Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging.

Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogs were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm-specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

Royal jelly extracellular vesicles promote wound healing by modulating underlying cellular responses.

Apis mellifera royal jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs), and, in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing-associated effects. Molecular analysis of RJEVs verified the presence of exosomal markers such as CD63 and syntenin, and cargo molecules MRJP1, defensin-1, and jellein-3. Furthermore, RJEVs were demonstrated to modulate mesenchymal stem cell (MSC) differentiation and secretome, as well as decrease LPS-induced inflammation in macrophages by blocking the mitogen-activated protein kinase (MAPK) pathway. In vivo studies confirmed antibacterial effects of RJEVs and demonstrated an acceleration of wound healing in a splinted mouse model. This study suggests that RJEVs play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing. Transfer of RJ into the clinics has been impeded by the high complexity of the raw material. Isolating EVs from the raw RJ decreases the complexity while allowing standardization and quality control, bringing a natural nano-therapy one step closer to the clinics.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. METHODS: Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. RESULTS: A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. CONCLUSION: Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

NOD2 Signaling Circuitry during Allergen Sensitization Does Not Worsen Experimental Neutrophilic Asthma but Promotes a Th2/Th17 Profile in Asthma Patients but Not Healthy Subjects.

Nucleotide-binding oligomerization domain 2 (NOD2) recognizes pathogens associated with the development of asthma. Moreover, NOD2 adjuvants are used in vaccine design to boost immune responses. Muramyl di-peptide (MDP) is a NOD2 ligand, which is able to promote Th2/Th17 responses. Furthermore, polymorphisms of the NOD2 receptor are associated with allergy and asthma development. This study aimed to evaluate if MDP given as an adjuvant during allergen sensitization may worsen the development of Th2/Th17 responses. We used a mouse model of Th2/Th17-type allergic neutrophil airway inflammation (AAI) to dog allergen, with in vitro polarization of human naive T cells by dendritic cells (DC) from healthy and dog-allergic asthma subjects. In the mouse model, intranasal co-administration of MDP did not modify the AAI parameters, including Th2/Th17-type lung inflammation. In humans, MDP co-stimulation of allergen-primed DC did not change the polarization profile of T cells in healthy subjects but elicited a Th2/Th17 profile in asthma subjects, as compared with MDP alone. These results support the idea that NOD2 may not be involved in the infection-related development of asthma and that, while care has to be taken in asthma patients, NOD2 adjuvants might be used in non-sensitized individuals.

NOD-like receptors in asthma.

Asthma is an extremely prevalent chronic inflammatory disease of the airway where innate and adaptive immune systems participate collectively with epithelial and other structural cells to cause airway hyperresponsiveness, mucus overproduction, airway narrowing, and remodeling. The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular innate immune sensors that detect microbe-associated molecular patterns and damage-associated molecular patterns, well-recognized for their central roles in the maintenance of tissue homeostasis and host defense against bacteria, viruses and fungi. In recent times, NLRs have been increasingly acknowledged as much more than innate sensors and have emerged also as relevant players in diseases classically defined by their adaptive immune responses such as asthma. In this review article, we discuss the current knowledge and recent developments about NLR expression, activation and function in relation to asthma and examine the potential interventions in NLR signaling as asthma immunomodulatory therapies.

A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4+ T cells and is defective in Crohn´s disease patients.

Immunosuppressive Interleukin (IL)-10 production by pro-inflammatory CD4+ T cells is a central self-regulatory function to limit aberrant inflammation. Still, the molecular mediators controlling IL-10 expression in human CD4+ T cells are largely undefined. Here, we identify a Notch/STAT3 signaling-module as a universal molecular switch to induce IL-10 expression across human naïve and major effector CD4+ T cell subsets. IL-10 induction was transient, jointly controlled by the transcription factors Blimp-1/c-Maf and accompanied by upregulation of several co-inhibitory receptors, including LAG-3, CD49b, PD-1, TIM-3 and TIGIT. Consistent with a protective role of IL-10 in inflammatory bowel diseases (IBD), effector CD4+ T cells from Crohn's disease patients were defective in Notch/STAT3-induced IL-10 production and skewed towards an inflammatory Th1/17 cell phenotype. Collectively, our data identify a Notch/STAT3-Blimp-1/c-Maf axis as a common anti-inflammatory pathway in human CD4+ T cells, which is defective in IBD and thus may represent an attractive therapeutic target.

Bacterial adhesion to collagens: implications for biofilm formation and disease progression in the oral cavity.

Collagen is the most abundant structural protein in the body and the main component of the extracellular matrix of most tissues, including dentine and periodontal tissues. Despite the well-characterized role of collagen and specifically type-I collagen, as a ligand for host cells, its role as a substrate for bacterial adhesion and biofilm formation is less explored. Therefore, the purpose of this review is to discuss recent findings regarding the adhesion of oral bacteria to collagen surfaces and its role in the progression and severity of oral and systemic diseases. Initial oral colonizers such as streptococci have evolved collagen-binding proteins (cbp) that are important for the colonization of dentine and periodontal tissues. Also, periodontal pathogens such as Porphyromonas gingivalis and Tannerella forsythia utilise cbps for tissue sensing and subsequent invasion. The implications of bacteria-collagen coupling in the context of collagen biomaterials and regenerative dentistry approaches are also addressed. Furthermore, the importance of interdisciplinary techniques such as atomic force microscopy for the nanocharacterization of bacteria-collagen interactions is also considered. Overall, understanding the process of oral bacterial adhesion onto collagen is important for developing future therapeutic approaches against oral and systemic diseases, by modulating the early stages of biofilm formation.

Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci.

PURPOSE: Recently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci. METHODS: A. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure. RESULTS: Molecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans. CONCLUSION: Honey is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilm-modulating strategy in dental caries.

A rapid test for the environmental detection of pigeon antigen.

INTRODUCTION: Avoidance of inhaled bird antigens is essential to prevent hypersensitivity pneumonitis disease progression. The aim of the present study was to develop a sandwich enzyme link immunoassay (ELISA) and an immunochromatographic test (ICT) and compare their ability to detect pigeon antigens in environmental samples. METHODS: An amplified sandwich ELISA using pigeon serum as a calibration standard and a ICT using gold-labeled anti-pigeon serum antibodies for the rapid detection of pigeon antigens in environmental samples were developed. Twenty-two different airborne samples were collected and analysed using both methods. Strip density values obtained with ICT were calculated and compared with the concentrations determined by the ELISA method for pigeon antigens. Strips results were also visually analysed by five independent evaluators. RESULTS: The ELISA method to quantify pigeon antigen had a broader range (58.4 and 10,112.2 ng/ml), compared to the ICT assay (420 to 3360 ng/ml). A kappa index of 0.736 (p < 0.0001) was obtained between the observers evaluating the ICT strips. The results of the ELISA and the relative density of the ICT showed a highly significant correlation (rs:0.935; p < 0.0001). Bland-Altman plot also confirmed excellent agreement between the two methods (mean difference: -1.626; p < 0.0001). CONCLUSIONS: Since there was a good correlation between both assays, we can conclude that the rapid and simple ICT assay is a good and valid alternative, which does not require expensive equipment, for the validated ELISA technique.

Desarrollo y validación interna de un modelo de predicción clínica del riesgo de infección bacteriana nosocomial en pacientes con lupus eritematoso sistémico / Development and internal validation of a clinical prediction model of the risk of nosocomial bacterial infection in patients with systemic lupus erythematosus

RESUMEN Introducción: Los pacientes con lupus eritematoso sistémico (LES) tienen un riesgo aumen tado de padecer infecciones tanto adquiridas en la comunidad como asociadas con el cuidado de la salud. Las infecciones bacterianas son las más frecuentes y graves durante la hospitalización de estos pacientes. Objetivo: Desarrollar y validar internamente un modelo de predicción clínica de pronóstico del riesgo de infección bacteriana adquirida en el hospital en pacientes con LES, usando datos clínicos y de laboratorio obtenidos durante las primeras horas de hospitalización. Métodos: Se analizó una cohorte retrospectiva de pacientes con LES mayores de 16 arios, hos pitalizados por motivos diferentes a infección bacteriana en 2 hospitales de alta complejidad de Medellín entre 2011 y 2016. Se compararon las características de los pacientes que des arrollaron el desenlace de infección bacteriana entre el día 3 y el día 15 de hospitalización con aquellos que no lo presentaron. Las variables significativas en el análisis bivariado fueron consideradas para la construcción del modelo por medio de regresión logística multivariada. Resultados: Se incluyeron 765 episodios, de los cuales 98 (12,8%) presentaron el desenlace de interés. Se consideraron 35 predictores candidatos. Las variables incorporadas en el modelo final fueron: edad, recuento de neutrófilos, puntaje de actividad lúpica SLEDAI, uso de sonda vesical, uso de catéter venoso central en las primeras 72 h, dosis de glucocorticoides en el mes previo y el uso de un antimalárico en los 3 meses previos. La capacidad de discrimi nación del modelo fue aceptable a buena (AUC-ROC 0,74; IC 95% 0,69-0,80). La prueba de bondad de ajuste de Hosmer-Lemeshow (p = 0,637) evidenció una adecuada calibración. Conclusión: Desarrollamos un modelo de predicción clínica de pronóstico del riesgo de infec ción bacteriana nosocomial en pacientes con LES. El modelo desarrollado está compuesto por variables clínicas y de laboratorio simples disponibles en el momento del ingreso al hospital. Se requieren estudios de validación externa y de impacto clínico antes de su implementación rutinaria.

ABSTRACT Introduction: Patients with systemic lupus erythematosus (SLE) have an increased risk of developing community-acquired infections, as well as those associated with health care. Bacterial infections are the most common and serious while these patients are in hospital. Objective: To develop, and internally validate, a clinical prediction model for the prognosis of the risk of hospital-acquired bacterial infection in SLE patients using clinical and laboratory data obtained during the first hours of hospital admission. Methods: An analysis was performed on retrospective cohort of patients with SLE older than 16 years and admitted for reasons other than bacterial infection in 2 highly complex hospitals in Medellín between 2011 and 2016. The characteristics of the patients who developed a bacterial infection were compared between day 3 and day 15 of hospital admission with those who did not develop one. The significant variables in the bivariate analysis were used for the construction of the model using multivariate logistic regression. Results: A total of 765 episodes were included, of which 98 (12.8%) presented the outcome of interest. Thirty-five candidate predictors were considered. The variables incorporated in the final model were: age, neutrophil count, SLEDAI lupus activity score, use of a bladder catheter, use of a central venous catheter in the first 72 h, glucocorticoid doses in the previous month, and use of an antimalarial drug in the 3 previous months. The discrimination capacity of the model was acceptable to good (AUC-ROC 0.74; 95% CI 0.69-0.80). The Hosmer-Lemeshow goodness of fit test (P = .637) suggested adequate calibration. Conclusion: A clinical prediction model of prognostic risk of nosocomial bacterial infection in patients with SLE has been developed. This model is made up of simple clinical and laboratory variables available at the time of hospital admission. External validation and clinical impact studies are required before routine implementation.

NOD1 sensing of house dust mite-derived microbiota promotes allergic experimental asthma.

BACKGROUND: Asthma severity has been linked to exposure to gram-negative bacteria from the environment that are recognized by NOD1 receptor and are present in house dust mite (HDM) extracts. NOD1 polymorphism has been associated with asthma. OBJECTIVE: We sought to evaluate whether either host or HDM-derived microbiota may contribute to NOD1-dependent disease severity. METHODS: A model of HDM-induced experimental asthma was used and the effect of NOD1 deficiency was evaluated. Contribution of host microbiota was evaluated by fecal transplantation. Contribution of HDM-derived microbiota was assessed by 16S ribosomal RNA sequencing, mass spectrometry analysis, and peptidoglycan depletion of the extracts. RESULTS: In this model, loss of the bacterial sensor NOD1 and its adaptor RIPK2 improved asthma features. Such inhibitory effect was not related to dysbiosis caused by NOD1 deficiency, as shown by fecal transplantation of Nod1-deficient microbiota to wild-type germ-free mice. The 16S ribosomal RNA gene sequencing and mass spectrometry analysis of HDM allergen, revealed the presence of some muropeptides from gram-negative bacteria that belong to the Bartonellaceae family. While such HDM-associated muropeptides were found to activate NOD1 signaling in epithelial cells, peptidoglycan-depleted HDM had a decreased ability to instigate asthma in vivo. CONCLUSIONS: These data show that NOD1-dependent sensing of HDM-associated gram-negative bacteria aggravates the severity of experimental asthma, suggesting that inhibiting the NOD1 signaling pathway may be a therapeutic approach to treating asthma.

Urinary levels of ceruloplasmin and monocyte chemoattractant protein-1 correlate with extra-capillary proliferation and chronic damage in patients with lupus nephritis.

BACKGROUND AND AIM: There are few studies of urinary biomarkers and histopathologic features in lupus nephritis (LN). The aim was to analyze the correlation between a wide panel of urinary biomarkers and serum concentrations of anti C1q antibodies with histological items of activity and chronicity on kidney biopsy in LN patients. METHODS: Patients with systemic lupus erythematosus (SLE) according to American College of Rheumatology (ACR) criteria were included. LN diagnosis was based on ACR criteria. Histologic features of activity and chronicity indices were analyzed according to the Austin classification. Serum Anti C1q levels were determined by commercial ELISA. Urinary levels of transferrin, ceruloplasmin (CP), VCAM-1, TWEAK, monocyte chemoattractant protein-1 (MCP-1), neutrophil gelatinase-associated lipocalin (NGAL), and alpha-1-acid glycoprotein were measured by commercial ELISA. RESULTS: We included 120 SLE patients (81% female, mean age 33.1 ± 9.3 years, 59.4% Mestizo, 37.8% Afro-Latin American): 64% had LN. Kidney biopsy was performed in 55 patients, but only 37 were made in our center. Anti C1q antibodies were associated with endocapillary proliferation. In patients with cellular crescents, urinary concentrations of CP were significantly higher. In patients with a chronicity index (CI) ≥ 4, fibrous crescents, tubular atrophy, and interstitial fibrosis, urinary MCP-1 levels were higher. CONCLUSIONS: In SLE patients, serum anti C1q antibodies and urinary CP were associated with activity on kidney biopsy and MCP-1 with chronic damage. This panel of biomarkers could be validated in larger, multi-ethnic population as a complementary tool for better stratification of LN patients. Key Points • Urinary biomarkers are complementary useful tools for the assessment of SLE patients. • Urinary levels of CP correlated with activity findings on kidney biopsy in LN patients. • Urinary levels of MCP-1 correlated with chronic damage, especially with fibrous crescents, tubular atrophy, and interstitial fibrosis.

Validation of an Asbestos Exposure Questionnaire (QEAS-7) for Clinical Practice.

INTRODUCTION: The seven-item QEAS-7 questionnaire (exposure to asbestos questionnaire) has been designed as a useful and simple tool to establish the probability of exposure to asbestos. The objective of the present study is to validate the QEAS-7 following the recommended methodology. METHODS: The QEAS-7 was prospectively administered to 90 subjects with and without asbestos-related disease (ARD), on two consecutive occasions by two independent researchers. Logical and content validity was evaluated by a committee of experts and construct validity through hypothesis testing. Intra- and interobserver reliability was assessed by calculating Cohen's Kappa index (κ), which was estimated as weak if below 0.40, moderate if between 0.41 and 0.60 and good/very good if above 0.60. The comparison between proportions was examined using Pearson's Chi-square test. RESULTS: The majority of participants (88.9%) were male. Mean age was 70.8 years (SD = 8.4) and most of the sample had completed primary education but had not progressed further (62.2%). Forty-three had ARD. The logical, content and construct validity of the QEAS-7 was considered adequate both by a committee of experts and by the users interviewed. The mean administration time was 9 min and 25 s (SD = 3 min and 49 s). The verification of the five hypotheses confirmed the construct validity and the intra- and interobserver reliability to be κ = 0.93 and κ = 0.50 respectively. The concordance in the estimation of asbestos exposure was κ = 0.65. CONCLUSIONS: The QEAS-7 is a simple, valid and reliable tool for estimating the probability of exposure to asbestos. Its application in clinical practice appears justified. What is already known about this subject? No studies have been published to date on the validation of specific questionnaires designed to determine asbestos exposure for routine use by healthcare staff in the clinical setting. What are the new findings? This questionnaire can be considered a comprehensible, viable, valid and reliable instrument for identifying exposure to asbestos. Its brevity and simplicity of administration make it ideally suited for use in daily clinical practice. How might this impact on policy or clinical practice in the foreseeable future? This questionnaire can be of help for physicians attending to patients with suspected asbestos-related diseases both in the hospital and in the primary care setting.

The Aryl Hydrocarbon Receptor in Asthma: Friend or Foe?

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has emerged as an important player in asthma control. AhR is responsive to environmental molecules and endogenous or dietary metabolites and regulates innate and adaptive immune responses. Binding of this receptor by different ligands has led to seemingly opposite responses in different asthma models. In this review, we present two sides of the same coin, with the beneficial and deleterious roles of AhR evaluated using known endogenous or exogenous ligands, deficient mice or antagonists. On one hand, AhR has an anti-inflammatory role since its activation in dendritic cells blocks the generation of pro-inflammatory T cells or shifts macrophages toward an anti-inflammatory M2 phenotype. On the other hand, AhR activation by particle-associated polycyclic aromatic hydrocarbons from the environment is pro-inflammatory, inducing mucus hypersecretion, airway remodelling, dysregulation of antigen presenting cells and exacerbates asthma features. Data concerning the role of AhR in cells from asthmatic patients are also reviewed, since AhR could represent a potential target for therapeutic immunomodulation.

Type I collagen hydrogels as a delivery matrix for royal jelly derived extracellular vesicles.

Throughout the last decade, extracellular vesicles (EVs) have become increasingly popular in several areas of regenerative medicine. Recently, Apis mellifera royal jelly EVs (RJ EVs) were shown to display favorable wound healing properties such as stimulation of mesenchymal stem cell migration and inhibition of staphylococcal biofilms. However, the sustained and effective local delivery of EVs in non-systemic approaches - such as patches for chronic cutaneous wounds - remains an important challenge for the development of novel EV-based wound healing therapies. Therefore, the present study aimed to assess the suitability of type I collagen -a well-established biomaterial for wound healing - as a continuous delivery matrix. RJ EVs were integrated into collagen gels at different concentrations, where gels containing 2 mg/ml collagen were found to display the most stable release kinetics. Functionality of released RJ EVs was confirmed by assessing fibroblast EV uptake and migration in a wound healing assay. We could demonstrate reliable EV uptake into fibroblasts with a sustained pro-migratory effect for up to 7 d. Integrating fibroblasts into the RJ EV-containing collagen gel increased the contractile capacity of these cells, confirming availability of RJ EVs to fibroblasts within the collagen gel. Furthermore, EVs released from collagen gels were found to inhibit Staphylococcus aureus ATCC 29213 biofilm formation. Overall, our results suggest that type I collagen could be utilized as a reliable, reproducible release system to deliver functional RJ EVs for wound healing therapies.

Development of a Dipstick Assay for Soy Allergens: Inexpensive Detection to Control Allergen Exposure.

Allergen measurements are use in the food industry and are also routinely performed as part of indoor air quality investigations and occupational health monitoring. In this chapter we describe how to develop a simple, convenient, rapid test to analyze soy allergens that can be used in production environments by non-skilled staff to facilitate immediate corrective action and minimize risk and that can be produced in laboratories not equipped with sophisticated instruments.The strip assay described consists of a membrane that is bonded to an adhesive support where an absorbent pad is also attached to absorb excess reagents. The membrane is stripped with specific antibodies against soy allergens in this case (test line) and a goat anti-rabbit IgG antibody (control line). The strip is exposed to a mix of sample and gold-conjugated specific antibody. Colored bands are read out visually or by densitometry.