquisition of oral tolerance., (© 2019 S. Karger AG, Basel.)

Background and Objective: Treg cells and dietetic factors may play a significant role in the natural acquisition of tolerance in children with cow's milk allergy (CMA). The best marker for Treg lymphocytes is the transcription factor forkhead boxP3 (FOXP3). Objective: We examine the relationship between FOXP3 mRNA expression and serum concentrations of vitamins D and C and the development of different phenotypes of tolerance in children with CMA., Material and Methods: The study group comprised 138 infants with CMA and 101 healthy infants. All children underwent oral food challenge, first with an extensively heated milk product and then with unheated products. FOXP3 mRNA expression and serum vitamin C and D concentrations were evaluated., Results: At 2 years of life, 54 children (39.1%) still had CMA, 43 (31.2%) were unheated milk-reactive and heated milk-tolerant, while 41 (29.7%) had outgrown their allergy. The mean (SD) level of FOXP3 expression in the study group was 2.07 (1.23), which was lower than the control group value of 2.98 (1.52) (P<.001). A value below 1.45 indicated allergy. The mean serum level of vitamin D in the study group was lower than in the control group (29.67 [7.09] vs 33.35 [4.13] ng/mL; P<.001). No significant differences were found in mean serum vitamin C content., Conclusions: Increased FOXP3 mRNA expression can predict faster acquisition of tolerance in infants with CMA. These children have lower serum vitamin D levels than healthy children. No relationship was found between the natural history of CMA and serum vitamin C concentration.

Antibacterial factors act as innate immune components, which respond as soon as bacteria enter a living organism. To prevent and treat mastitis in cattle, understanding the concentrations of these substances inside the udder is important; however, they remain to be studied. In this investigation, the concentration of lingual antimicrobial peptide (LAP), S100 protein (S100A7), lactoferrin (LF), and immunoglobulin antibody were measured in the different fractions of foremilk. Lactating Holstein cows were examined, and 10 foremilk fractions were obtained from sequential samples up to 150 ml. The LAP concentrations in milk samples increased until 25 ml. The LF concentrations increased up to the 10 ml fraction, then stabilized at low level after the 50 ml fraction. For S100A7, some fractions had significantly higher (p < .05) concentrations than the 5 or 10 ml fractions. The IgA antibody concentration increased up to the 5 ml fraction, then after 50 ml fraction showed relatively low concentrations. This investigation determined the concentration patterns of LAP, LF, S100A7, and IgA antibody secreted in milk inside the udders of healthy lactating cows as baseline data. These distinct concentration patterns might indicate various protective responses., (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Introduction: Oral immunotherapy (OIT) has been reported to be effective but associated with a risk of severe symptoms. Thus, an OIT method with decreased risk is required., Objectives: We aimed to evaluate the efficacy and safety of low- and high-dose OIT regimens in children with severe milk allergy., Methods: Overall, 33 participants (median age, 9 years; median final dose of the milk oral food challenge [OFC], 2 mL) were included. The participants were randomly assigned to groups that received either a low (20 mL; n = 19) or high (100 mL; n = 14) maintenance target dose of OIT. The dose was gradually increased to the target dose in the rush escalation phase and was then maintained daily at home. The primary endpoint was the final OFC dose at 6 months of OIT. Adverse events during OIT were evaluated., Results: The final OFC dose after OIT was significantly higher than that before OIT in both groups (low-dose, p = 0.000; high-dose, p = 0.006), but there was no significant difference in the final OFC dose between the 2 groups (p = 0.767). In the maintenance phase, the high-dose group had significantly more severe symptoms than did the low-dose group (0.5%, 11/2,355 total intake events vs. 0.1%, 4/3,230 total intake events; p = 0.018)., Conclusions: An equally increased dose effect was observed for maintenance OIT doses of 20 and 100 mL in children with severe milk allergy. The risk of severe symptoms in the maintenance phase was lower in the low-dose group. A low-dose OIT regimen is recommended for severe milk allergy., (© 2020 S. Karger AG, Basel.)

Milk beverages derived from plant-based protein have attracted the interest of consumers and researchers as a health-promoting functional food. It can also be considered as a substitute for animal milk due to various allergy concerns associated with dairy milk. The plant-based emulsions are directed to prevent diet-related chronic diseases including diabetes, cardiovascular, obesity and other disorders due to the presence of healthy long-chain unsaturated fatty acids as compared to bovine milk. Further, associations between nutritional contents (vitamins, minerals and low fat) and pharmacological properties of plant-based protein may have extra beneficial effects. The review aims to summarize the four different groups of plant sources (nuts, cereals, seeds and legumes) used for the preparation of plant-based milk beverages. In addition, it also provides a detailed review of the general characteristics and functional properties of these plant sources. Physicochemical composition, protein and fats quality, functional properties, effect of heat and high-pressure treatment is also provided in detail. It also covers fats digestibility, protein stability, protein solubility and digestibility. Furthermore, the effect of processing, possible comparative study and potential applications in healthcare have been discussed.

The incidence of bovine mastitis and the bulk milk somatic cell count (BMSCC) are influenced by season, which may be associated with innate immune functions, including antimicrobial components in mammary glands. Therefore, the present study was conducted to examine the effect of season on antimicrobial components in milk. Rectal temperature and plasma cortisol, thyroxine, and derivatives of reactive oxygen metabolites (d-ROMs) were measured as stress parameters. Concentrations of lactoferrin (LF), lingual antimicrobial peptide (LAP), psoriasin (S100A7), and Immunoglobulin A (IgA) in milk were measured as indicators of innate immune function. LF and LAP concentrations were significantly lower in summer than in winter and spring, respectively, whereas the concentration of S100A7 was significantly lower in winter than in spring and autumn. The rectal temperature was significantly higher in summer than in other seasons, whereas plasma cortisol, thyroxine, and d-ROMs did not exhibit any seasonal variation. In conclusion, even though stress parameters were not changed, the concentration of antimicrobial components, such as LF and LAP, decreased in summer, which may explain the frequent occurrence of mastitis during this season., (© 2020 Japanese Society of Animal Science.)

Cow's milk protein allergy (CMPA) is one of the most common food allergies in infancy. Clinical food allergy guidelines recommend an extensively hydrolyzed formula (EHF) as the first-line treatment in nonbreastfed infants with CMPA. Designing and commercializing EHF poses both technical and regulatory challenges. Each manufacturing step, from sourcing of raw materials to release of the final product, needs to be managed in accordance with comprehensive quality systems. To avoid cross-contamination via externally sourced ingredients, suppliers should be carefully selected based on quality requirements. Strict zoning of the manufacturing areas according to contamination risk and air flow control are effective strategies to prevent accidental allergen contamination. Furthermore, dedicated manufacturing lines for hypoallergenic products are used to prevent potential cross-contamination from other products produced on the same line. The enzymatic hydrolysis, heat treatment and ultrafiltration used are specific to each manufacturer. Consequently, EHF are a heterogenous group of products with differences in the molecular weight profile of peptides, content of residual immunogenic cow's milk allergens, and residual in-vitro allergenicity. These differences are likely to affect clinical efficacy and safety. As not all commercialized EHF products have undergone formal testing in the laboratory and clinical trials, there is a need to develop guidelines for minimum technical and regulatory requirements for EHF products, including validated assays for ongoing quality control. Clinical trials assessing new EHF products for their hypoallergenicity and ability to support normal growth remain the definitive proof of efficacy and safety in infants and young children with CMPA., (© 2020 Elsevier Inc. All rights reserved.)

Introduction: Cow's milk allergy (CMA) is one of the most common food allergies especially early in life. A mixture of nondigestible short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides, and pectin-derived acidic-oligosaccharides (GFA) may reduce allergy development and allergic symptoms in murine CMA. Recently, vitamin D (VitD) has been suggested to have beneficial effects in reducing allergy as well., Objective: In this study, the immune modulatory effect on allergy prevention using the combination of GFA and VitD was investigated., Methods: Female C3H/HeOuJ mice were fed a control or GFA-containing diet with depleted, standard (1,000 IU/kg), or supplemented (5,000 IU/kg) VitD content for 2 weeks before and during whey sensitization (n = 10-15). Mice were sensitized 5 times intragastrically with PBS as a control, whey as cow's milk allergen, and/or cholera toxin as adjuvant on a weekly interval. One week after the last sensitization, mice were intradermally challenged in both ear pinnae and orally with whey, subsequently the acute allergic skin response and shock symptoms were measured. After 18 h, terminal blood samples, mesenteric lymph nodes, and spleens were collected. Whey-specific immunoglobulin (Ig) E and IgG1 levels were measured by means of ELISA. T cell subsets and dendritic cells (DCs) were studied using flow cytometry., Results: Additional VitD supplementation did not lower the allergic symptoms compared to the standard VitD diet. CMA mice fed the GFA diet supplemented with VitD (GFA VitD+) significantly decreased the acute allergic skin response of whey sensitized mice when compared to the CMA mice fed VitD (VitD+) group (p < 0.05). The effect of GFA was not improved by extra VitD supplementation even though the CMA mice fed the GFA VitD+ diet had a significantly increased percentage of CD103+ DCs compared to the VitD+ group (p < 0.05). The VitD-deprived mice showed a high percentage of severe shock and many reached the humane endpoint; therefore, these groups were not further analyzed., Conclusions: High-dose VitD supplementation in mice does not protect against CMA development in the presence or absence of GFA., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Objectives: Human milk-based fortifiers have shown a protective effect on major complications for very low birth weight newborns. The current study aimed to estimate the cost-effectiveness of an exclusive human milk diet (EHMD) compared to the current approach using cow's milk-based fortifiers in very low birth weight newborns., Methods: A decision tree model using the health states of necrotising enterocolitis (NEC), sepsis, NEC + sepsis and no complication was used to calculate the cost-effectiveness of an EHMD. For each health state, bronchopulmonary dysplasia (BPD), retinopathy of prematurity (RoP) and neurodevelopmental problems were included as possible complications; additionally, short-bowel syndrome (SBS) was included as a complication for surgical treatment of NEC. The model was stratified into birth weight categories. Costs for inpatient treatment and long-term consequences were considered from a third party payer perspective for the reference year 2017. Deterministic and probabilistic sensitivity analyses were performed, including a societal perspective, discounting rate and all input parameter-values., Results: In the base case, the EHMD was estimated to be cost-effective compared to the current nutrition for very low birth weight newborns with an incremental cost-effectiveness ratio (ICER) of €28,325 per Life-Year-Gained (LYG). From a societal perspective, the ICER is €27,494/LYG using a friction cost approach and €16,112/LYG using a human capital approach. Deterministic sensitivity analyses demonstrated that the estimate was robust against changes in the input parameters and probabilistic sensitivity analysis suggested that the probability EHMD was cost-effective at a threshold of €45,790/LYG was 94.8 percent., Conclusion: Adopting EHMD as the standard approach to nutrition is a cost-effective intervention for very low birth weight newborns in Germany., Competing Interests: Both authors, SMS and WG, wish to disclose that the research on which our manuscript is based was funded by an unrestricted research grant by Prolacta BioSciences (https://www.prolacta.com/). The presence of the funding from a commercial source does not alter our adherence to PLOS ONE policies on sharing data and materials.

Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients' blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization.

Natural antibodies (NAb) are produced without any antigenic stimulation as a part of the innate immune system and provide a first line of defense against pathogens. Hence, they may be a useful trait when estimating an animal's potential immune competence and in selection for disease resistance. The aim of this study was to identify genomic regions associated with different NAb traits in milk and potentially describe candidate genes. Milk samples from 1,695 first-lactation Holstein Friesian cows with titer measurements for keyhole limpet hemocyanin, lipopolysaccharide, lipoteichoic acid, and peptidoglycan-binding total NAb and isotypes IgG 1 , IgM, and IgA were used. Genome-wide association study analyses were performed using imputed 777K SNP genotypes, accounting for relationships using pedigree information. Functional enrichment analysis was performed on the significantly associated genomic regions to look for candidate genes. For IgM NAb, significant associations (false discovery rate <0.05) were found on Bos taurus autosome (BTA) 17, 18, and 21 with candidate genes related to immunoglobulin structure and early B cell development. For IgG 1 , associations were found on BTA3, and we confirmed a quantitative trait loci on BTA21 previously reported for IgG NAb in serum. Our results provide new insights into the regulation of milk NAb that will help unravel the complex relationship between milk immunoglobulins and disease resistance in dairy cattle., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Neospora caninum is known to cause reproductive disturbances in several animal species, such as cattle, sheep, and goats. However, research on the effects of N. caninum on reproduction in pigs is limited. The objective of this study was to verify the transplacental transmission of N. caninum in pigs during several gestational stages. Twelve healthy Toxoplasma gondii and N. caninum seronegative female pigs were selected and separated into four groups of three animals each. Group A was maintained as a control group. Groups B, C, and D were inoculated intravenously with 2.9 × 10 7 tachyzoites of the N. caninum strain Nc1, 30 days before conception and at 45 and 90 days of gestation, respectively. Blood samples were collected from females periodically through IFAT for IgG and IgM screening to confirm the infection. At birth, after blood samples were collected from the piglets, they were then euthanized for the collection of the brain, heart, lung, liver, and diaphragm, which were then subjected to PCR. All inoculated gilts seroconverted (IgG) from the seventh day after inoculation. Nine of the 12 females expelled 24 mummified fetuses at the time of delivery, two in group A (eight), two in group B (four), three in group C (nine), and two in group D (three). Of the 24 mummified fetuses, nine were positive for N. caninum (one (25%) fetus of group B, seven (77.8%) of group C, and one (33.3%) of group D). A total of 126 live piglets were born. When the organs of the piglets from the inoculated females were analyzed by PCR for N. caninum, 88 (93.61%) were positive. All gilts inoculated produced at least one positive piglet. This demonstrates that there is transplacental transmission of N. caninum in all phases of gestation, regardless of the time of infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) are involved in cell-to-cell communication and modulation of numerous physiological and pathological processes. EVs are found in large quantities in milk and contain several inflammation- and immunity-modulating proteins and microRNAs, through which they exert beneficial effects in several inflammatory disease models. Here, we investigated the effects of two EV subsets, concentrated from commercial cow's milk, on a murine model of colitis induced with dextran sodium sulfate (DSS). P35K EVs, isolated by ultracentrifugation at 35,000 g, and P100K EVs, isolated at 100,000 g, were previously characterized and administered by gavage to healthy and DSS-treated mice. P35K EVs and, to a lesser extent, P100K EVs improved several outcomes associated to DSS-induced colitis, modulated the gut microbiota, restored intestinal impermeability and replenished mucin secretion. Also, P35K EVs modulated innate immunity, while P100K EVs decreased inflammation through the downregulation of colitis-associated microRNAs, especially miR-125b, associated with a higher expression of the NFκB inhibitor TNFAIP3 (A20). These results suggest that different milk EV subsets may improve colitis outcomes through different, and possibly complementary, mechanisms. Further unveiling of these mechanisms might offer new opportunities for improving the life of patients with colitis and be of importance for milk processing, infant milk formulation and general public health.

The diagnosis of cow's milk allergy (CMA) is particularly challenging in infants, especially with non-Immunoglobulin E (IgE)-mediated manifestations, and inaccurate diagnosis may lead to unnecessary dietary restrictions. The aim of this study was to assess the accuracy of the cow's milk-related symptom score (CoMiSS TM ) in response to a cow's milk-free diet (CMFD). We prospectively recruited 47 infants (median age three months) who had been placed on a CMFD due to persisting unexplained gastrointestinal symptoms. We compared data with 94 healthy controls (median age three months). The CoMiSS TM score was completed at recruitment and while on the exclusion diet. In 19/47 (40%) cases a response to the diet occurred. At recruitment CoMiSS TM was significantly higher in cases compared to controls (median score 8 vs. 3; p -value: <0.05), 9 cases had a score ≥12 and 8/9 normalized on CMFD. An oral milk challenge was performed in all 19 responders and six of these had a positive reaction to cow's milk (CM). In eight infants IgE allergy tests were positive. The receiver operation characteristic (ROC) curve identified a CoMISS TM score of 9 to be the best cut-off value (84% sensitivity, 85% specificity, 80% positive (PPV) and 88% negative predictive value (NPV)) for the response to CMFD. We found CoMiSS TM to be a useful tool to help identify infants with persisting gastrointestinal symptoms and suspected CMA that would benefit from CMFD.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO + cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)