Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs.

Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair.

Restoration of barrier-tissue integrity after injury is dependent on the function of immune cells and stem cells (SCs) residing in the tissue. In response to skin injury, hair-follicle stem cells (HFSCs), normally poised for hair generation, are recruited to the site of injury and differentiate into cells that repair damaged epithelium. We used a SC fate-mapping approach to examine the contribution of regulatory T (Treg) cells to epidermal-barrier repair after injury. Depletion of Treg cells impaired skin-barrier regeneration and was associated with a Th17 inflammatory response and failed HFSC differentiation. In this setting, damaged epithelial cells preferentially expressed the neutrophil chemoattractant CXCL5, and blockade of CXCL5 or neutrophil depletion restored barrier function and SC differentiation after epidermal injury. Thus, Treg-cell regulation of localized inflammation enables HFSC differentiation and, thereby, skin-barrier regeneration, with implications for the maintenance and repair of other barrier tissues.

Early-life inflammation primes a T helper 2 cell-fibroblast niche in skin.

Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.

Layilin Anchors Regulatory T Cells in Skin.

Regulatory T cells (Tregs) reside in nonlymphoid tissues where they carry out unique functions. The molecular mechanisms responsible for Treg accumulation and maintenance in these tissues are relatively unknown. Using an unbiased discovery approach, we identified LAYN (layilin), a C-type lectin-like receptor, to be preferentially and highly expressed on a subset of activated Tregs in healthy and diseased human skin. Expression of layilin on Tregs was induced by TCR-mediated activation in the presence of IL-2 or TGF-ß. Mice with a conditional deletion of layilin in Tregs had reduced accumulation of these cells in tumors. However, these animals somewhat paradoxically had enhanced immune regulation in the tumor microenvironment, resulting in increased tumor growth. Mechanistically, layilin expression on Tregs had a minimal effect on their activation and suppressive capacity in vitro. However, expression of this molecule resulted in a cumulative anchoring effect on Treg dynamic motility in vivo. Taken together, our results suggest a model whereby layilin facilitates Treg adhesion in skin and, in doing so, limits their suppressive capacity. These findings uncover a unique mechanism whereby reduced Treg motility acts to limit immune regulation in nonlymphoid organs and may help guide strategies to exploit this phenomenon for therapeutic benefit.

Prediction of clinical anxious and depressive problems in mid childhood amongst temperamentally inhibited preschool children: a population study.

Shy/inhibited young children are at risk for internalising difficulties; however, for many, this temperamental style does not result in mental health problems. This study followed a population-based sample of temperamentally inhibited preschool children into mid childhood to explore the aetiology of clinical-level anxious and depressive problems. Amongst inhibited preschool children, we aimed to predict each of clinical child anxiety and depressive problems in mid childhood from a broad range of potential risks (demographics, traumatic events and broader recent stressors, parents' well-being, and parenting practices). This study is based on data from a wider population trial of Cool Little Kids that recruited a representative sample of inhibited preschool children enrolled in their year before starting school. In 2011-2012, an inhibition screen was universally distributed to parents of children in their year before school (age 4 years) across eight diverse government areas in Melbourne, Australia. Participants were 545 parents of inhibited preschoolers (78% uptake, 545/703) who were followed to mid childhood (three annual waves 2015-2017, age 7-10 years) with 84% retention (456/545). Parents completed questionnaires spanning child ages 4-10 years, along with diagnostic interviews for child anxiety. Children also completed questionnaires in mid childhood. The questionnaires encompassed a variety of potential risks including sociodemographics, traumatic events, recent life stressors, parent wellbeing and parenting practices. In mid childhood, 57% (246/430) of inhibited preschoolers had a clinical level of anxiety problems while 22% (95/432) had depressive problems (by one or more sources). The aetiology analyses highlighted parent distress and parenting practices (overinvolved/protective, harsh discipline) as key predictors of inhibited preschoolers' internalising problems by mid childhood. Some high-risk families may not have participated. Child depression was not assessed with a diagnostic interview. The measures did not include every possible risk factor. The findings lend support to parenting programs for shy/inhibited young children that aim to prevent the development of anxiety and depression as they grow.

Follow-up of the Cool Little Kids translational trial into middle childhood.

BACKGROUND: Public health advocates have highlighted internalising problems as a leading cause of global burden of disease. Internalising problems (anxiety/depression) affect up to 20% of school-age children and can impact peer relations, school engagement and later employment and mortality. This translational trial aimed to determine whether a selective/indicated parenting group programme to prevent internalising distress in shy/inhibited preschool children had sustained effects in middle childhood. Translational design aspects were a brief parent-report screening tool for child inhibition offered universally across the population via preschools in the year before school, followed by an invitation to parents of all inhibited children to attend the parenting programme at venues in their local community. METHODS: Design of the study was a randomised controlled trial. The setting was 307 preschool services across eight socioeconomically diverse government areas in Melbourne, Australia. Participants were 545 parents of inhibited four-year-old children of which 456 (84%) were retained during middle childhood (age of seven to 10 years). Early intervention was the Cool Little Kids parenting group programme, and control was 'usual care' access to available support services in the community. Primary outcomes were child anxiety and depression symptoms (parent and child report) and DSM-IV anxiety disorders (assessor masked). Secondary outcomes were parenting practices and parent mental health. RESULTS: There was no significant difference in anxiety disorders between the intervention and control group during the three annual follow-ups of the cohort in middle childhood (2015 43% vs. 41%, 2016 40% vs. 36%, 2017 27% vs. 30%, respectively; p's > .05). There were also no significant differences in child anxiety or depression symptoms (by child or parent report), parenting practices or parent mental health, between the intervention and control group during middle childhood. However, a priori interaction tests suggested that for children with anxious parents, early intervention attenuated risk for middle childhood internalising problems. CONCLUSIONS: An issue for population translation is low levels of parent engagement in preventive interventions. Initial effects of the Cool Little Kids parenting group programme in reducing shy/inhibited preschool children's internalising distress at school entry dissipated over time, perhaps due to low engagement. Future translational research on early prevention of internalising problems could benefit from screening preschool children in the population at higher risk (combining temperamental inhibition and parent distress) and incorporating motivational techniques to facilitate family engagement. Trial registration ISRCTN30996662 http://www.isrctn.com/ISRCTN30996662.

Histologic progression of acne inversa/hidradenitis suppurativa: Implications for future investigations and therapeutic intervention.

Since first recognized in 1839, the pathogenesis of acne inversa (AI) has undergone repeated revisions. Although there is agreement that AI involves occlusion of hair follicles with subsequent inflammation and the formation of tracts, the histologic progression of this disease still requires refinement. The objective of this study was to examine the histologic progression of AI based on the examination of a large cohort of punch biopsies and excisional samples that were examined first by hematoxylin and eosin staining. The most informative of these samples were step-sectioned and stained by immunohistochemistry for epithelial and inflammatory markers. Based on this examination, the following observations were made: 1) AI arises from the epithelium of the infundibulum of terminal and vellus hairs; 2) These form cysts and epithelial tendrils that extend into soft tissue; 3) Immunohistochemical staining demonstrates the epithelium of AI is disordered with infundibular and isthmic differentiation and de novo expression of stem cell markers; 4) The inflammatory response in AI is heterogeneous and largely due to cyst rupture. The conclusions of this investigation were that AI is an epithelial-driven disease caused by infiltrative, cyst forming tendrils and most of the inflammation is due to cyst rupture and release of cornified debris and bacteria. Cyst rupture often occurs below the depths of punch biopsy samples indicating their use for analysis may give an incomplete picture of the disease. Finally, our data suggest that unless therapies inhibit tendril development, it is unlikely they will cause prolonged treatment-induced remission in AI.

Patient-Clinician Discordance in Perceptions of Treatment Risks and Benefits in Older Patients with Acute Myeloid Leukemia.

BACKGROUND: Older patients (≥60 years) with acute myeloid leukemia (AML) face difficult decisions regarding treatment with "intensive" chemotherapy that carries significant toxicity for a small chance of a cure versus "nonintensive" chemotherapy to control the disease, but with fewer side effects. However, studies of how these patients understand the risks and benefits of such treatments are lacking. METHODS: We conducted a longitudinal study of older patients newly diagnosed with AML assessing patients' (n = 100) and oncologists' (n = 11) perceptions of treatment-related mortality at enrollment and prognosis at 1 month. We examined concordance between patients' and oncologists' perceptions using Cohen's kappa (κ < 0.10 indicates little/no concordance). RESULTS: We enrolled patients within 72 hours of initiating intensive (n = 50) or nonintensive (n = 50) chemotherapy. Whereas 91% of patients reported that they were "somewhat" to "extremely likely" to die from treatment, oncologists estimated that only 12% were at high risk of dying because of treatment (κ = -0.09). Ninety percent of patients reported that they were "somewhat" or "very likely" to be cured of their AML, whereas oncologists estimated this chance of cure for only 31% of patients (κ = 0.05). Among patients receiving intensive chemotherapy, 98% reported that they were "somewhat" or "very likely" to be cured, whereas their oncologists estimated this likelihood of cure for only 49% (κ = 0.04); among those receiving nonintensive chemotherapy and their clinicians, these proportions were 82% and 13%, respectively (κ = 0.03). Patients who indicated a lower likelihood of cure reported significantly higher depression symptoms (p = .03). CONCLUSION: Older patients with AML overestimate the risks and benefits of treatment. Interventions to facilitate communication and enhance patients' understanding of the goals of therapy and treatment risk are needed. IMPLICATIONS FOR PRACTICE: Older patients with acute myeloid leukemia (AML) are confronted with challenging decisions regarding treatment with "intensive" chemotherapy that carries significant toxicity for a small chance of a cure versus "nonintensive" chemotherapy to control the disease, but with fewer side effects. A clear understanding of the likely outcome and risks of the various treatment strategies is essential for these patients to make informed decisions about their care. This article reports that older patients with AML overestimate both the risks and benefits of treatment and have substantial misperceptions about their prognosis. Interventions to enhance patients' understanding of their prognosis and treatment risk are needed.

Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche.

Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.

Tertiary lymphoid structures sustain cutaneous B cell activity in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic skin condition affecting approximately 1% of the US population. HS skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu, the biology and the contribution of these cells in HS pathogenesis are unclear. We aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions. Combining histological analysis, single-cell RNA sequencing, and spatial transcriptomics profiling of HS lesions, we defined the tissue microenvironment relative to B cell activity within this disease. Our findings identified tertiary lymphoid structures (TLSs) within HS lesions and described organized interactions among T cells, B cells, antigen-presenting cells, and skin stroma. We found evidence that B cells within HS TLSs actively underwent maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells were primed to support the formation of TLSs and facilitate B cell recruitment during HS. Our data definitively demonstrated the presence of TLSs in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed nonlymphoid tissue.

Tertiary Lymphoid Structures Sustain Cutaneous B cell Activity in Hidradenitis Suppurativa.

Background: Hidradenitis suppurativa (HS) skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu the biology and contribution of these cells in HS pathogenesis is unclear. Objective: We aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions. Methods: We combined histological analysis, single-cell RNA-sequencing (scRNAseq), and spatial transcriptomic profiling of HS lesions to define the tissue microenvironment relative to B cell activity within this disease. Results: Our findings identify tertiary lymphoid structures (TLS) within HS lesions and describe organized interactions between T cells, B cells, antigen presenting cells and skin stroma. We find evidence that B cells within HS TLS actively undergo maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells are primed to support the formation of TLS and facilitate B cell recruitment during HS. Conclusion: Our data definitively demonstrate the presence of TLS in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed non-lymphoid tissue.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

The catalytic subunit of human telomerase is a unique caspase-6 and caspase-7 substrate.

Telomerase is a ribonucleoprotein complex that is essential for persistent cellular proliferation. The catalytic subunit of human telomerase, hTERT, functions as a reverse transcriptase and promotes vitality by maintaining telomeric DNA length. hTERT is tightly regulated with complex but poorly understood positive and negative regulation at several levels including transcription, protein-protein interactions, and post-translation modifications. Because evidence implicates hTERT as an apoptosis inhibitor and because telomerase activity tends to decrease during apoptosis, we hypothesized that hTERT is a caspase substrate leading to down regulation during apoptosis. Caspases are proteases that initiate and execute apoptosis by cleaving target proteins. Indeed, we found that caspases-6 and -7 cleave hTERT during apoptosis in cultured cells. Caspase-6 cleaves at residues D129 and D637, and caspase-7 cleaves at E286 and D628. Three of the caspase cleavage sites are unique motifs. All four caspase motifs appear conserved in TERTs from Old World monkeys and apes, and the caspase-6 sites appear conserved in all primates. The caspase site that cleaves at D129 appears conserved in amniotes. hTERT fragments generated by cleavage were remarkably persistent, lasting hours after caspase activation. These results reveal a new biologically relevant mechanism for telomerase down regulation through caspase-mediated cleavage of hTERT and expand the list of known caspase motifs.

Application of the sexual health model in the long-term treatment of hypoactive sexual desire and female orgasmic disorder.

Using the Sexual Health Model as a framework, this case study illustrates the treatment of female orgasmic and low desire disorder in a long-term case with numerous complexities and other co-morbid mental health diagnoses. Derived from a sexological approach to education, the Sexual Health Model defines 10 key components posited to be essential aspects of healthy human sexuality: talking about sex, culture and sexual identity, sexual anatomy and functioning, sexual health care and safer sex, challenges to sexual health, body image, masturbation and fantasy, positive sexuality, intimacy and relationships, and spirituality. The client was selected because of the commonality of her initial presenting concerns and the etiological and treatment complexity of the case, which necessitated the use of all the sexual health treatment modalities provided at our center-individual, couple, and group therapy, sexual medicine, and psychiatric care. Her case is distinct in that her sexual dysfunctions and negative cognitions, while common, occurred in the context of serious relational, family sexual abuse, depression, and life-threatening medical problems, which necessitated long-term treatment. This case illustrates the multifactoral etiology of complex sexual dysfunctions requiring treatment that deals with varied psychosocial and biological factors.

B Home or You Are In Trouble: B Cell Integrin-Mediated Recruitment Attenuates Skin Inflammation.

B cells were long presumed to be a minor and functionally unimportant component of cutaneous immunobiology. However, it is now clear that these lymphocytes are present in healthy skin and accumulate during inflammatory disease. Aira and Debes (2021) identify ⍺4ꞵ1 integrin-mediated recruitment of IL-10+ B cells as a key pathway in attenuating skin inflammation. Their work provides valuable insight into the potential for B cells to regulate skin pathology.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-ß activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-ß pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvß8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-ß, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvß8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Single-cell analyses identify circulating anti-tumor CD8 T cells and markers for their enrichment.

The ability to monitor anti-tumor CD8+ T cell responses in the blood has tremendous therapeutic potential. Here, we used paired single-cell RNA and TCR sequencing to detect and characterize "tumor-matching" (TM) CD8+ T cells in the blood of mice with MC38 tumors or melanoma patients using the TCR as a molecular barcode. TM cells showed increased activation compared with nonmatching T cells in blood and were less exhausted than matching cells in tumors. Importantly, PD-1, which has been used to identify putative circulating anti-tumor CD8+ T cells, showed poor sensitivity for identifying TM cells. By leveraging the transcriptome, we identified candidate cell surface markers for TM cells in mice and patients and validated NKG2D, CD39, and CX3CR1 in mice. These data show that the TCR can be used to identify tumor-relevant cells for characterization, reveal unique transcriptional properties of TM cells, and develop marker panels for tracking and analysis of these cells.

An Injectable Cytokine Trap for Local Treatment of Autoimmune Disease.

Systemic cytokine therapy is limited by toxicity due to activation of unwanted immune cells in off-target tissues. Injectable nanomaterials that interact with the immune system have potential to offer improved pharmacokinetics and cell specificity compared to systemic cytokine therapy by instead capturing and potentiating endogenous cytokine. Here we demonstrate the use of high aspect ratio polycaprolactone nanowires conjugated to cytokine-binding antibodies that assemble into porous matrices when injected into the subcutaneous space. Nanowires are well tolerated in vivo over several weeks, incite minimal foreign body response and resist clearance. Nanowires conjugated with JES6-1, an anti-interleukin-2 (IL-2) antibody, were designed to capture endogenous IL-2 and selectively activate tissue resident regulatory T cells (Tregs). Together these nanowire-antibody matrices were capable of sequestering endogenous IL-2 in the skin and were successful in rebalancing local immune compartments to a more suppressive, Treg-mediated phenotype in both wild type and transgenic murine autoimmune disease models.

Neurology clinicians' views on palliative care communication: "How do you frame this?"

BACKGROUND: The communication process of preparing patients and families facing progressive neurodegenerative diseases for future illness has not been empirically elucidated; the goal of this qualitative study was to explore neurology interdisciplinary health professionals' communication experiences, including current approaches, facilitators, and challenges. METHODS: Three focus groups were conducted with 22 clinicians representing a range of health professions from several multidisciplinary neurology outpatient clinics at a large academic medical center. A thematic analysis approach was used to develop a coding structure and identify overarching themes. RESULTS: Neurology clinicians highlighted that in their practice, (1) conversations are triggered by acute events and practical needs; (2) conversations occur routinely but are rarely documented; (3) loss of patient capacity and resultant surrogate decision-making can be ethically fraught, especially in times of family conflict; (4) prognostic uncertainty, unfamiliarity with disease trajectories, and patient or surrogate avoidance pose communication challenges; and (5) generalist- and specialty-level palliative care roles should be better defined. CONCLUSIONS: There is a need for a systematic, structured approach to communication that can be applied early in the disease trajectory and considered when developing integrated neuro-palliative care programs.

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αß T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy.