Development and dysfunction of structural cells in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a disorder characterized by dysfunction and chronic local inflammation of the esophagus. The incidence and prevalence of EoE are increasing worldwide. The mechanisms responsible are poorly understood, and effective treatment options are limited. From the lumen outward, the esophagus comprises stratified squamous epithelium, lamina propria, and muscle. The tissue-specific nature of EoE strongly suggests that structural cells in the esophagus are involved in the EoE diathesis. Epithelial basal cell hyperplasia and dilated intercellular spaces are cardinal features of EoE. Some patients with EoE develop lamina propria fibrosis, strictures, or esophageal muscle dysmotility. Clinical symptoms of EoE are only weakly correlated with peak eosinophil count, implying that other cell types contribute to EoE pathogenesis. Epithelial, endothelial, muscle, and fibroblast cells can each initiate inflammation and repair, regulate tissue resident immune cells, recruit peripheral leukocytes, and tailor adaptive immune cell responses. A better understanding of how structural cells maintain tissue homeostasis, respond to cell-intrinsic and cell-extrinsic stressors, and exacerbate and/or resolve inflammatory responses in the esophagus is needed. This knowledge will facilitate the development of more efficacious treatment strategies for EoE that can restore homeostasis of both hematopoietic and structural elements in the esophagus.

Natural history study of patients with familial platelet disorder with associated myeloid malignancy.

ABSTRACT: Deleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

Severe food allergy reactions are associated with α-tryptase.

BACKGROUND: Increased TPSAB1 copy numbers encoding ⍺-tryptase are associated with severe reactions in adults with Hymenoptera venom allergy, systemic mastocytosis, and idiopathic anaphylaxis. OBJECTIVE: The primary objective was to assess the association between ⍺-tryptase and severity of food allergy. METHODS: A total of 119 subjects underwent tryptase genotyping; 82 of them were from an observational food allergy cohort at the National Institute of Allergy and Infectious Disease (NIAID), and 37 were from a cohort of children who reacted to peanut oral food challenge (OFC) at Lurie Children's Hospital of Chicago. The primary predictor was presence or absence of ⍺-tryptase. The primary outcomes for both cohorts were measures of severity of food allergy reaction. Secondary outcomes included OFC symptom scores (Bock/Practical Allergy [PRACTALL] and Severity Grading Score for Acute Reactions [SGSAR]). Correlation between total α-tryptase isoforms and OFC scores was also assessed to account for gene dosage effects. RESULTS: Among the subjects in the NIAID cohort, the presence of ⍺-tryptase was associated with a higher prevalence of food-triggered anaphylaxis than in those with only ß-tryptase (P = .026). Similarly, only 1 of 6 subjects in the OFC cohort with only ß-tryptase (17%) had a severe reaction, whereas 20 of 31 of subjects with α-tryptase (65%) had a severe reaction (P = .066). Subjects with ⍺-tryptase also had higher total SGSAR scores than did the subjects with no ⍺-tryptase (P = .003). In addition, there were also significant positive correlations between ⍺-tryptase isoform copy numbers and both higher total SGSAR and Bock/PRACTALL OFC scores (P = .008 and P = .003, respectively). CONCLUSION: The presence of α-tryptase in subjects is correlated with a higher prevalence of anaphylaxis or severe reaction to food than in subjects without any α-tryptase.

The Risk of Allergic Reaction to SARS-CoV-2 Vaccines and Recommended Evaluation and Management: A Systematic Review, Meta-Analysis, GRADE Assessment, and International Consensus Approach.

Concerns for anaphylaxis may hamper severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization efforts. We convened a multidisciplinary group of international experts in anaphylaxis composed of allergy, infectious disease, emergency medicine, and front-line clinicians to systematically develop recommendations regarding SARS-CoV-2 vaccine immediate allergic reactions. Medline, EMBASE, Web of Science, the World Health Organizstion (WHO) global coronavirus database, and the gray literature (inception, March 19, 2021) were systematically searched. Paired reviewers independently selected studies addressing anaphylaxis after SARS-CoV-2 vaccination, polyethylene glycol (PEG) and polysorbate allergy, and accuracy of allergy testing for SARS-CoV-2 vaccine allergy. Random effects models synthesized the data to inform recommendations based on the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) approach, agreed upon using a modified Delphi panel. The incidence of SARS-CoV-2 vaccine anaphylaxis is 7.91 cases per million (n = 41,000,000 vaccinations; 95% confidence interval [95% CI] 4.02-15.59; 26 studies, moderate certainty), the incidence of 0.15 cases per million patient-years (95% CI 0.11-0.2), and the sensitivity for PEG skin testing is poor, although specificity is high (15 studies, very low certainty). We recommend vaccination over either no vaccination or performing SARS-CoV-2 vaccine/excipient screening allergy testing for individuals without history of a severe allergic reaction to the SARS-CoV-2 vaccine/excipient, and a shared decision-making paradigm in consultation with an allergy specialist for individuals with a history of a severe allergic reaction to the SARS-CoV-2 vaccine/excipient. We recommend further research to clarify SARS-CoV-2 vaccine/vaccine excipient testing utility in individuals potentially allergic to SARS-CoV2 vaccines or their excipients.

Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism.

Differences between female and male immunity may contribute to variations in response to infections and predisposition to autoimmunity. We previously reported that neutrophils from reproductive-age males are more immature and less activated than their female counterparts. To further characterize the mechanisms that drive differential neutrophil phenotypes, we performed RNA sequencing on circulating neutrophils from healthy adult females and males. Female neutrophils displayed significant up-regulation of type I IFN (IFN)-stimulated genes (ISGs). Single-cell RNA-sequencing analysis indicated that these differences are neutrophil specific, driven by a distinct neutrophil subset and related to maturation status. Neutrophil hyperresponsiveness to type I IFNs promoted enhanced responses to Toll-like receptor agonists. Neutrophils from young adult males had significantly increased mitochondrial metabolism compared to those from females and this was modulated by estradiol. Assessment of ISGs and neutrophil maturation genes in Klinefelter syndrome (47, XXY) males and in prepubescent children supported that differences in neutrophil phenotype between adult male and female neutrophils are hormonally driven and not explained by X chromosome gene dosage. Our results indicate that there are distinct sex differences in neutrophil biology related to responses to type I IFNs, immunometabolism, and maturation status that may have prominent functional and pathogenic implications.

Corneal thinning and cornea guttata in patients with mutations in TGFB2.

OBJECTIVE: Human genome-wide association studies and animal models suggest a role for TGFB2 in contributing to the corneal thickness phenotype. No specific mutations, however, have been reported in this gene that affect corneal thickness. We sought to determine if haploinsufficiency of TGFB2 in humans associated with Loeys-Dietz syndrome type 4 is associated with corneal thinning. DESIGN: Observational cohort study of families with Loeys-Dietz syndrome type 4, caused specifically by TGFB2 mutations, in a tertiary care setting. PARTICIPANTS: Three probands with pathogenic mutations in TGFB2 and family members underwent comprehensive ophthalmic examination. METHODS: Clinical assessment included Scheimpflug imaging, specular microscopy, and slit-lamp biomicroscopy. We measured visual acuity, axial length, refractive error, and central corneal thickness. RESULTS: Clinical evaluation of 2 probands identified corneal thinning and cornea guttata, despite a young age and distinct mutations in TGFB2 (c.905G>A, p.Arg302His; c.988C>A, p.Arg330Ser). In the third family, corneal thinning co-segregated with a TGFB2 mutation (c.1103G>A, p.Gly368Glu), although without apparent guttae. CONCLUSIONS: In this series, participants with TGFB2 mutations associated with Loeys-Dietz syndrome type 4 demonstrated decreased corneal thickness, and in 2 cases with splice site mutations, also demonstrated cornea guttata. The data demonstrate the importance of considering distinct phenotype-genotype correlations within this condition.

Severity of oro-dental anomalies in Loeys-Dietz syndrome segregates by gene mutation.

Background Loeys-Dietz syndrome (LDS), an autosomal dominant rare connective tissue disorder, has multisystemic manifestations, characterised by vascular tortuosity, aneurysms and craniofacial manifestations. Based on the associated gene mutations along the transforming growth factor-beta (TGF-ß) pathway, LDS is presently classified into six subtypes. Methods We present the oro-dental features of a cohort of 40 patients with LDS from five subtypes. Results The most common oro-dental manifestations were the presence of a high-arched and narrow palate, and enamel defects. Other common characteristics included bifid uvula, submucous cleft palate, malocclusion, dental crowding and delayed eruption of permanent teeth. Both deciduous and permanent teeth had enamel defects in some individuals. We established a grading system to measure the severity of enamel defects, and we determined that the severity of the enamel anomalies in LDS is subtype-dependent. In specific, patients with TGF-ß receptor II mutations (LDS2) presented with the most severe enamel defects, followed by patients with TGF-ß receptor I mutations (LDS1). LDS2 patients had higher frequency of oro-dental deformities in general. Across all five subtypes, as well as within each subtype, enamel defects exhibited incomplete penetrance and variable expression, which is not associated with the location of the gene mutations. Conclusion This study describes, in detail, the oro-dental manifestations in a cohort of LDS, and we conclude that LDS2 has the most severely affected phenotype. This extensive characterisation, as well as some identified distinguishing features can significantly aid dental and medical care providers in the diagnosis and clinical management of patients with this rare connective tissue disorder.

Hematopoietic reconstitution of neonatal immunocompetent mice to study conditions with a perinatal window of susceptibility.

Efficient hematopoietic reconstitution of wild type mice requires preconditioning. Established experimental protocols exist to transplant hematopoietic stem cells into lethally irradiated or chemically myeloablated adult mice or unirradiated immunodeficient mice. We sought to develop a protocol to reconstitute immuno-replete neonatal mice. We describe irradiation and injection procedures for two-day old mice that lead to efficient long-term reconstitution of primary and secondary lymphoid organs. We demonstrate that the frequencies of lymphoid and myeloid cells in primary and secondary lymphoid organs are indistinguishable from unirradiated uninjected sex- and age-matched control animals by 5 weeks post-reconstitution. Thus, this system will facilitate studies aimed at understanding the developmental and environmental mechanisms that contribute to conditions that have a window of susceptibility during the perinatal period.

Mechanistic correlates of clinical responses to omalizumab in the setting of oral immunotherapy for milk allergy.

BACKGROUND: In our recent clinical trial, the addition of omalizumab to oral immunotherapy (OIT) for milk allergy improved safety, but no significant clinical benefit was detected. OBJECTIVE: We sought to investigate mechanisms by which omalizumab modulates immunity in the context of OIT and to identify baseline biomarkers that predict subgroups of patients most likely to benefit from omalizumab. METHODS: Blood was obtained at baseline and multiple time points during a placebo-controlled trial of OIT for milk allergy in which subjects were randomized to receive omalizumab or placebo. Immunologic outcomes included measurement of basophil CD63 expression and histamine release and casein-specific CD4+ regulatory T-cell proliferation. Biomarkers were analyzed in relationship to measurements of safety and efficacy. RESULTS: Milk-induced basophil CD63 expression was transiently reduced in whole blood samples from both omalizumab- and placebo-treated subjects. However, IgE-dependent histamine release increased in washed cell preparations from omalizumab- but not placebo-treated subjects. No increase in regulatory T-cell frequency was evident in either group. Subjects with lower rates of adverse reactions, regardless of arm, experienced better clinical outcomes. Pre-OIT basophil reactivity positively associated with occurrence of symptoms during OIT, whereas the baseline milk IgE/total IgE ratio correlated with the likelihood of achieving sustained unresponsiveness. A combination of baseline basophil and serologic biomarkers defined a subset of patients in which adjunctive therapy with omalizumab was associated with attainment of sustained unresponsiveness and a reduction in adverse reactions. CONCLUSIONS: Combining omalizumab therapy with milk OIT led to distinct alterations in basophil reactivity but not T-cell responses. Baseline biomarkers can identify subjects most likely to benefit from adjunctive therapy with omalizumab.

Suppression of the immunologic response to peanut during immunotherapy is often transient.

BACKGROUND: Studies suggest that oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) for food allergy hold promise; however, the immunologic mechanisms underlying these therapies are not well understood. OBJECTIVE: We sought to generate insights into the mechanisms and duration of suppression of immune responses to peanut during immunotherapy. METHODS: Blood was obtained from subjects at baseline and at multiple time points during a placebo-controlled trial of peanut OIT and SLIT. Immunologic outcomes included measurement of spontaneous and stimulated basophil activity by using automated fluorometry (histamine) and flow cytometry (activation markers and IL-4), measurement of allergen-induced cytokine expression in dendritic cell (DC)-T-cell cocultures by using multiplexing technology, and measurement of MHC II and costimulatory molecule expression on DCs by using flow cytometry. RESULTS: Spontaneous and allergen-induced basophil reactivity (histamine release, CD63 expression, and IL-4 production) were suppressed during dose escalation and after 6 months of maintenance dosing. Peanut- and dust mite-induced expression of TH2 cytokines was reduced in DC-T-cell cocultures during immunotherapy. This was associated with decreased levels of CD40, HLA-DR, and CD86 expression on DCs and increased expression of CD80. These effects were most striking in myeloid DC-T-cell cocultures from subjects receiving OIT. Many markers of immunologic suppression reversed after withdrawal from immunotherapy and in some cases during ongoing maintenance therapy. CONCLUSION: OIT and SLIT for peanut allergy induce rapid suppression of basophil effector functions, DC activation, and TH2 cytokine responses during the initial phases of immunotherapy in an antigen-nonspecific manner. Although there was some interindividual variation, in many patients suppression appeared to be temporary.

TGFß receptor mutations impose a strong predisposition for human allergic disease.

Transforming growth factor-ß (TGFß) is a multifunctional cytokine that plays diverse roles in physiologic processes as well as human disease, including cancer, heart disease, and fibrotic disorders. In the immune system, TGFß regulates regulatory T cell (Treg) maturation and immune homeostasis. Although genetic manipulation of the TGFß pathway modulates immune tolerance in mouse models, the contribution of this pathway to human allergic phenotypes is not well understood. We demonstrate that patients with Loeys-Dietz syndrome (LDS), an autosomal dominant disorder caused by mutations in the genes encoding receptor subunits for TGFß, TGFBR1 and TGFBR2, are strongly predisposed to develop allergic disease, including asthma, food allergy, eczema, allergic rhinitis, and eosinophilic gastrointestinal disease. LDS patients exhibited elevated immunoglobulin E levels, eosinophil counts, and T helper 2 (TH2) cytokines in their plasma. They had an increased frequency of CD4(+) T cells that expressed both Foxp3 and interleukin-13, but retained the ability to suppress effector T cell proliferation. TH2 cytokine-producing cells accumulated in cultures of naïve CD4(+) T cells from LDS subjects, but not controls, after stimulation with TGFß, suggesting that LDS mutations support TH2 skewing in naïve lymphocytes in a cell-autonomous manner. The monogenic nature of LDS demonstrates that altered TGFß signaling can predispose to allergic phenotypes in humans and underscores a prominent role for TGFß in directing immune responses to antigens present in the environment and foods. This paradigm may be relevant to nonsyndromic presentations of allergic disease and highlights the potential therapeutic benefit of strategies that inhibit TGFß signaling.

The safety and efficacy of sublingual and oral immunotherapy for milk allergy.

BACKGROUND: Oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) are potential therapies for food allergy, but the optimal method of administration, mechanism of action, and duration of response remain unknown. OBJECTIVE: We sought to explore the safety and efficacy of OIT and SLIT for the treatment of cow's milk (CM) allergy. METHODS: We randomized children with CM allergy to SLIT alone or SLIT followed by OIT. After screening double-blind, placebo-controlled food challenges and initial SLIT escalation, subjects either continued SLIT escalation to 7 mg daily or began OIT to either 1000 mg (the OITB group) or 2000 mg (the OITA group) of milk protein. They were challenged with 8 g of milk protein after 12 and 60 weeks of maintenance. If they passed the 60-week challenge, therapy was withdrawn, with challenges repeated 1 and 6 weeks later. Mechanistic correlates included end point titration skin prick testing and measurement of CM-specific IgE and IgG(4) levels, basophil histamine release, constitutive CD63 expression, CD203c expression, and intracellular spleen tyrosine kinase levels. RESULTS: Thirty subjects with CM allergy aged 6 to 17 years were enrolled. After therapy, 1 of 10 subjects in the SLIT group, 6 of 10 subjects in the SLIT/OITB group, and 8 of 10 subjects in the OITA group passed the 8-g challenge (P = .002, SLIT vs OIT). After avoidance, 6 of 15 subjects (3 of 6 subjects in the OITB group and 3 of 8 subjects in the OITA group) regained reactivity, 2 after only 1 week. Although the overall reaction rate was similar, systemic reactions were more common during OIT than during SLIT. By the end of therapy, titrated CM skin prick test results and CD63 and CD203c expression decreased and CM-specific IgG(4) levels increased in all groups, whereas CM-specific IgE and spontaneous histamine release values decreased in only the OIT group. CONCLUSION: OIT was more efficacious for desensitization to CM than SLIT alone but was accompanied by more systemic side effects. Clinical desensitization was lost in some cases within 1 week off therapy.