VEGF-D-mediated signaling in tendon cells is involved in degenerative processes.

Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

Allergy-induced systemic inflammation impairs tendon quality.

BACKGROUND: Treatment of degenerating tendons still presents a major challenge, since the aetiology of tendinopathies remains poorly understood. Besides mechanical overuse, further known predisposing factors include rheumatoid arthritis, diabetes, obesity or smoking all of which combine with a systemic inflammation. METHODS: To determine whether the systemic inflammation accompanying these conditions contributes to the onset of tendinopathy, we studied the effect of a systemic inflammation induced by an allergic episode on tendon properties. To this end, we induced an allergic response in mice by exposing them to a timothy grass pollen allergen and subsequently analysed both their flexor and Achilles tendons. Additionally, we analysed data from a health survey comprising data from more than 10.000 persons for an association between the occurrence of an allergy and tendinopathy. FINDINGS: Biomechanical testing and histological analysis revealed that tendons from allergic mice not only showed a significant reduction of both elastic modulus and tensile stress, but also alterations of the tendon matrix. Moreover, treatment of 3D tendon-like constructs with sera from allergic mice resulted in a matrix-remodelling expression profile and the expression of macrophage-associated markers and matrix metalloproteinase 2 (MMP2) was increased in allergic Achilles tendons. Data from the human health study revealed that persons suffering from an allergy have an increased propensity to develop a tendinopathy. INTERPRETATION: Our study demonstrates that the presence of a systemic inflammation accompanying an allergic condition negatively impacts on tendon structure and function. FUNDING: This study was financially supported by the Fund for the Advancement of Scientific Research at Paracelsus Medical University (PMU-FFF E-15/22/115-LEK), by the Land Salzburg, the Salzburger Landeskliniken (SALK, the Health Care Provider of the University Hospitals Landeskrankenhaus and Christian Doppler Klinik), the Paracelsus Medical University, Salzburg and by unrestricted grants from Bayer, AstraZeneca, Sanofi-Aventis, Boehringer-Ingelheim.

Load-induced regulation of tendon homeostasis by SPARC, a genetic predisposition factor for tendon and ligament injuries.

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc -/- mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc -/- mice. Tendon hypoplasia was attenuated in Sparc -/- mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc -/- three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc -/- tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

Macromechanics and polycaprolactone fiber organization drive macrophage polarization and regulate inflammatory activation of tendon in vitro and in vivo.

Appropriate macrophage response to an implanted biomaterial is crucial for successful tissue healing outcomes. In this work we investigated how intrinsic topological cues from electrospun biomaterials and extrinsic mechanical loads cooperate to guide macrophage activation and macrophage-tendon fibroblast cross-talk. We performed a series of in vitro and in vivo experiments using aligned or randomly oriented polycaprolactone nanofiber substrates in both mechanically loaded and unloaded conditions. Across all experiments a disorganized biomaterial fiber topography was alone sufficient to promote a pro-inflammatory signature in macrophages, tendon fibroblasts, and tendon tissue. Extrinsic mechanical loading was found to strongly regulate the character of this signature by reducing pro-inflammatory markers both in vitro and in vivo. We observed that macrophages generally displayed a stronger response to biophysical cues than tendon fibroblasts, with dominant effects of cross-talk between these cell types observed in mechanical co-culture models. Collectively our data suggest that macrophages play a potentially important role as mechanosensory cells in tendon repair, and provide insight into how biological response might be therapeutically modulated by rational biomaterial designs that address the biomechanical niche of recruited cells.

MicroRNA Profiling Reveals Distinct Signatures in Degenerative Rotator Cuff Pathologies.

MicroRNAs (miRNAs) have emerged as key regulators orchestrating a wide range of inflammatory and fibrotic diseases. However, the role of miRNAs in degenerative shoulder joint disorders is poorly understood. The aim of this explorative case-control study was to identify pathology-related, circulating miRNAs in patients with chronic rotator cuff tendinopathy and degenerative rotator cuff tears (RCT). In 2017, 15 patients were prospectively enrolled and assigned to three groups based on the diagnosed pathology: (i) no shoulder pathology, (ii) chronic rotator cuff tendinopathy, and (iii) degenerative RCTs. In total, 14 patients were included. Venous blood samples ("liquid biopsies") were collected from each patient and serum levels of 187 miRNAs were determined. Subsequently, the change in expression of nine candidate miRNAs was verified in tendon biopsy samples, collected from patients who underwent arthroscopic shoulder surgery between 2015 and 2018. Overall, we identified several miRNAs to be progressively deregulated in sera from patients with either chronic rotator cuff tendinopathy or degenerative RCTs. Importantly, for the several of these miRNAs candidates repression was also evident in tendon biopsies harvested from patients who were treated for a supraspinatus tendon tear. As similar expression profiles were determined for tendon samples, the newly identified systemic miRNA signature has potential as novel diagnostic or prognostic biomarkers for degenerative rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. Inc. J Orthop Res 38:202-211, 2020.

Tenophages: a novel macrophage-like tendon cell population expressing CX3CL1 and CX3CR1.

Tendon disorders frequently occur and recent evidence has clearly implicated the presence of immune cells and inflammatory events during early tendinopathy. However, the origin and properties of these cells remain poorly defined. Therefore, the aim of this study was to determine the presence of cells in healthy rodent and human tendon tissue fulfilling macrophage-like functions. Using various transgenic reporter mouse models, we demonstrate the presence of tendon-resident cells in the dense matrix of the tendon core expressing the fractalkine (Fkn) receptor CX3CR1 and its cognate ligand CX3CL1/Fkn. Pro-inflammatory stimulation of 3D tendon-like constructs in vitro resulted in a significant increase in the expression of IL-1ß, IL-6, Mmp3, Mmp9, CX3CL1 and epiregulin, which has been reported to contribute to inflammation, wound healing and tissue repair. Furthermore, we demonstrate that inhibition of the Fkn receptor blocked tendon cell migration in vitro, and show the presence of CX3CL1/CX3CR1/EREG-expressing cells in healthy human tendons. Taken together, we demonstrate the presence of CX3CL1+/CX3CR1+ 'tenophages' within the healthy tendon proper, which potentially fulfill surveillance functions in tendons.This article has an associated First Person interview with the first author of the paper.

Global Responses of Il-1ß-Primed 3D Tendon Constructs to Treatment with Pulsed Electromagnetic Fields.

Tendinopathy is accompanied by a cascade of inflammatory events promoting tendon degeneration. Among various cytokines, interleukin-1ß plays a central role in driving catabolic processes, ultimately resulting in the activation of matrix metalloproteinases and a diminished collagen synthesis, both of which promote tendon extracellular matrix degradation. Pulsed electromagnetic field (PEMF) therapy is often used for pain management, osteoarthritis, and delayed wound healing. In vitro PEMF treatment of tendon-derived cells was shown to modulate pro-inflammatory cytokines, potentially limiting their catabolic effects. However, our understanding of the underlying cellular and molecular mechanisms remains limited. We therefore investigated the transcriptome-wide responses of Il-1ß-primed rat Achilles tendon cell-derived 3D tendon-like constructs to high-energy PEMF treatment. RNASeq analysis and gene ontology assignment revealed various biological processes to be affected by PEMF, including extracellular matrix remodeling and negative regulation of apoptosis. Further, we show that members of the cytoprotective Il-6/gp130 family and the Il-1ß decoy receptor Il1r2 are positively regulated upon PEMF exposure. In conclusion, our results provide fundamental mechanistic insight into the cellular and molecular mode of action of PEMF on tendon cells and can help to optimize treatment protocols for the non-invasive therapy of tendinopathies.

3D-Embedded Cell Cultures to Study Tendon Biology.

Tendons harbor various cell populations, including cells displaying classical adult mesenchymal stromal cell criteria. Previous studies have shown that a tenogenic phenotype is more effectively maintained in a 3D cell culture model under mechanical load. This chapter describes a method to isolate tendon-derived cells from rat Achilles tendons and the subsequent formation of 3D-embedded cell cultures. These tendon-like constructs can then be analyzed by various means, including histology, immunohistochemistry, qPCR, or standard protein analysis techniques.

Limiting angiogenesis to modulate scar formation.

Angiogenesis, the process of new blood vessel formation from existing blood vessels, is a key aspect of virtually every repair process. During wound healing an extensive, but immature and leaky vascular plexus forms which is subsequently reduced by regression of non-functional vessels. More recent studies indicate that uncontrolled vessel growth or impaired vessel regression as a consequence of an excessive inflammatory response can impair wound healing, resulting in scarring and dysfunction. However, in order to elucidate targetable factors to promote functional tissue regeneration we need to understand the molecular and cellular underpinnings of physiological angiogenesis, ranging from induction to resolution of blood vessels. Especially for avascular tissues (e.g. cornea, tendon, ligament, cartilage, etc.), limiting rather than boosting vessel growth during wound repair potentially is beneficial to restore full tissue function and may result in favourable long-term healing outcomes.

Bevacizumab Improves Achilles Tendon Repair in a Rat Model.

BACKGROUND/AIMS: Effective wound-healing generally requires efficient re-vascularization after injury, ensuring sufficient supply with oxygen, nutrients, and various cell populations. While this applies to most tissues, tendons are mostly avascular in nature and harbor relatively few cells, probably contributing to their poor regenerative capacity. Considering the minimal vascularization of healthy tendons, we hypothesize that controlling angiogenesis in early tendon healing is beneficial for repair tissue quality and function. METHODS: To address this hypothesis, Bevacizumab, a monoclonal antibody blocking VEGF-A signaling, was locally injected into the defect area of a complete tenotomy in rat Achilles tendon. At 28 days post-surgery, the defect region was investigated using immunohistochemistry against vascular and lymphatic epitopes. Polarization microscopy and biomechanical testing was used to determine tendon integrity and gait analysis for functional testing in treated vs non-treated animals. RESULTS: Angiogenesis was found to be significantly reduced in the Bevacizumab treated repair tissue, accompanied by significantly reduced cross sectional area, improved matrix organization, increased stiffness and Young's modulus, maximum load and stress. Further, we observed an improved gait pattern when compared to the vehicle injected control group. CONCLUSION: Based on the results of this study we propose that reducing angiogenesis after tendon injury can improve tendon repair, potentially representing a novel treatment-option.

A high-glucose diet affects Achilles tendon healing in rats.

Chronic and acute tendinopathies are difficult to treat and tendon healing is generally a very slow and incomplete process and our general understanding of tendon biology and regeneration lags behind that of muscle or bone. Although still largely unexplored, several studies suggest a positive effect of nutritional interventions on tendon health and repair. With this study, we aim to reveal effects of a high-glucose diet on tendon neoformation in a non-diabetic rat model of Achilles tenotomy. After surgery animals received either a high-glucose diet or a control diet for 2 and 4 weeks, respectively. Compared to the control group, tendon repair tissue thickness and stiffness were increased in the high-glucose group after 2 weeks and gait pattern was altered after 1 and 2 weeks. Cell proliferation was up to 3-fold higher and the expression of the chondrogenic marker genes Sox9, Col2a1, Acan and Comp was significantly increased 2 and 4 weeks post-surgery. Further, a moderate increase in cartilage-like areas within the repair tissue was evident after 4 weeks of a high-glucose diet regimen. In summary, we propose that a high-glucose diet significantly affects tendon healing after injury in non-diabetic rats, potentially driving chondrogenic degeneration.

Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

Expression of tight junction proteins and transporters for xenobiotic metabolism at the blood-CSF barrier during development in the nonhuman primate (P. hamadryas).

The choroid plexus (CP) is rich in barrier mechanisms including transporters and enzymes which can influence drug disposition between blood and brain. We have limited knowledge of their state in fetus. We have studied barrier mechanisms along with metabolism and transporters influencing xenobiotics, using RNAseq and protein analysis, in the CP during the second-half of gestation in a nonhuman primate (Papio hamadryas). There were no differences in the expression of the tight-junctions at the CP suggesting a well-formed fetal blood-CSF barrier during this period of gestation. Further, the fetal CP express many enzymes for phase I-III metabolisms as well as transporters suggesting that it can greatly influence drug disposition and has a significant machinery to deactivate reactive molecules with only minor gestational changes. In summary, the study suggests that from, at least, midgestation, the CP in the nonhuman primate is restrictive and express most known genes associated with barrier function and transport.

Brain barrier properties and cerebral blood flow in neonatal mice exposed to cerebral hypoxia-ischemia.

Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood-brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.

Presence of lymphatics in a rat tendon lesion model.

Tendons lack sufficient blood supply and represent a bradytroph tissue with prolonged healing time under pathological conditions. While the role of lymphatics in wound/defect healing in tissues with regular blood supply is well investigated, its involvement in tendon defects is not clear. We here try to identify the role of the lymphatic system in a tendon lesion model with morphological methods. A rat Achilles tendon lesion model (n = 5) was created via surgical intervention. Two weeks after surgery, animals were killed and lesioned site removed and prepared for polarization microscopy (picrosirius red) and immunohistochemistry using the lymphatic markers PROX1, VEGFR3, CCL21, LYVE-1, PDPN, and the vascular marker CD31. Additionally, DAPI was applied. Untreated tendons served as controls, confocal laser-scanning microscopy was used for documentation. At the lesion site, polarization microscopy revealed a structural reintegration while immunohistochemistry detected band-like profiles immunoreactive for PDPN, VEGFR3, CCL21, LYVE1, and CD31, surrounding DAPI-positive nuclei. PROX1-positive nuclei were detected within the lesion forming lines and opposed to each other. These PROX1-positive nuclei were surrounded by LYVE-1- or VEGFR3-positive surfaces. Few CD31-positive profiles contained PROX1-positive nuclei, while the majority of CD31-positive profiles lacked PROX1-positive nuclei. VEGFR3-, PDPN-, and LYVE-1-positive profiles were numerous within the lesion site, but absent in control tissue. Within 2 weeks, a structural rearrangement takes place in this lesion model, with dense lymphatic supply. The role of lymphatics in tendon wound healing is unclear, and proposed model represents a good possibility to study healing dynamics and lymphangiogenesis in a tissue almost completely lacking lymphatics in physiological conditions.

Perinatal influences on the development of asthma and atopy in childhood.

BACKGROUND: In most children with asthma and atopy, onset of disease occurs early in life, indicating a crucial role of in utero and early childhood environment. However, only a small part of this burden of disease established early in life has been explained. OBJECTIVE: To examine the effects of early environmental exposures on the development of asthma and atopy within the setting of an affluent urban population. METHODS: The authors followed 526 German children from birth to 5 years of age. Parental interviews in pregnancy and then yearly assessed the health of the child and environmental characteristics. Endotoxin and allergens in house dust were measured at 3 months. Atopic sensitization was assessed at 1 and 5 years. RESULTS: In atopic mothers, acute atopic symptoms during pregnancy were associated with increased risk of early atopic dermatitis (adjusted odds ratio [aOR] 1.74, 95% confidence interval [CI] 1.00-3.02) and allergic rhinitis at 5 years (aOR 2.11, 95% CI 1.01-4.41). Further, maternal illnesses during pregnancy (ie, repeated common colds) increased the risk of asthma at 5 years (aOR 2.31, 95% CI 1.12-4.78). Endotoxin in the child's mattress was inversely associated with atopic sensitization (aOR 0.79, 95% CI 0.64-0.97) and asthma (aOR 0.71, 95% CI 0.55-0.93). A contrasting effect of early endotoxin and mite exposure was observed for mite sensitization: mite exposure increased the risk of mite sensitization at 5 years (aOR 1.30, 95% CI 1.11-1.53), whereas endotoxin exposure was inversely associated with mite sensitization (aOR 0.73, 95% CI 0.57-0.95). CONCLUSION: Factors affecting the in utero environment, such as maternal atopy and infections, and bacterial exposure in pregnancy or early life may act as immunomodulators enhancing or inhibiting the development of asthma and atopy in childhood.

Bupivacaine induces short-term alterations and impairment in rat tendons.

BACKGROUND: Toxicity of the local anesthetic bupivacaine (BV) has been a matter of debate across medical fields. Numerous in vitro studies demonstrate considerable toxicity of BV on various cell types. PURPOSE: This study addresses the question of how tendon tissue responds to BV in vivo and in vitro. STUDY DESIGN: Controlled laboratory study. METHODS: In vitro studies on cultured rat Achilles tendon-derived cells were performed with cell viability assays and cleaved caspase 3 immunocytochemistry. Quantitative reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, and a biomechanical testing routine were applied on rat Achilles tendons at 1 and 4 weeks after a single unilateral peritendinous injection of 0.5% BV. The BV-mediated cell death in tendons was estimated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunohistochemical detection of cleaved caspase 3. RESULTS: Treatment of rat tendon-derived cells with 0.5% bupivacaine for 10 minutes had detrimental effects on cell viability, which can be reduced by N-acetyl-L-cysteine or reduction of extracellular calcium. In vivo, single peritendinous injections of BV caused apoptosis in endotenon cells and an increase of pro-matrix metalloproteinase-9 after 6 hours. The collagen ratio shifted toward collagen type III after 6 hours and 2 days; scleraxis messenger RNA (mRNA) expression was reduced by 87%. Maximum tensile load was reduced by 17.6% after 1 week. CONCLUSION: Bupivacaine exerts a severe, reactive oxygen species-mediated effect on tendon cell viability in vitro in a time- and dose-dependent manner, depending on extracellular calcium concentration. Culture conditions need to be taken into account when in vitro data are translated into the in vivo situation. In vivo, administration of BV elicits a marked but temporary functional damage. CLINICAL RELEVANCE: Local anesthetics cause short-term alterations in rat tendons, which, if occurring in humans to a similar extent, may be relevant regarding decreased biomechanical properties and increased vulnerability to tendon overload or injury.

Beyond cell-cell adhesion: Emerging roles of the tight junction scaffold ZO-2.

Zonula occludens proteins (ZO-1, ZO-2, ZO-3), which belong to the family of membrane-associated guanylate kinase (MAGUK) homologs, serve as molecular hubs for the assembly of multi-protein networks at the cytoplasmic surface of intercellular contacts in epithelial and endothelial cells. These multi-PDZ proteins exert crucial functions in the structural organization of intercellular contacts and in transducing intracellular signals from the plasma membrane to the nucleus. The junctional MAGUK protein ZO-2 not only associates with the C-terminal PDZ-binding motif of various transmembrane junctional proteins but also transiently targets to the nucleus and interacts with a number of nuclear proteins, thereby modulating gene expression and cell proliferation. Recent evidence suggests that ZO-2 is also involved in stress response and cytoprotective mechanisms, which further highlights the multi-faceted nature of this PDZ domain-containing protein. This review focuses on ZO-2 acting as a molecular scaffold at the cytoplasmic aspect of tight junctions and within the nucleus and discusses additional aspects of its cellular activities. The multitude of proteins interacting with ZO-2 and the heterogeneity of proteins either influencing or being influenced by ZO-2 suggests an exceptional functional capacity of this protein far beyond merely serving as a structural component of cellular junctions.

Protection from childhood asthma and allergy in Alpine farm environments-the GABRIEL Advanced Studies.

BACKGROUND: Studies on the association of farm environments with asthma and atopy have repeatedly observed a protective effect of farming. However, no single specific farm-related exposure explaining this protective farm effect has consistently been identified. OBJECTIVE: We sought to determine distinct farm exposures that account for the protective effect of farming on asthma and atopy. METHODS: In rural regions of Austria, Germany, and Switzerland, 79,888 school-aged children answered a recruiting questionnaire (phase I). In phase II a stratified random subsample of 8,419 children answered a detailed questionnaire on farming environment. Blood samples and specific IgE levels were available for 7,682 of these children. A broad asthma definition was used, comprising symptoms, diagnosis, or treatment ever. RESULTS: Children living on a farm were at significantly reduced risk of asthma (adjusted odds ratio [aOR], 0.68; 95% CI, 0.59-0.78; P< .001), hay fever (aOR, 0.43; 95% CI, 0.36-0.52; P< .001), atopic dermatitis (aOR, 0.80; 95% CI, 0.69-0.93; P= .004), and atopic sensitization (aOR, 0.54; 95% CI, 0.48-0.61; P< .001) compared with nonfarm children. Whereas this overall farm effect could be explained by specific exposures to cows, straw, and farm milk for asthma and exposure to fodder storage rooms and manure for atopic dermatitis, the farm effect on hay fever and atopic sensitization could not be completely explained by the questionnaire items themselves or their diversity. CONCLUSION: A specific type of farm typical for traditional farming (ie, with cows and cultivation) was protective against asthma, hay fever, and atopy. However, whereas the farm effect on asthma could be explained by specific farm characteristics, there is a link still missing for hay fever and atopy.

The GABRIEL Advanced Surveys: study design, participation and evaluation of bias.

Exposure to farming environments has been shown to protect substantially against asthma and atopic disease across Europe and in other parts of the world. The GABRIEL Advanced Surveys (GABRIELA) were conducted to determine factors in farming environments which are fundamental to protecting against asthma and atopic disease. The GABRIEL Advanced Surveys have a multi-phase stratified design. In a first-screening phase, a comprehensive population-based survey was conducted to assess the prevalence of exposure to farming environments and of asthma and atopic diseases (n = 103,219). The second phase was designed to ascertain detailed exposure to farming environments and to collect biomaterial and environmental samples in a stratified random sample of phase 1 participants (n = 15,255). A third phase was carried out in a further stratified sample only in Bavaria, southern Germany, aiming at in-depth respiratory disease and exposure assessment including extensive environmental sampling (n = 895). Participation rates in phase 1 were around 60% but only about half of the participating study population consented to further study modules in phase 2. We found that consenting behaviour was related to familial allergies, high parental education, wheeze, doctor diagnosed asthma and rhinoconjunctivitis, and to a lesser extent to exposure to farming environments. The association of exposure to farm environments with asthma or rhinoconjunctivitis was not biased by participation or consenting behaviour. The GABRIEL Advanced Surveys are one of the largest studies to shed light on the protective 'farm effect' on asthma and atopic disease. Bias with regard to the main study question was able to be ruled out by representativeness and high participation rates in phases 2 and 3. The GABRIEL Advanced Surveys have created extensive collections of questionnaire data, biomaterial and environmental samples promising new insights into this area of research.