Environmental Antigens Research Articles

Antibodies in breast milk: Pro-bodies designed for healthy newborn development.

This manuscript sheds light on the impact of maternal breast milk antibodies on infant health. Milk antibodies prepare and protect the newborn against environmental exposure, guide and regulate the offspring's immune system, and promote transgenerational adaptation of the immune system to its environment. While the transfer of IgG across the placenta ceases at birth, milk antibodies are continuously replenished by the maternal immune system. They reflect the mother's real-time adaptation to the environment to which the infant is exposed. They cover the infant's upper respiratory and digestive mucosa and are perfectly positioned to control responses to environmental antigens and might also reach their circulation. Maternal antibodies in breast milk play a key role in the immune defense of the developing child, with a major impact on infectious disease susceptibility in both HIC and LMIC. They also influence the development of another major health burden in children-allergies. Finally, emerging evidence shows that milk antibodies also actively shape immune development. Much of this is likely to be mediated by their effect on the seeding, composition and function of the microbiota, but not only. Further understanding of the bridge that maternal antibodies provide between the child and its environment should enable the best interventions to promote healthy development.

Occurrence of carbapenemase producing NAG Vibriocholerae (NAG-CPV.c) in aquatic environment before the recommendation of carbapenem antibiotics

Recent research updates and advances have shown an upsurge of antibiotic resistance reports amongst bacterial species with increasing spread/distribution especially in the water nexus. Such has been the trend among Vibrio cholerae strain as it is observed to be emerging with diverse resistant determinants. The current study presents the occurrence of carbapenemase producing NAG Vibriocholerae (NAG-CPV.c) in aquatic environment before the recommendation of Carbapenem antibiotics. It focused on carbapenem resistant phenotype/genotype among environmental and somatic antigen nonagglutinating V. cholerae (SANAG-Vc) strains recovered from water sources, applying standard microbiological, serological and molecular biology techniques. Domestic water samples were collected for isolation of V. cholerae strains in Eastern Cape Province, South Africa. Carbapenem and specific β-lactamase inhibitors were employed for antibiotic susceptibility testing using K-B disc diffusion technique in addition to the Modified-Hodge-Test (MHT). Our results revealed 61 strains of environmental and SANAG-Vc serogroups. Amongst these confirmed SANAG-Vc strains recovered, 25 possess carbapenemase phenotype or NDM-1 phenotype (40.89%), whereas 24 (39.34%) were MHT positive phenotype. Further gene-based detection revealed 20 (32.79%) PCR confirmed as NDM-1 resistant gene positive strains. It is important to note that the carbapenem members of antibiotics are not readily employed in the therapeutic control of cholera cases as recommended by CLSI. Observing such resistant phenotypes/genotypes indicates a possible transfer/dissemination and emergence of such resistant determinants in the environment as the coastal water serves as a potential breeding hub for such resistant genes among potential pathogens. This is a serious threat to environmental wellness/public health especially those in the sub-urban and poor living localities, who source the analyzed water as their major source of water for domestic activities. The need for adroit and continuous monitoring of released water effluents of domestic and clinical sources remains a control strategy for environmental wellness and water bodies safety.

The Role of Serum IgG Precipitins against Six Typical Organic Antigens Involved in Hypersensitivity Pneumonitis: A 10-Year Retrospective Study of a Referral Interstitial Lung Disease Centre

Hypersensitivity pneumonitis (HP) represents the third common interstitial lung disease caused by an exaggerated immune response following the inhalation of organic and/or chemical environmental antigens. The aim of this study was to determine the cut-off values of specific IgG antibodies (named precipitins) and their association with clinical data in the diagnosis of HP. In this 10-year retrospective study, the IgG concentrations against six antigens, Penicillium chrysogenum/notatum, Aspergillus fumigatus, Alternaria alternata, Aspergillus niger, Micropolyspora faeni, and pigeon droppings, were retrieved. The controlled group was made of 1516 healthy subjects without diagnosis of lung pathologies, while the case group consisted of 54 individuals affected by HP. Considering all six IgG antibodies together and the 97.5% percentiles determined in the control group, 30 of 54 subjects (56%) had one or more positive precipitins. In these patients, the major frequencies found were IgG antibodies against pigeon droppings, followed by Penicillium chrysogenum/notatum and Aspergillus niger. Although the sensitivity of serum precipitins depends on the population enrolled and the method used, the cut-off values determined in this study can be a valuable tool for clinicians in the diagnosis of HP, in eliminating the antigens responsible from the environment, and in establishing more specific IgG panels.

Immunohistochemistry of the nasal cavity-associated lymphoid tissue in the dolphin (Stenella coeruleoalba, Meyen 1833).

The striped dolphin (Stenella coeruleoalba) is a medium-sized pelagic dolphin with a single external nasal opening (blowhole) located in the rostral and dorsal regions of the skull. The nasal cavity is divided into three sections: the olfactory, respiratory, and vestibular areas. The surface epithelium lining the regio vestibularis is the first tissue in the nose to be directly affected by environmental antigens. Cetaceans have a significant amount of mucosa-associated lymphoid tissue (MALT) located throughout their bodies. The lymphoid tissue found in the nasal mucosa is known as nose- or nasopharynx-associated lymphoid tissue (NALT). NALT has not yet been studied in dolphins, but it has been identified and documented in humans and laboratory rodents. This study utilized toll-like receptor 2 (TLR2), CD4, Langerin/CD207, and inducible nitric oxide synthase to characterize, for the first time, immune cells in the mucosal regio vestibularis of the S. coeruleoalba nasal cavity using confocal microscopy immunofluorescence techniques. The findings revealed scattered immune cells immunoreactive to the tested antibodies, present in both the epithelial tissue lining the nasal cavity vestibulum and the underlying connective tissue. This study enhances our comprehension of the immune system of cetaceans. RESEARCH HIGHLIGHTS: This study provides new insights into NALT in S. coeruleoalba. This research deepens the knowledge of the skin of cetaceans.

Sounding the alarm in the lung with TL1A.

Environmental airborne antigens are central to the development of allergic asthma, but the cellular processes that trigger disease remain incompletely understood. In this report, Schmitt et al. (https://doi.org/10.1084/jem.20231236) identify TNF-like protein 1A (TL1A) as an epithelial alarmin constitutively expressed by a subset of lung epithelial cells, which is released in response to airborne microbial challenge and synergizes with IL-33 to drive allergic disease.

Early life imprinting of intestinal immune tolerance and tissue homeostasis.

Besides its canonical role in protecting the host from pathogens, the immune system plays an arguably equally important role in maintaining tissue homeostasis. Within barrier tissues that interface with the external microenvironment, induction of immune tolerance to innocuous antigens, such as commensal, dietary, and environmental antigens, is key to establishing immune homeostasis. The early postnatal period represents a critical window of opportunity in which parallel development of the tissue, immune cells, and microbiota allows for reciprocal regulation that shapes the long-term immunological tone of the tissue and subsequent risk of immune-mediated diseases. During early infancy, the immune system appears to sacrifice pro-inflammatory functions, prioritizing the establishment of tissue tolerance. In this review, we discuss mechanisms underlying early life windows for intestinal tolerance with a focus on newly identified RORγt+ antigen-presenting cells-Thetis cells-and highlight the role of the intestinal microenvironment in shaping intestinal immune system development and tolerance.

Multiple environmental antigens may trigger autoimmunity in psoriasis through T-cell receptor polyspecificity.

Psoriasis is a T-cell mediated autoimmune skin disease. HLA-C*06:02 is the main psoriasis-specific risk gene. Using a Vα3S1/Vβ13S1 T-cell receptor (TCR) from a lesional psoriatic CD8+ T-cell clone we had discovered that, as an underlying pathomechanism, HLA-C*06:02 mediates an autoimmune response against melanocytes in psoriasis, and we had identified an epitope from ADAMTS-like protein 5 (ADAMTSL5) as a melanocyte autoantigen. The conditions activating the psoriatic autoimmune response in genetically predisposed individuals throughout life remain incompletely understood. Here, we aimed to identify environmental antigens that might trigger autoimmunity in psoriasis because of TCR polyspecificity. We screened databases with the peptide recognition motif of the Vα3S1/Vβ13S1 TCR for environmental proteins containing peptides activating this TCR. We investigated the immunogenicity of these peptides for psoriasis patients and healthy controls by lymphocyte stimulation experiments and peptide-loaded HLA-C*06:02 tetramers. We identified peptides from wheat, Saccharomyces cerevisiae, microbiota, tobacco, and pathogens that activated both the Vα3S1/Vβ13S1 TCR and CD8+ T cells from psoriasis patients. Using fluorescent HLA-C*06:02 tetramers loaded with ADAMTSL5 or wheat peptides, we find that the same CD8+ T cells may recognize both autoantigen and environmental antigens. A wheat-free diet could alleviate psoriasis in several patients. Our results show that due to TCR polyspecificity, several environmental antigens corresponding to previously suspected psoriasis risk conditions converge in the reactivity of a pathogenic psoriatic TCR and might thus be able to stimulate the psoriatic autoimmune response against melanocytes. Avoiding the corresponding environmental risk factors could contribute to the management of psoriasis.

Novel adjuvants in allergen-specific immunotherapy: where do we stand?

Type I hypersensitivity, or so-called type I allergy, is caused by Th2-mediated immune responses directed against otherwise harmless environmental antigens. Currently, allergen-specific immunotherapy (AIT) is the only disease-modifying treatment with the potential to re-establish clinical tolerance towards the corresponding allergen(s). However, conventional AIT has certain drawbacks, including long treatment durations, the risk of inducing allergic side effects, and the fact that allergens by themselves have a rather low immunogenicity. To improve AIT, adjuvants can be a powerful tool not only to increase the immunogenicity of co-applied allergens but also to induce the desired immune activation, such as promoting allergen-specific Th1- or regulatory responses. This review summarizes the knowledge on adjuvants currently approved for use in human AIT: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine, and MPLA, as well as novel adjuvants that have been studied in recent years: oil-in-water emulsions, virus-like particles, viral components, carbohydrate-based adjuvants (QS-21, glucans, and mannan) and TLR-ligands (flagellin and CpG-ODN). The investigated adjuvants show distinct properties, such as prolonging allergen release at the injection site, inducing allergen-specific IgG production while also reducing IgE levels, as well as promoting differentiation and activation of different immune cells. In the future, better understanding of the immunological mechanisms underlying the effects of these adjuvants in clinical settings may help us to improve AIT.

Microbiota-accessible fiber activates short-chain fatty acid and bile acid metabolism to improve intestinal mucus barrier in broiler chickens.

The intestinal mucus barrier, located at the interface of the intestinal epithelium and the microbiota, is the first line of defense against pathogenic microorganisms and environmental antigens. Dietary polysaccharides, which act as microbiota-accessible fiber, play a key role in the regulation of intestinal microbial communities. However, the mechanism via which dietary fiber affects the intestinal mucus barrier through targeted regulation of the gut microbiota is not clear. This study provides fundamental evidence for the benefits of dietary fiber supplementation in broiler chickens through improvement in the intestinal mucus barrier by targeted regulation of the gut ecosystem. Our findings suggest that the microbiota-accessible fiber-gut microbiota-short-chain fatty acid/bile acid axis plays a key role in regulating intestinal function.

Persistent Anterior Thoracalgia in a Patient with Mediastinal - Pulmonary Sarcoidosis

Abstract Sarcoidosis is a systemic condition characterized by the formation of non-caseating granulomas in any organ or tissue, with a predominant impact on the lungs and intrathoracic lymph nodes(1). The heterogeneity of the disease is reflected in the broad possibilities of the clinical and paraclinical evolution, which can range from the incidental identification of the disease in an asymptomatic patient to the progressive chronic involvement of the affected organs. Commonly, sarcoidosis initially manifests through nonspecific respiratory symptoms, characterized by dry cough, dyspnea, chest pain, and fatigue(2,3). Generally, it is considered that this condition occurs in individuals with a susceptible genetic background, exposed to certain environmental factors or antigens that remain unknown, despite numerous existing hypotheses(4). Cardiac involvement in sarcoidosis has a significant negative impact on prognosis. Although this location of the disease is predominantly silent, 5% of cases present with dilated or restrictive cardiomyopathy, progressive heart failure, high-grade atrioventricular blocks, and ventricular arrhythmias(5). The authors present the case of a young patient who, although at the onset of the disease, exhibited a typical clinical and imaging profile of sarcoidosis, myocardial involvement - a rare localization of this pathology - was detected during the course of the condition. Patients with sarcoidosis may have normal spirometry, may present with a restrictive syndrome (similar to diffuse interstitial lung diseases), or an obstructive syndrome, secondary to the involvement of small bronchi and bronchial distortions caused by the disease, often with an unfavorable prognosis.

A missing jigsaw within the hygiene hypothesis: Low-dose bisphenol A exposure attenuates lipopolysaccharide-induced asthma protection.

The rising occurrence of allergic asthma in early life across industrialized countries suggests that environmental factors play a crucial role in determining asthma susceptibility and severity. While prior exposure to microbial lipopolysaccharides (LPSs) has been found to offer protection against allergic asthma, infants residing in urban environments are increasingly exposed to environmental pollutants. Utilizing limulus lysate test screens and virtual screening models, we identified pollutants that can modulate LPS bioactivity. This investigation revealed that bisphenol A (BPA), a chemical commonly used in numerous household items and previously implicated in obesity and cancer, effectively neutralizes LPS. In-depth mechanistic analyses showed that BPA specifically binds to the lipid A component of LPS, leading to inactivation. This interaction eliminates the immunostimulatory activity of LPS, making mice more susceptible to house dust mite (HDM)-induced allergic asthma. BPA reactivates lung epithelial cells, consequently amplifying type 2 responses to HDMs in dendritic cells. This chemical interplay provides new insights into the pathophysiology of asthma in relation to human exposure. Understanding the intricate relationships between environmental chemicals and microbial antigens, as well as their impacts on innate immunity, is critical for the development of intervention strategies to address immune disorders resulting from urbanization.

Diacylglycerol kinase zeta deficiency attenuates papain-induced type 2 airway inflammation

Allergic airway diseases are caused by inappropriate immune responses directed against inhaled environmental antigens. We previously reported that the inhibition of diacylglycerol (DAG) kinaseζ (DGKζ),an enzyme that terminates DAG-mediated signaling,protects against T cell-mediated allergic airway inflammation by blocking Th2 cell differentiation.In this study, we tested whether DGKζ deficiency also affects allergic airway disease mediated by type 2 innate lymphoid cells (ILC2)s. DGKζ-deficient mice displayed diminished ILC2 function and reduced papain-induced airway inflammation compared to wildtype mice. Unexpectedly, however, mice with hematopoietic cell-specific deletion ofDGKζ displayed intact airway inflammation upon papain challenge. Rather, bone marrow chimera studies revealed thatDGKζ deficiency in the non-hematopoietic compartment was responsible for the reduction in papain-induced airway inflammation. These data suggest that DGK might represent a novel therapeutic target not only for T cell-dependent but also ILC2-dependent allergic airway inflammation by affecting non-hematopoietic cells.

Evaluation of the Suppressive Capacity of Regulatory T Cells in Food Allergy Research.

Regulatory T cells (Treg) exert a crucial role in the suppression of exacerbated T helper (Th) cell responses, including those of type 2 Th (Th2) cells, and in the maintenance of tolerance to environmental antigens and food allergens. The functional capacity of Tregs to suppress Th2 responses has been studied through activation and immunosuppression assays using cells from mice and humans. The immunosuppression assay is an essential in vitro tool that allows the evaluation of the Treg capacity to limit the proliferation and expansion of conventional T cells. This approach enables the determination of the suppressive ability of different Treg subsets. In this chapter, we describe a basic and well-established immunosuppression protocol for human and murine Treg that has been widely applied in food allergy research.

Neferine Attenuates HDM-Induced Allergic Inflammation by Inhibiting the Activation of Dendritic Cell.

House dust mite (HDM) acts as an environmental antigen that might cause chronic allergic diseases. Neferine (NEF) shows anti-inflammation therapeutic effects. This study is to explore the protection role of NEF against HDM-induced allergic inflammation. HDM-induced allergic asthmatic C57BL/6J mice models were established. Differential histological staining was used to analyze lung tissue pathological scores. Flow cytometry was used to analyze subtypes and biomarker expression of immune cells. RT-PCR and ELISA were used to test cytokines-related gene and/or protein expression levels. Western blot was performed to investigate the signaling pathway that mediates allergic inflammation from mice lung tissue and bone marrow-derived dendritic cells (BMDCs). H&E and PAS staining results indicate NEF significantly attenuated inflammatory index and the percentage of goblet cells in the lung tissue induced by HDM. The HDM-elevated TH2 and TH17 cells were significantly decreased by NEF; inflammatory cytokines Il-4, Il-13 and Il-17 were dramatically downregulated in the NEF plus HDM group compared with HDM alone. CD40+ and CD86+ DCs, eosinophils and mast cells, and ILC2 cells were decreased by NEF which was elevated under HDM stimulation. In vivo and ex vivo investigations indicated NEF can attenuate the activated NF-κB signaling induced by HDM is involved in allergic inflammatory immune response and regulates cytokines-related gene expression. HDM-activated DCs promoted differentiation of TH2 and TH17 cells but were attenuated by NEF. This study suggests NEF interrupts the overexpression of some cytokines released by DCs, TH2, and TH17 cells; NEF attenuates HDM-induced allergic inflammation via inhibiting NF-κB signaling of DCs.

Allergen exposure functionally alters influenza-specific CD4+ Th1 memory cells in the lung.

CD4+ lung-resident memory T cells (TRM) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 TRM in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naïve influenza-specific CD4+ T cells in the lymph nodes do not respond to HDM, influenza-specific CD4+ TRM in the lungs do respond to a subsequent allergen exposure by decreasing expression of the transcription factor T-bet. This functional alteration is associated with decreased IFN-γ production upon restimulation and improved disease outcomes following heterosubtypic influenza challenge. Further investigation revealed that ST2 signaling in CD4+ T cells during allergic challenge is necessary to induce these changes in lung-resident influenza-specific CD4+ TRM. Thus, heterologous antigen exposure or ST2-signaling can drive persistent changes in CD4+ Th1 TRM populations and impact protection upon reinfection.

Mining Autoimmune-Disorder-Linked Molecular-Mimicry Candidates in Clostridioides difficile and Prospects of Mimic-Based Vaccine Design: An In Silico Approach.

Molecular mimicry, a phenomenon in which microbial or environmental antigens resemble host antigens, has been proposed as a potential trigger for autoimmune responses. In this study, we employed a bioinformatics approach to investigate the role of molecular mimicry in Clostridioides difficile-caused infections and the induction of autoimmune disorders due to this phenomenon. Comparing proteomes of host and pathogen, we identified 23 proteins that exhibited significant sequence homology and were linked to autoimmune disorders. The disorders included rheumatoid arthritis, psoriasis, Alzheimer's disease, etc., while infections included viral and bacterial infections like HIV, HCV, and tuberculosis. The structure of the homologous proteins was superposed, and RMSD was calculated to find the maximum deviation, while accounting for rigid and flexible regions. Two sequence mimics (antigenic, non-allergenic, and immunogenic) of ≥10 amino acids from these proteins were used to design a vaccine construct to explore the possibility of eliciting an immune response. Docking analysis of the top vaccine construct C2 showed favorable interactions with HLA and TLR-4 receptor, indicating potential efficacy. The B-cell and T-helper cell activity was also simulated, showing promising results for effective immunization against C. difficile infections. This study highlights the potential of C. difficile to trigger autoimmunity through molecular mimicry and vaccine design based on sequence mimics that trigger a defensive response.

Prescribing Patterns of Dupilumab for Atopic Dermatitis in Adults: Retrospective, Observational Cohort Study.

Atopic dermatitis (AD) is a common inflammatory disease caused by a type 2 T helper cell-mediated immune response to environmental antigens. Approximately 1 in 5 patients with AD presents with moderate to severe disease, and treatments approved by the Food and Drug Administration include emollients, topical glucocorticoids, and calcineurin inhibitors. Dupilumab, a fully human monoclonal antibody, improves AD via inhibition of interleukin-4 and interleukin-13. Our aim was to characterize the prescribing patterns of dupilumab for AD in adults at a large university-affiliated health system. A retrospective, observational cohort study was conducted using electronic data from the Observational Health Data Sciences and Informatics database, assessing data from the University of Colorado Medical Campus and its affiliates. The outcome measured was the prevalence of dupilumab prescribed for adults with AD (n=6421), between March 28, 2013, and March 28, 2021. We assessed whether the characteristics of patients who received dupilumab were different from those who did not. Each patient characteristic was assessed using a univariate logistic regression with the binary outcome of receiving or not receiving dupilumab. We found a population prevalence of 5.6% (6421/114,476) for AD. In our cohort, Black patients with AD were more than twice as likely to have received dupilumab compared to White patients (odds ratio 2.352, 95% CI 1.58-3.39). Patients with a diagnosis of atopic neurodermatitis were approximately twice as likely to have received dupilumab compared to those with other diagnostic variants of AD (odds ratio 1.87, 95% CI 1.01-3.22). Our results demonstrate that both patient racial characteristics and specific AD diagnoses were associated with variations in dupilumab prescription patterns.

Immunologic, genetic, and ecological interplay of factors involved in allergic diseases.

An allergic or type I hypersensitivity reaction involves a misdirected immune overreaction to innocuous environmental and dietary antigens called allergens. The genetic predisposition to allergic disease, referred to as atopy, can be expressed as a variety of manifestations-e.g., allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergic asthma, anaphylaxis. Globally, allergic diseases are one the most common types of chronic conditions. Several factors have been identified to contribute to the pathogenesis and progression of the disease, leading to distinctively variable clinical symptoms. The factors which can attenuate or exacerbate allergic reactions can range from genetic heterozygosity, the prominence of various comorbid infections, and other factors such as pollution, climate, and interactions with other organisms and organism-derived products, and the surrounding environment. As a result, the effective prevention and control of allergies remains to be one of the most prominent public health problems. Therefore, to contextualize the current knowledge about allergic reactions, this review paper attempts to synthesize different aspects of an allergic response to describe its significance in the global health scheme. Specifically, the review shall characterize the biomolecular mechanisms of the pathophysiology of the disease based on underlying disease theories and current findings on ecologic interactions and describe prevention and control strategies being utilized. An integrated perspective that considers the underlying genetic, immunologic, and ecologic aspects of the disease would enable the development of more effective and targeted diagnostic tools and therapeutic strategies for the management and control of allergic diseases.

Antibody responses to dietary antigens are accompanied by specific plasma cells in the infant thymus

Human infants develop IgG responses to dietary antigens during the first 2 years of life. Yet, the source of these antibodies is unclear. In previous studies we reported on the thymus as a unique functional niche for plasma cells (PCs) specific to environmental antigens. We sought to examine whether PCs specific to dietary antigens are detected in the infant thymus. We tested IgG reactivity to 112 food antigens and allergens in the serum of 20 neonates and infants using microarrays. The presence of PC-secreting IgG specific to the most prominent antigens was then assessed among thymocytes in the same cohort. Using an LC-MS proteomics approach, we looked for traces of these antigens in the thymus. Our studies first confirmed that cow's milk proteins are prevalent targets of serum IgG in early life. Subjects with the highest serum IgG titers to cow's milk proteins also harbored IgG-producing PCs specific to the same antigens in the thymic niche. Furthermore, we detected multiple peptide fragments of cow's milk antigens in the thymus. Lastly, we verified that both serum IgG and IgG secreted by thymic PCs recognized the peptide epitopes found in the thymus. Our studies reveal the presence of antibody-secreting PCs specific to common dietary antigens in the infant thymus. The presence of these antigens in the thymus suggested that activation and differentiation of specific PCs occurred in this organ. Further studies are now warranted to evaluate the possible implication of these cells in tolerance to dietary antigens.

The Role of Lactoferrin in Intestinal Health.

The intestine represents one of the first barriers where microorganisms and environmental antigens come into tight contact with the host immune system. A healthy intestine is essential for the well-being of humans and animals. The period after birth is a very important phase of development, as the infant moves from a protected environment in the uterus to one with many of unknown antigens and pathogens. In that period, mother's milk plays an important role, as it contains an abundance of biologically active components. Among these components, the iron-binding glycoprotein, lactoferrin (LF), has demonstrated a variety of important benefits in infants and adults, including the promotion of intestinal health. This review article aims to provide a compilation of all the information related to LF and intestinal health, in infants and adults.