Distinct Type 1 Immune Networks Underlie the Severity of Restrictive Lung Disease after COVID-19.

The variable etiology of persistent breathlessness after COVID-19 have confounded efforts to decipher the immunopathology of lung sequelae. Here, we analyzed hundreds of cellular and molecular features in the context of discrete pulmonary phenotypes to define the systemic immune landscape of post-COVID lung disease. Cluster analysis of lung physiology measures highlighted two phenotypes of restrictive lung disease that differed by their impaired diffusion and severity of fibrosis. Machine learning revealed marked CCR5+CD95+ CD8+ T-cell perturbations in mild-to-moderate lung disease, but attenuated T-cell responses hallmarked by elevated CXCL13 in more severe disease. Distinct sets of cells, mediators, and autoantibodies distinguished each restrictive phenotype, and differed from those of patients without significant lung involvement. These differences were reflected in divergent T-cell-based type 1 networks according to severity of lung disease. Our findings, which provide an immunological basis for active lung injury versus advanced disease after COVID-19, might offer new targets for treatment.

ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients.

Many mechanisms responsible for COVID-19 pathogenesis are well-established, but COVID-19 includes features with unclear pathogenesis, such as autonomic dysregulation, coagulopathies, and high levels of inflammation. The receptor for the SARS-CoV-2 spike protein receptor-binding domain (RBD) is angiotensin-converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop antibodies that have a negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity-ACE2-like abzymes. To explore this hypothesis, we studied patients hospitalized with COVID-19 who had plasma samples available obtained about 7 days after admission. ACE2 is a metalloprotease that requires Zn2+ for activity. However, we found that the plasma from some patients studied could specifically cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using disodium EDTA anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+ was added or the plasma was diluted to decrease EDTA concentration. After processing samples by 100 kDa size exclusion columns and protein A/G adsorption, which depleted immunoglobulin by >99.99%, the plasma samples did not cleave the ACE2 substrate peptide. The data suggest that some patients with COVID-19 develop antibodies with abzyme-like activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate many physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure COVID-19 pathogenesis. IMPORTANCE: We provide what we believe to be the first description of angiotensin-converting enzyme 2 (ACE2)-like enzymatic activity associated with immunoglobulin in COVID-19 patients. COVID-19 includes many puzzling clinical features that have unclear pathogenesis, including a hyperinflammatory state, abnormalities of the clotting cascade, and blood pressure instability. We hypothesized that some patients with COVID-19 patients may produce antibodies against SARS-CoV-2 with enzymatic activity, or abzymes, that target important proteolytic regulatory cascades. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein binds ACE2 on the surface of the future host cell. This means that the RBD has a negative molecular image of ACE2. We hypothesized that some antibodies produced against the RBD would have, in turn, a negative molecular image of the RBD sufficiently similar to ACE2 to have ACE2-like catalytic activity. In other words, some anti-RBD antibodies would be ACE2-like abzymes. Abzymes elicited by SARS-CoV-2 infection have the potential to affect host physiology.

Tick bites, IgE to galactose-alpha-1,3-galactose and urticarial or anaphylactic reactions to mammalian meat: The alpha-gal syndrome.

The recent recognition of a syndrome of tick-acquired mammalian meat allergy has transformed the previously held view that mammalian meat is an uncommon allergen. The syndrome, mediated by IgE antibodies against the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal), can also involve reactions to visceral organs, dairy, gelatin and other products, including medications sourced from non-primate mammals. Thus, fittingly, this allergic disorder is now called the alpha-gal syndrome (AGS). The syndrome is strikingly regional, reflecting the important role of tick bites in sensitization, and is more common in demographic groups at risk of tick exposure. Reactions in AGS are delayed, often by 2-6 h after ingestion of mammalian meat. In addition to classic allergic symptomatology such as urticaria and anaphylaxis, AGS is increasingly recognized as a cause of isolated gastrointestinal morbidity and alpha-gal sensitization has also been linked with cardiovascular disease. The unusual link with tick bites may be explained by the fact that allergic cells and mediators are mobilized to the site of tick bites and play a role in resistance against ticks and tick-borne infections. IgE directed to alpha-gal is likely an incidental consequence of what is otherwise an adaptive immune strategy for host defense against endo- and ectoparasites, including ticks.

IgE to common food allergens is associated with cardiovascular mortality in the National Health and Examination Survey and the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: In individuals without symptomatic food allergy, food-specific IgE is considered clinically irrelevant. However, recent studies have suggested that galactose-α-1,3-galactose (alpha-gal) IgE is associated with cardiovascular (CV) disease. OBJECTIVE: We sought to determine whether sensitization to common food allergens is associated with CV mortality. METHODS: The association between IgE sensitization to foods and CV mortality ascertained to 2019 was examined in the National Health and Examination Survey (NHANES) 2005-2006 and the Wake Forest site of the Multi-Ethnic Study of Atherosclerosis (MESA) cohort; MESA enrolled adults without baseline clinical CV diseases between 2000 and 2002. Total and specific IgE was measured to cow's milk, egg, peanut, shrimp, and a panel of aeroallergens (NHANES), and to cow's milk, alpha-gal, peanut, dust mite, and timothy grass (MESA). Cox proportional hazard models were constructed, adjusting for sex, age, race/ethnicity, smoking, education, and asthma. RESULTS: A total of 4414 adults from NHANES (229 CV deaths) and 960 from MESA (56 CV deaths) were included. In NHANES, sensitization to at least 1 food was associated with higher CV mortality (hazard ratio [HR], 1.7 [95% confidence interval (CI), 1.2-2.4], P = .005). Milk sensitization was particularly associated (HR, 2.0 [95% CI, 1.1-3.8], P = .026), a finding replicated in MESA (HR, 3.8 [95% CI, 1.6-9.1], P = .003). Restricting analyses in NHANES to consumers of the relevant allergen strengthened food sensitization relationships, unmasking shrimp and peanut sensitization as additional risk factors for CV mortality. CONCLUSIONS: The finding that food sensitization is associated with increased risk of CV mortality challenges the current paradigm that sensitization without overt allergy is benign. Further research is needed to clarify mechanisms of this association.

Alpha-Gal IgE Prevalence Patterns in the United States: An Investigation of 3,000 Military Recruits.

BACKGROUND: IgE to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal) is an important cause of allergic reactions to mammalian meat. The "alpha-gal syndrome" is strongly associated with a preceding history of tick bites and in the United States is most commonly reported in parts of the southeast, but there has been limited investigation into national alpha-gal sensitization patterns and the relevance of other risk factors. OBJECTIVE: To systematically investigate alpha-gal IgE prevalence, regional patterns, and risk factors. METHODS: Alpha-gal IgE was measured by ImmunoCAP in biobanked serum samples collected from 3000 service members who presented for intake to 1 of 10 military bases in the central/eastern United States. Alpha-gal IgE sensitization (cutoff 0.1 international units/mL) was related to home of record at enlistment. RESULTS: Of the cohort, 2456 (81.9%) subjects were male, median age was 19 years (interquartile range: 18-22 years), and alpha-gal IgE was detected in 179 (6.0%). Home of record spanned all 50 states, with a median of 36 recruits per state (range: 3-261). The highest prevalence rates were in Arkansas (39%), Oklahoma (35%), and Missouri (29%), with several other southeastern states >10%. Granular mapping revealed sensitization patterns that closely mimicked county-level Amblyomma americanum reports and Ehrlichia chaffeensis infections. Sensitization was associated with male sex, rural residence, and White race in univariate and multivariable models. CONCLUSIONS: In this systematic survey, the prevalence of alpha-gal IgE among incoming military personnel was 6.0%. There were significant regional differences, with an overall pattern consistent with the known range of the lone star tick (A. americanum) and highest frequency in an area including Arkansas, Oklahoma, and Missouri.

Immunoglobulin G4 in eosinophilic esophagitis: Immune complex formation and correlation with disease activity.

BACKGROUND: Recent studies have shown deposition of immunoglobulin G4 (IgG4) and food proteins in the esophageal mucosa of eosinophilic esophagitis (EoE) patients. Our aims were to assess whether co-localization of IgG4 and major cow's milk proteins (CMPs) was associated with EoE disease activity and to investigate the proteins enriched in proximity to IgG4 deposits. METHODS: This study included adult subjects with EoE (n = 13) and non-EoE controls (n = 5). Esophageal biopsies were immunofluorescence stained for IgG4 and CMPs. Co-localization in paired samples from active disease and remission was assessed and compared to controls. The proteome surrounding IgG4 deposits was evaluated by the novel technique, AutoSTOMP. IgG4-food protein interactions were confirmed with co-immunoprecipitation and mass spectrometry. RESULTS: IgG4-CMP co-localization was higher in the active EoE group compared to paired remission samples (Bos d 4, p = .02; Bos d 5, p = .002; Bos d 8, p = .002). Co-localization was also significantly higher in the active EoE group compared to non-EoE controls (Bos d 4, p = .0013; Bos d 5, p = .0007; Bos d 8, p = .0013). AutoSTOMP identified eosinophil-derived proteins (PRG 2 and 3, EPX, RNASE3) and calpain-14 in IgG4-enriched areas. Co-immunoprecipitation and mass spectrometry confirmed IgG4 binding to multiple food allergens. CONCLUSION: These findings further contribute to the understanding of the interaction of IgG4 with food antigens as it relates to EoE disease activity. These data strongly suggest the immune complex formation of IgG4 and major cow's milk proteins. These immune complexes may have a potential role in the pathophysiology of EoE by contributing to eosinophil activation and disease progression.

Enhanced SARS-CoV-2 IgG durability following COVID-19 mRNA booster vaccination and comparison of BNT162b2 with mRNA-1273.

BACKGROUND: BNT162b2 (Pfizer/BioNTech, Comirnaty) and mRNA-1273 (Moderna, Spikevax) are messenger RNA (mRNA) vaccines that elicit antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (S-RBD) and have been approved by the US Food and Drug Administration to combat the coronavirus disease 2019 (COVID-19) pandemic. Because vaccine efficacy and antibody levels waned over time after the 2-shot primary series, the US Food and Drug Administration authorized a booster (third) dose for both mRNA vaccines to adults in the fall of 2021. OBJECTIVE: To evaluate the magnitude and durability of S-RBD immunoglobulin (Ig)G after the booster mRNA vaccine dose in comparison to the primary series. We also compared S-RBD IgG levels after BNT162b2 and mRNA-1273 boosters and explored effects of age and prior infection. METHODS: Surrounding receipt of the second and third homologous mRNA vaccine doses, adults in an employee-based cohort provided serum and completed questionnaires, including information about previous COVID-19 infection. The IgG to S-RBD was measured using an ImmunoCAP-based system. A subset of samples were assayed for IgG to SARS-CoV-2 nucleocapsid by commercial assay. RESULTS: There were 228 subjects who had samples collected between 7 and 150 days after their primary series vaccine and 117 subjects who had samples collected in the same time frame after their boost. Antibody levels from 7 to 31 days after the primary series and booster were similar, but S-RBD IgG was more durable over time after the boost, regardless of prior infection status. In addition, mRNA-1273 post-boost antibody levels exceeded BNT162b2 out to 5 months. CONCLUSION: The COVID-19 mRNA vaccine boosters increase antibody durability, suggesting enhanced long-term clinical protection from SARS-CoV-2 infection compared with the 2-shot regimen.

Food antigen consumption and disease activity affect food-specific IgG4 levels in patients with eosinophilic esophagitis (EoE).

INTRODUCTION: High levels of serum food-specific IgG4 (sIgG4) have been reported in patients with EoE. The objective of this study was to examine whether serum sIgG4 levels to foods and aeroallergens are higher in EoE patients than allergic controls and to investigate the association between sIgG4 and EoE clinical characteristics. METHODS: This was a case-control study nested in a prospective EoE Cohort. EoE cases were defined per consensus guidelines, and controls were individuals with symptoms who were confirmed to be EoE-negative on upper endoscopy. Demographic and clinical information was prospectively collected. Serum IgE and sIgG4 were measured to foods and aeroallergens by ImmunoCAP. Mean levels of sIgG4 were compared between cases and controls, and logistic regression models were used to examine predictors of elevated milk sIgG4 levels. RESULTS: The analysis included 123 individuals (EoE n = 93, control n = 30) with a similar distribution of allergic disease between EoE patients and controls (86% vs. 93%; p = .30). EoE patients had significantly higher sIgG4 levels to all allergens evaluated, with the exception of birch (p = .24). Milk sIgG4 levels were independently associated with milk consumption (OR 4.95; p = .01) and the presence of sIgE to milk (OR 4.23; p = .008). CONCLUSION: Serum sIgG4 levels to food and aeroallergen proteins were higher in patients with EoE than non-EoE controls, and higher levels of milk sIgG4 were independently associated with milk consumption and the presence of sIgE to milk proteins. Whether sIgG4 plays a pathogenic role in EoE or could be used as an EoE biomarker remains unknown and warrants further study.

Trajectory of IgG to SARS-CoV-2 After Vaccination With BNT162b2 or mRNA-1273 in an Employee Cohort and Comparison With Natural Infection.

Three COVID-19 vaccines have received FDA-authorization and are in use in the United States, but there is limited head-to-head data on the durability of the immune response elicited by these vaccines. Using a quantitative assay we studied binding IgG antibodies elicited by BNT162b2, mRNA-1273 or Ad26.COV2.S in an employee cohort over a span out to 10 months. Age and sex were explored as response modifiers. Of 234 subjects in the vaccine cohort, 114 received BNT162b2, 114 received mRNA-1273 and six received Ad26.COV2.S. IgG levels measured between seven to 20 days after the second vaccination were similar in recipients of BNT162b2 and mRNA-127 and were ~50-fold higher than in recipients of Ad26.COV2.S. However, by day 21 and at later time points IgG levels elicited by BNT162b2 were lower than mRNA-1273. Accordingly, the IgG decay curve was steeper for BNT162b2 than mRNA-1273. Age was a significant modifier of IgG levels in recipients of BNT162b2, but not mRNA-1273. After six months, IgG levels elicited by BNT162b2, but not mRNA-1273, were lower than IgG levels in patients who had been hospitalized with COVID-19 six months earlier. Similar findings were observed when comparing vaccine-elicited antibodies with steady-state IgG targeting seasonal human coronaviruses. Differential IgG decay could contribute to differences observed in clinical protection over time between BNT162b2 and mRNA-1273.

Optimal umbilical cord blood collection, processing and cryopreservation methods for sustained public cord blood banking.

BACKGROUND AIMS: Umbilical cord blood is an established source of stem cells in patients with hematologic malignancies who do not have HLA-compatible matched related or unrelated donors. The success of an umbilical cord blood transplant depends on the dose of total nucleated and CD34+ cells infused. Therefore, collecting, banking and listing high-quality cord blood units with high total nucleated and CD34+ cell dose are essential. METHODS: Here the authors describe their cord blood bank's novel collection technique, which involves both in utero and ex utero collection of a single cord blood unit. The authors also evaluated maternal, neonatal and collection parameters that may impact the cell dose. RESULTS: Maternal gestational age and race, and neonatal weight and sex correlated with the total nucleated cell dose. CONCLUSIONS: The optimized collection of umbilical cord blood is critical for its use as a source of stem cells for transplantation.

An Overview of the Relevance of IgG4 Antibodies in Allergic Disease with a Focus on Food Allergens.

Antibodies of the IgG4 isotype are strongly associated with allergic disease but have several properties such as not precipitating with allergens, not activating complement and poor binding to Fcγ receptors that argue against a pro-inflammatory role. In keeping with that, IgG4 antibodies are a striking feature of the response to immunotherapy. In two naturally occurring situations IgG4 antibodies are common with low or absent IgE antibodies. The first example is children raised in a house with a cat and the second is eosinophilic esophagitis (EoE). In many population-based cohorts, the ownership of a cat in early childhood is associated with a decreased prevalence of a cat allergy at age 10. The second example (i.e., EoE) is a novel form of food allergy that is not mediated by IgE and is related to consuming cow's milk or wheat. In EoE, patients have IgG4 to milk proteins in high > 10 µg/mL or very high > 100 µg/mL titers. Enigmatically these patients are found to have deposits of IgG4 in the wall of their inflamed esophagus. The factors that have given rise to EoE remain unclear; however, changes in food processing over the past 50 years, particularly ultra-heat treatment and the high pressure homogenization of milk, represent a logical hypothesis.

Quantitative Measurement of IgG to Severe Acute Respiratory Syndrome Coronavirus-2 Proteins Using ImmunoCAP.

BACKGROUND: Detailed understanding of the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the cause of coronavirus disease 2019 (CO-VID-19) has been hampered by a lack of quantitative antibody assays. OBJECTIVE: The objective was to develop a quantitative assay for IgG to SARS-CoV-2 proteins that could be implemented in clinical and research laboratories. METHODS: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding domain (RBD) or nucleocapsid protein to the solid phase of the ImmunoCAP. Plasma and serum samples from patients hospitalized with COVID-19 (n = 60) and samples from donors banked before the emergence of COVID-19 (n = 109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform. RESULTS: At a cutoff of 2.5 µg/mL, the assay demonstrated sensitivity and specificity exceeding 95% for IgG to both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples, there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG. CONCLUSIONS: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to mutated COVID-19 proteins, has good performance characteristics, and has a readout in standardized units.

α-Gal specific-IgE prevalence and levels in Ecuador and Kenya: Relation to diet, parasites, and IgG4.

BACKGROUND: IgE to α-Gal is a cause of mammalian meat allergy and has been linked to tick bites in North America, Australia, and Eurasia. Reports from the developing world indicate that α-Gal sensitization is prevalent but has been little investigated. OBJECTIVE: We sought evidence for the cause(s) of α-Gal sensitization and lack of reported meat allergy among children in less developed settings in Ecuador and Kenya. METHODS: IgE to α-Gal and total IgE were assessed in children from Ecuador (n = 599) and Kenya (n = 254) and compared with children with (n = 42) and without known (n = 63) mammalian meat allergy from the southeastern United States. Information on diet, potential risk factors, and helminth infections was available for children from Ecuador. IgG4 to α-Gal and antibodies to regionally representative parasites were assessed in a subset of children. RESULTS: In Ecuador (32%) and Kenya (54%), α-Gal specific IgE was prevalent, but levels were lower than in children with meat allergy from the United States. Sensitization was associated with rural living, antibody markers of Ascaris exposure, and total IgE, but not active infections with Ascaris or Trichuris species. In Ecuador, 87.5% reported consuming beef at least once per week, including 83.9% of those who had α-Gal specific IgE. Levels of α-Gal specific IgG4 were not high in Ecuador, but were greater than in children from the United States. CONCLUSIONS: These results suggest that in areas of the developing world with endemic parasitism, α-Gal sensitization is (1) common, (2) associated with Ascaris exposure, and (3) distinguished by a low percentage of specific/total IgE compared with individuals with meat allergy in the United States.

The use of microarray and other multiplex technologies in the diagnosis of allergy.

OBJECTIVE: To give an overview and describe the strengths and weaknesses of immunoglobulin E (IgE) microarray and other multiplex assays that have been developed and are being used for allergy diagnostics. DATA SOURCES: Queries for IgE microarray and multiplex assays were conducted with PubMed and Google Scholar, searching for primary articles and review papers. STUDY SELECTIONS: We focused on articles written in English on commercially available IgE multiplex assays that were reported in the allergy and immunology literature. RESULTS: Several commercial IgE assays that use microarray or other multiplex technology have been developed, and some have been implemented into clinical practice in Europe and Asia, with the Immuno Solid-Phase Allergen Chip being the most widely studied. Results of these assays generally correlate with results using "singleplex" IgE assays (eg, ImmunoCAP), though there can be variability among products and among allergens. A strength of the microarray technology is that IgE to a large number of allergens can be detected simultaneously in a single test, and only a small amount of patient serum is required. Cost, inadequate sensitivity under some scenarios, and difficulties with data interpretation, in some cases of 100 or more allergens, can be limitations. CONCLUSION: IgE microarray assays are already a valuable tool in research applications. These assays, and also other forms of IgE multiplex assays, are likely to play an important role in the clinical practice of allergy in the future. Additional studies focused on clinical outcomes, and the development of more targeted allergen panels could facilitate increased clinical use.

Chemokine Receptor Activation Enhances Memory B Cell Class Switching Linked to IgE Sensitization to Alpha Gal and Cardiovascular Disease.

Background: Recent studies have suggested that IgE sensitization to α-gal is associated with coronary artery disease (CAD). However, the B cell subtype(s) responsible for production of IgE to α-gal and mechanisms mediating this production remain elusive. Methods: Single cell multi-omics sequencing, was utilized to phenotype B cells obtained from 60 subjects that had undergone coronary angiography in whom serum IgE was evaluated by ImmunoCAP. Bioinformatics approaches were used to identify B cell subtype(s) and transcriptomic signatures associated with α-gal sensitization. In vitro characterization of chemokine/chemokine receptor pairs on switched memory B cells associated with IgE to α-gal was performed. Results: Of the 60 patients, 17 (28%) were positive for IgE to α-gal. CITESeq identified CCR6+ class-switched memory (SWM) B cells and CXCR4 expresssion on these CCR6+ SWM B cells as significantly associated with IgE sensitization to α-gal but not to other common allergens (peanut or inhalants). In vitro studies of enriched human B cells revealed significantly greater IgE on SWM B cells with high CCR6 and CXCR4 expression 10 days after cells were treated with IL-4 and CD40 to stimulate class switch recombination. Both CCL20 (CCR6 ligand) and CXCL12 (ligand for CXCR4) increased the expression of IgE on SWM B cells expressing their receptors. However, they appeared to have unique pathways mediating this effect as only CCL20 increased activation-induced cytidine deaminase (AID), while CXCL12 drove proliferation of CXCR4+ SWM B cells. Lastly, correlation analysis indicated an association between CAD severity and the frequency of both CCR6+ SWM and CXCR4+ SWM B cells. Conclusions: CCR6+ SWM B cells were identified as potential producers of IgE to α-gal in CAD patients. Additionally, our findings highlighted non-chemotaxis roles of CCL20/CCR6 and CXCL12/CXCR4 signaling in mediating IgE class switching and cell proliferation of SWM B cells respectively. Results may have important implications for a better understanding and better therapeutic approaches for subjects with IgE sensitization to α-gal.

Lessons in Innate and Allergic Immunity From Dust Mite Feces and Tick Bites.

Allergic diseases represent a major cause of morbidity in modern industrialized and developing countries. The origins and development of allergic immune responses have proven difficult to unravel and remain an important scientific objective. House dust mites (HDM) and ticks represent two important causes of allergic disease. Investigations into HDM fecal particles and tick bites have revealed insights which have and will continue to shape our understanding of allergic immunity. In the present review, focus is given to the role of innate immunity in shaping the respective responses to HDM and ticks. The HDM fecal particle represents a rich milieu of molecules that can be recognized by pathogen-recognition receptors of the innate immune system. Factors in tick saliva and/or tissue damage resultant from tick feeding are thought to activate innate immune signaling that promotes allergic pathways. Recent evidence indicates that innate sensing involves not only the direct recognition of allergenic agents/organisms, but also indirect sensing of epithelial barrier disruption. Although fecal particles from HDM and bites from ticks represent two distinct causes of sensitization, both involve a complex array of molecules that contribute to an innate response. Identification of specific molecules will inform our understanding of the mechanisms that contribute to allergic immunity, however the key may lie in the combination of molecules delivered to specific sites in the body.