From Amish farm dust to bacterial lysates: The long and winding road to protection from allergic disease.

Allergic diseases typically begin in early life and can impose a heavy burden on children and their families. Effective preventive measures are currently unavailable but may be ushered in by studies on the "farm effect", the strong protection from asthma and allergy found in children born and raised on traditional farms. Two decades of epidemiologic and immunologic research have demonstrated that this protection is provided by early and intense exposure to farm-associated microbes that target primarily innate immune pathways. Farm exposure also promotes timely maturation of the gut microbiome, which mediates a proportion of the protection conferred by the farm effect. Current research seeks to identify allergy-protective compounds from traditional farm environments, but standardization and regulation of such substances will likely prove challenging. On the other hand, studies in mouse models show that administration of standardized, pharmacological-grade lysates of human airway bacteria abrogates allergic lung inflammation by acting on multiple innate immune targets, including the airway epithelium/IL-33/ILC2 axis and dendritic cells whose Myd88/Trif-dependent tolerogenic reprogramming is sufficient for asthma protection in adoptive transfer models. To the extent that these bacterial lysates mimic the protective effects of natural exposure to microbe-rich environments, these agents might provide an effective tool for prevention of allergic disease.

Asthma-protective agents in dust from traditional farm environments.

BACKGROUND: Growing up on traditional European or US Amish dairy farms in close contact with cows and hay protects children against asthma, and airway administration of extracts from dust collected from cowsheds of those farms prevents allergic asthma in mice. OBJECTIVES: This study sought to begin identifying farm-derived asthma-protective agents. METHODS: Our work unfolded along 2 unbiased and independent but complementary discovery paths. Dust extracts (DEs) from protective and nonprotective farms (European and Amish cowsheds vs European sheep sheds) were analyzed by comparative nuclear magnetic resonance profiling and differential proteomics. Bioactivity-guided size fractionation focused on protective Amish cowshed DEs. Multiple in vitro and in vivo functional assays were used in both paths. Some of the proteins thus identified were characterized by in-solution and in-gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzymatic digestion/peptide mapping followed by liquid chromatography/mass spectrometry. The cargo carried by these proteins was analyzed by untargeted liquid chromatography-high-resolution mass spectrometry. RESULTS: Twelve carrier proteins of animal and plant origin, including the bovine lipocalins Bos d 2 and odorant binding protein, were enriched in DEs from protective European cowsheds. A potent asthma-protective fraction of Amish cowshed DEs (≈0.5% of the total carbon content of unfractionated extracts) contained 7 animal and plant proteins, including Bos d 2 and odorant binding protein loaded with fatty acid metabolites from plants, bacteria, and fungi. CONCLUSIONS: Animals and plants from traditional farms produce proteins that transport hydrophobic microbial and plant metabolites. When delivered to mucosal surfaces, these agents might regulate airway responses.

Protection against allergies: Microbes, immunity, and the farming effect.

The prevalence of asthma and other allergic diseases has rapidly increased in "Westernized" countries over recent decades. This rapid increase suggests the involvement of environmental factors, behavioral changes or lifestyle, rather than genetic drift. It has become increasingly clear that the microbiome plays a key role in educating the host immune system and, thus, regulation of disease susceptibility. This review will focus on recent advances uncovering immunological and microbial mechanisms that protect against allergies, in particular, within the context of a farming environment. A whole body of epidemiological data disclosed the nature of the protective exposures in a farm. Current evidence points toward an important role of the host microbiome in setting an immunological equilibrium that determines progression toward, or protection against allergic diseases. Conclusive mechanistic insights on how microbial exposures prevent from developing allergic diseases in humans are still lacking but findings from experimental models reveal plausible immunological mechanisms. Gathering further knowledge on these mechanisms and confirming their relevance in humans is of great importance to develop preventive strategies for children at risk of developing allergies.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

Environmental influences on childhood allergies and asthma - The Farm effect.

Asthma and allergies are major health problems and exert an enormous socioeconomic burden. Besides genetic predisposition, environmental factors play a crucial role in the development of these diseases in childhood. Multiple worldwide epidemiological studies have shown that children growing up on farms are immune to allergic diseases and asthma. Farm-related exposures shape children's immune homeostasis, via mediators such as N-glycolylneuraminic acid or arabinogalactan, or by diverse environmental microbes. Moreover, nutritional factors, such as breastfeeding or farm milk and food diversity, inducing short-chain fatty acids-producing bacteria in the intestine, contribute to farm-related effects. All farm-related exposures induce an anti-inflammatory response of the innate immunity and increase the differentiation of regulatory T cells and T helper cell type 1. A better understanding of the components of the farm environment, that are protective to the development of allergy and asthma, and their underlying mechanisms, will help to develop new strategies for the prevention of allergy and asthma.

Patterns of farm exposure are associated with reduced incidence of atopic dermatitis in early life.

BACKGROUND: Farm exposures may reduce the risk of atopic dermatitis (AD) in children, but this is controversial and US data are limited. OBJECTIVE: This study was conducted to identify patterns of farm exposure in Wisconsin family farms that modify AD incidence and prevalence in early childhood. METHODS: Environmental exposures, health history, and clinical outcomes were prospectively recorded for 111 farm families and 129 non-farm families enrolled in the Wisconsin Infant Study Cohort birth cohort study. Exposures from the prenatal and early postnatal (2-month) visits were evaluated together with parental report of AD diagnosis by a health care provider through age 24 months. Latent class analysis was performed with prenatal and early postnatal farm-exposure variables to assign farm children to 3 classes. RESULTS: Overall, children of farm families had reduced AD incidence (P = .03). Within farm families, exposures including poultry (3% vs 28%; P = .003), pig (4% vs 25%; P = .04), feed grain (13% vs 34%; P = .02), and number of animal species were inversely associated with AD incidence. Among the latent class groups, children in families with diverse or more intense farm exposures (classes A and B) had reduced AD incidence, whereas low-exposure (class C) infants had AD incidence similar to that in nonfarm children. CONCLUSIONS: Infants in Wisconsin farm families had reduced AD incidence, and patterns of farm exposures further defined AD risk. These findings suggest that exposure to diverse farm animals, feed, and bedding during the prenatal period and in early infancy reduce the risk of early-onset AD, a phenotype associated with multiple other atopic diseases.

The role of innate immunity in asthma development and protection: Lessons from the environment.

Asthma, a complex, chronic disease characterized by airway inflammation, hyperresponsiveness and remodelling, affects over 300 million people worldwide. While the disease is typically associated with exaggerated allergen-induced type 2 immune responses, these responses are strongly influenced by environmental exposures that stimulate innate immune pathways capable of promoting or protecting from asthma. The dual role played by innate immunity in asthma pathogenesis offers multiple opportunities for both research and clinical interventions and is the subject of this review.

The rural-urban enigma of allergy: what can we learn from studies around the world?

Childhood asthma and related allergic conditions have become the most common chronic disorders in the Western world. Many studies from around the world have demonstrated an increasing trend of asthma prevalence over the last few decades (Lancet, 368, 2004, 733). A few recent reports also suggested that childhood asthma prevalence may be showing a plateau or even a decline in few developed countries. Given the rapid changes in the prevalence over a short period of time, environmental factors are the more likely candidates explaining such trend. One of the most consistent epidemiological findings was that subjects living in the rural areas had lower prevalence of allergies when compared to those from urban areas (Clin Exp Allergy 30, 2000, 187; Pediatr Pulmonol 44, 2009, 793). Clear understanding of the mechanisms of how the environmental determinants in the rural environment may affect the early immune system resulting in lower risk of allergies and asthma will facilitate the development of future primary preventive strategies. In this study, we review recent data from around the world and explore the epidemiology and mechanistic studies that may explain the rural-urban difference of allergies.

The extended farm effect: The milk protein ß-lactoglobulin in stable dust protects against allergies.

BACKGROUND: The allergy- and asthma-protective farm effect is mediated by numerous factors. Especially dust from cattle stables and raw cow's milk show beneficial properties, suggesting a bovine protein to be involved. As a major milk protein and member of the lipocalin family, ß-lactoglobulin (BLG) binds small, hydrophobic ligands and thereby modulates the immune response. Empty BLG promotes allergy development, whereas BLG in association with ligands shows allergy-preventive as well as allergy-reducing effects in vivo and in vitro. RESULTS: BLG has been identified as a major protein in stable dust (therein bound to zinc) as well as in the air around cattle stables. This association with zinc favors an allergy-protective immune profile. CONCLUSION: Its immune-modulating, allergy-protective characteristics together with its presence in raw cow's milk as well as in stable dust and ambient air render BLG an essential contributor to the farm effect.

The role of LPS and CpG in the farm effect against allergies, and beyond.

The prevalence of allergic disease has increased significantly over the past decades. Although allergies are inherently multifactorial and heterogenous; environmental, maternal, and early-life microbial exposures could strongly modify disease risk. The effects of environmental microbiota are illustrated by the "farm effect", showing protection against asthma when children grow up on traditional farms. Recent studies have further revealed an important role for early-life exposure to a microbe-rich environment imposing lung and gut microbiome maturation and immune education, preventing allergic disease in childhood. Advances are made in the field of immunology and microbiome research, which identified entire microbial taxa, as well as specific microbial metabolites and bacterial products associated with reducing disease risk. Here we discuss the cross-talk between the microbiota and the pathogenesis of allergic disease, including bacterial products as lipopolysaccharide and CpG, in the farm effect.

Secretory protein beta-lactoglobulin in cattle stable dust may contribute to the allergy-protective farm effect.

BACKGROUND: Growing up on a cattle farm and consuming raw cow's milk protects against asthma and allergies. We expect a cattle-specific protein as active component in this farm effect. METHODS: Dust was collected from cattle and poultry stables and from mattresses of households. Urine was obtained from cattle, and ambient aerosols were sampled. Samples were analysed for BLG by SDS PAGE/immunoblot and mass spectrometry, and for association with metals by SEC-ICP-MS. PBMC of healthy donors were incubated with BLG +/- zinc, and proliferation and cytokines determined. BALB/c mice were pre-treated intranasally with stable dust extract containing BLG or depleted of BLG, and subsequent allergy response after sensitization was evaluated on antibody and symptom level. RESULTS: A major protein in dust from cattle farms and ambient air was identified as BLG. Urine from female and male cattle is a major source of BLG. In dust samples, BLG was associated with zinc. In vitro, zinc-BLG provoked significantly lower proliferation of CD4+ and CD8+ cells while inducing significantly higher levels of IFN-γ and IL-6 than the apo-BLG devoid of zinc. In vivo, pre-treatment of mice with dust extract containing BLG resulted in lower allergy symptom scores to BLG and unrelated Bet v 1 than pre-treatment with extract depleted of BLG. These in vitro and in vivo effects were independent of endotoxin. CONCLUSION: The lipocalin BLG is found in large amounts in cattle urine, accumulates in bovine dust samples and is aerosolized around farms. Its association with zinc favorably shapes the human cellular immune response towards Th1-cytokines in vitro. BLG together with zinc in stable dust protects mice from allergic sensitization. BLG with its associated ligands may in an innate manner contribute to the allergy-protective farm effect.

Ameliorating Atopy by Compensating Micronutritional Deficiencies in Immune Cells: A Double-Blind Placebo-Controlled Pilot Study.

BACKGROUND: Functional iron deficiency facilitates allergy development and amplifies the symptom burden in people experiencing allergies. Previously we selectively delivered micronutrients to immune cells with ß-lactoglobulin as carrier (holoBLG), resulting in immune resilience and allergy prevention. OBJECTIVE: The clinical efficacy of a food for special medical purposes-lozenge containing ß-lactoglobulin with iron, polyphenols, retinoic acid, and zinc (holoBLG lozenge) was assessed in allergic women. METHODS: In a randomized, double-blind, placebo-controlled pilot study, grass- and/or birch pollen-allergic women (n = 51) were given holoBLG or placebo lozenges over 6 months. Before and after dietary supplementation, participants were nasally challenged and the blood was analyzed for immune and iron parameters. Daily symptoms, medications, pollen concentrations, and well-being were recorded by an electronic health application. RESULTS: Total nasal symptom score after nasal provocations improved by 42% in the holoBLG group versus 13% in the placebo group. The combined symptom medication score during the birch peak and entire season as well as the entire grass pollen season improved in allergic subjects supplemented with the holoBLG lozenge by 45%, 31%, and 40%, respectively, compared with the placebo arm. Participants ingesting the holoBLG lozenge had improved iron status with increased hematocrit values, decreased red cell distribution width, and higher iron levels in circulating CD14+ cells compared with the placebo group. CONCLUSIONS: Targeted micronutrition with the holoBLG lozenge seemed to be effective in elevating the labile iron levels in immune cells and reducing the symptom burden in allergic women in this pilot study. The underlying allergen-independent mechanism provides evidence that dietary nutritional supplementation of the immune system is one of the ways to combat atopy.

Allergic diseases in infancy: I - Epidemiology and current interpretation.

Objective: Among non-communicable diseases, the prevalence of allergic diseases has increased significantly in the new millennium. The increase of allergic diseases is linked to the changing environment of infants. Methods: This narrative review summarizes the discussions and conclusions from the 8th Human Milk Workshop. Information from the fields of pediatrics, epidemiology, biology, microbiology, and immunology are summarized to establish a framework describing potential avenues for the prevention of allergic diseases in the future. Results: Several environmental circumstances are linked to the development of allergic diseases. While cesarean section is increasing the risk of allergies, early childhood exposure to a farm environment has a protective effect. From their analysis, nutritive and non-nutritive factors influencing the allergy risk in later life have been identified. The effect of breastfeeding on food allergy development is non-univocal. Human milk components including immunoglobulins, cytokines, and prebiotics have been indicated as important for allergy prevention. Conclusion: Many factors linked to the western lifestyle have been associated with the development of allergic diseases. This suggests several theories that may serve as a basis for new protective interventions. While it is indubitable that mother's milk protects from infectious diseases, its role in the prevention of allergic diseases is to be elucidated.

Bovine Holo-Beta-Lactoglobulin Cross-Protects Against Pollen Allergies in an Innate Manner in BALB/c Mice: Potential Model for the Farm Effect.

The lipocalin beta-lactoglobulin (BLG) is a major protein compound in cow's milk, and we detected it in cattle stable dust. BLG may be a novel player in the farm protective effect against atopic sensitization and hayfever. In previous studies, we demonstrated that only the ligand-filled holo-form of BLG prevented sensitization to itself. Here, we investigated whether holo-BLG could, in an innate manner, also protect against allergic sensitization to unrelated birch pollen allergens using a murine model. BALB/c mice were nasally pretreated four times in biweekly intervals with holo-BLG containing quercetin-iron complexes as ligands, with empty apo-BLG, or were sham-treated. Subsequently, mice were intraperitoneally sensitized two times with apo-BLG or with the unrelated birch pollen allergen apo-Bet v 1, adjuvanted with aluminum hydroxide. After subsequent systemic challenge with BLG or Bet v 1, body temperature drop was monitored by anaphylaxis imaging. Specific antibodies in serum and cytokines of BLG- and Bet v 1-stimulated splenocytes were analyzed by ELISA. Additionally, human peripheral blood mononuclear cells of pollen allergic subjects were stimulated with apo- versus holo-BLG before assessment by FACS. Prophylactic treatment with the holo-BLG resulted in protection against allergic sensitization and clinical reactivity also to Bet v 1 in an unspecific manner. Pretreatment with holo-BLG resulted in significantly lower BLG-as well as Bet v 1-specific antibodies and impaired antigen-presentation with significantly lower numbers of CD11c+MHCII+ cells expressing CD86. Pretreatment with holo-BLG also reduced the release of Th2-associated cytokines from Splenocytes in BLG-sensitized mice. Similarly, in vitro stimulation of PBMCs from birch pollen allergic subjects with holo-BLG resulted in a relative decrease of CD3+CD4+ and CD4+CRTh2 cells, but not of CD4+CD25+CD127- Treg cells, compared to apo-BLG stimulation. In conclusion, prophylactic treatment with holo-BLG protected against allergy in an antigen-specific and -unspecific manner by decreasing antigen presentation, specific antibody production and abrogating a Th2-response. Holo-BLG therefore promotes immune resilience against pollen allergens in an innate manner and may thereby contribute to the farm protective effect against atopic sensitization.

High-performing farms exploit reproductive potential of high and low prolific sows better than low-performing farms.

BACKGROUND: Our objective was to examine the impact of farm effects and sow potential on various aspects of sow performance. We examined the interaction between sow prolificacy groups categorized at parity 1 and farm productivity groups for reproductive performance across parities, and lifetime performance. Data included 419,290 service records of 85,096 sows, on 98 Spanish farms, from first-service as gilts to removal, that were served between 2008 and 2013. Farms were categorized into three productivity groups based on the upper and lower 25th percentiles of the farm means of annualized lifetime piglets weaned per sow over the 6 years: high-performing (HP), intermediate-performing (IP), and low-performing (LP) farms. Also, parity 1 sows were categorized into three groups based on the upper and lower 10th percentiles of piglets born alive (PBA) as follows: 15 piglets or more (H-prolific), 8 to 14 piglets, and 7 piglets or fewer (L-prolific). The farm groups represent farm effects, whereas the sow groups represent sow potential. Linear mixed effects models were performed with factorial arrangements and repeated measures. RESULTS: Mean parity at removal (4.8 ± 0.01) was not associated with three farm productivity groups (P = 0.43). However, HP farms had 7.7% higher farrowing rates than LP farms (P <  0.05). As a result, H-prolific and L-prolific sows on HP farms had 29.7 and 30.7 fewer non-productive days during lifetime than the respective sows on LP farms (P <  0.05). Furthermore, the H-prolific and L-prolific sows on HP farms had 4.9 and 6.2 more annualized piglets weaned than respective H-prolific and L-prolific sows on LP farms (P <  0.05), which was achieved by giving birth to 0.8-1.0 and 1.4-1.7 more PBA per litter, respectively, than on HP farms during parities 2-6 (P <  0.05). During the first parity, HP farms had 18.8% H-prolific sows compared to 6.2% on LP farms. CONCLUSION: Farm effects substantially affected lifetime performance of sows. Higher lifetime productivity of sows on HP farms was achieved by higher farrowing rate, fewer non-productive days, more PBA and more piglets weaned per sow, regardless of prolific category of the sows.

Sensitivity of Caligus rogercresseyi (Boxshall and Bravo 2000) to pyrethroids and azamethiphos measured using bioassay tests-A large scale spatial study.

The variety of antiparasitics that can be used against caligid copepods is limited and efforts are needed to maintain their efficacies. The objective of this study was to monitor the sensitivity of Caligus rogercresseyi, populations towards antiparasitics based on deltamethrin, cypermethrin and azamethiphos within and across geographic regions. The bioassay design consisted of exposing parasites collected from 23 farms to the different chemotherapeutants at the concentration and exposure times recommended for field treatment, under laboratory conditions, and evaluating the number of dead and live parasites 48h after exposure. Parasites were collected from 23 farms distributed in four macrozones in the Los Lagos region and three macrozones in the Aysén region. Parasite sensitivity was evaluated using a Generalized Linear Mixed Model of the Binomial family (Logit) fit by the maximum likelihood, using the lme4 package in R. Parasite gender, macrozone, and antiparasitics were used as fixed factors and farm was the random factor. The model including all the factors proved to be a useful tool for predicting parasite sensitivity. This approach identified (i) those macrozones with a greater likelihood of finding parasite populations which are more or less sensitive to the three antiparasitics, (ii) cases in which parasite sensitivity to the different antiparasitics varied within a given macrozone, (iii) differences in sensitivity between females and males and (iv) an important random effect associated with farm. The results indicate a spatial variability of parasite sensitivity to antiparasitics which, added to the continuous treatments applied on farms, suggest it is necessary to regularly update the sensitivity status in the macrozones. This would allow managers to improve their decision making processes regarding the type of antiparasitic to be used in a given situation. The one-concentration type bioassay performed in this study allowed us to perform a large spatial study with replicated tests of the sensitivity of C. rogercresseyi to pyrethroids and azamethiphos. Further studies should focus on the farm effects, the relationship between the sensitivity of parasites and field efficacy, as well as parasite population structure and connectivity with regard to parasite transmission between farms.