Determinants of immunoglobulin G responses to respiratory syncytial virus and rhinovirus in children and adults.

Introduction: Exposure to respiratory viruses is a significant cause of morbidity and affects virus-specific antibody levels. Little is known about determinants associated with immune response to these viruses. We aimed to investigate the determinants of respiratory syncytial virus (RSV)- and rhinovirus (RV)- specific IgG responses in both children and adults. Methods: The study is based on the EGEA cohort, composed of 530 samples of children in EGEA1 (1991-95) and 1241 samples of adults in EGEA2 (2003-07). Cumulative RV-specific IgG levels (species A, B and C) and IgG levels to RSV-G protein were measured by using micro-array technoloy. Multiple linear mixed models (random effect to account for familial dependence) were performed to assess associations between age, sex, body mass index (BMI), tobacco smoke exposure and season of blood sampling with RSV-and RV-specific IgG levels. Results: In children (11.1 ± 2.8 years old, 57% boys), higher RV-specific IgG levels were associated with older age (only for RV-B), female sex and lower BMI, while only older age was associated with higher RSV-specific IgG levels. In adults (43.5 ± 16.7 years old, 48% men), younger age, female sex, lower BMI, active smoking and all seasons except summer were associated with higher RV-specific IgG levels. Older age, active smoking and all seasons except summer were associated with higher RSV-specific IgG levels. Conclusion: Personal and seasonal determinants of RSV- and RV-specific IgG levels seem to vary according to the respiratory virus type and between children and adults, suggesting different patterns of responses along the life course.

Antibody Conjugates Bispecific for Pollen Allergens and ICAM-1 with Potential to Prevent Epithelial Allergen Transmigration and Rhinovirus Infection.

Allergy and rhinovirus (RV) infections are major triggers for rhinitis and asthma, causing a socioeconomic burden. As RVs and allergens may act synergistically to promote airway inflammation, simultaneous treatment strategies for both causative agents would be innovative. We have previously identified the transmembrane glycoprotein intercellular adhesion molecule 1 (ICAM-1) as an anchor for antibody conjugates bispecific for ICAM-1 and Phleum pratense (Phl p) 2, a major grass pollen allergen, to block allergen transmigration through the epithelial barrier. Since ICAM-1 is a receptor for the major group RVs, we speculated that our bispecific antibody conjugates may protect against RV infection. Therefore, we created antibody conjugates bispecific for ICAM-1 and the major grass pollen allergen Phl p 5 and analyzed their capacity to affect allergen penetration and RV infection. Bispecific antibody conjugates significantly reduced the trans-epithelial migration of Phl p 5 and thus the basolateral Phl p 5 concentration and allergenic activity as determined by humanized rat basophilic leukemia cells and inhibited RV infection of cultured epithelial cells. A reduction in allergenic activity was obtained only through the prevention of allergen transmigration because the Phl p 5-specific IgG antibody did not block the allergen-IgE interaction. Our results indicate the potential of allergen/ICAM-1-specific antibody conjugates as a topical treatment strategy for allergy and RV infections.

Cumulative IgE-levels specific for respiratory allergens as biomarker to predict efficacy of anti-IgE-based treatment of severe asthma.

Molecular therapies, including anti-IgE, biologicals and small molecules are increasingly used for treatment of asthma. The effectiveness of these therapies may be increased with biomarkers. Aim of this study was to assess the value of measuring cumulative IgE levels specific for respiratory allergens to increase the efficacy of anti-IgE therapy for severe bronchial asthma. One hundred and thirty seven patients with severe asthma were recruited from 2016 to 2022. Standard empirical allergy diagnosis (i.e., anamnesis, skin testing, allergen-specific IgE measurement), blood eosinophil counting, measurement of total IgE and of cumulative IgE-specific for respiratory allergens by Phadiatop™ were performed. Thirty four patients with severe allergic asthma, for whom all three diagnostic methods were performed, were then used to analyze the efficacy of anti-IgE treatment in patients stratified in two groups according to cumulative IgE levels specific for respiratory allergens determined by Phadiatop™. Group #1 patients (n = 8) had cumulative specific IgE values ≥ 0.35 and < 1.53 PAU/l while in group #2 patients (n = 26) they were ≥ 1.53 PAU/l. Treatment with Omalizumab was performed for at least 12 months. The level of asthma control (ACT questionnaire), the number of asthma exacerbations, the quality of life (AQLQ questionnaire), the need for systemic corticosteroids, and the respiratory function (FEV1) was determined by "before-after" analysis for each group, followed by a comparison of the dynamics between groups. In group 2 patients with an initial allergen-specific IgE level ≥ 1.53 kUA/L, the efficacy of Omalizumab treatment was better regarding asthma control, number of exacerbations, and quality of life than in group 1 patients. Our study provides evidence that measuring cumulative levels of IgE specific for respiratory allergens could be a useful screening method for detecting an allergic phenotype of severe asthma and may serve as biomarker to enhance the success of IgE-targeted therapy.

Microarray-Based Analyses of Rhinovirus Species-Specific Antibody Responses in Exacerbated Pediatric Asthma in a German Pediatric Cohort.

Rhinoviruses (RV) account for a significant number of asthma exacerbations, and RV species C may be associated with a severe course in vulnerable patient groups. Despite important evidence on the role of RV reported by clinicians and life scientists, there are still unanswered questions regarding their influence on asthma exacerbation in young patients. Thus, we measured the RVspecies-specific IgG titers in our German pediatric exacerbation cohort using a microarray-based technology. For this approach, human sera of patients with exacerbated asthma and wheeze, as well as healthy control subjects (n = 136) were included, and correlation analyses were performed. Concordantly with previously published results, we observed significantly higher cumulative levels of RV species A-specific IgG (p = 0.011) and RV-C-specific IgG (p = 0.051) in exacerbated asthma group compared to age-matched controls. Moreover, atopic wheezers had increased RV-specific IgG levels for species A (p = 0.0011) and species C (p = 0.0009) compared to non-atopic wheezers. Hypothesizing that bacterial infection positively correlates with immune memory against RV, we included nasopharyngeal swab results in our analyses and detected limited correlations. Interestingly, the eosinophil blood titer positively correlated with RV-specific IgG levels. With these observations, we add important observations to the existing data regarding exacerbation in pediatric and adolescent medicine. We propose that scientists and clinicians should pay more attention to the relevance of RV species in susceptible pediatric patients.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Identification of Epitopes on Rhinovirus 89 Capsid Proteins Capable of Inducing Neutralizing Antibodies.

Rhinoviruses (RVs) are major causes of the common cold, but they can also trigger exacerbations of asthma. More than 160 different RV strains exist and can be classified into three genetic species (RV-A, RV-B and RV-C) which bind to different receptors on human cells including intracellular adhesion molecule 1 (ICAM-1), the low-density lipoprotein receptor (LDLR) or the cadherin-related family member 3 (CDHR3). Epitopes located in the RV capsid have mainly been determined for RV2, a minor-group RV-A strain binding to LDLR, and for RV14, a major-group RV-B strain binding to ICAM-1. In order to study epitopes involved in the neutralization of RV89, an ICAM-1-binding RV-A strain which is highly different from RV2 and RV14 in terms of receptor specificity and sequence, respectively, we analyzed the specificity and epitopes of a highly neutralizing antiserum using recombinantly produced RV89 capsid proteins (VP1, VP2, VP3 and VP4), recombinant fragments and synthetic overlapping peptides thereof. We found that the antiserum which neutralized in vitro RV89 infection up to a dilution of 1:24,000 reacted with the capsid proteins VP1 and VP2 but not with VP3 and VP4. The neutralizing antibodies recognized recombinant fragments comprising approximately 100 amino acids of the N- and C-terminus of VP1 and the middle part of VP2, in particular, three peptides which, according to molecular modeling based on the three-dimensional structure of RV16, were surface-exposed on the viral capsid. Two recombinant fusion proteins containing the identified peptides fused to hepatitis B (HBV)-derived preS as a carrier protein induced upon immunization of rabbits antibodies capable of neutralizing in vitro RV89 infections. Interestingly, the virus-neutralizing epitopes determined for RV89 corresponded to those determined for minor-group RV2 binding to LDL and major-group RV14 belonging to the RV-B species, which are highly different from RV89. Our results indicate that highly different RV strains, even when reacting with different receptors, seem to engage similar parts of their capsid in the infection process. These results may be important for the design of active and passive immunization strategies for RV.

Vaccine based on folded receptor binding domain-PreS fusion protein with potential to induce sterilizing immunity to SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in. METHODS: We report the construction and in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine (PreS-RBD) based on a structurally folded recombinant fusion protein consisting of two SARS-CoV-2 Spike protein receptor-binding domains (RBD) fused to the N- and C-terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS-RBD, but not RBD alone, induced a robust and uniform RBD-specific IgG response in rabbits. Currently available genetic SARS-CoV-2 vaccines induce mainly transient IgG1 responses in vaccinated subjects whereas the PreS-RBD vaccine induced RBD-specific IgG antibodies consisting of an early IgG1 and sustained IgG4 antibody response in a SARS-CoV-2 naive subject. PreS-RBD-specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS-CoV-2 variants, including the omicron variant of concern and the HBV receptor-binding sites on PreS of currently known HBV genotypes. PreS-RBD-specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus-neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS-CoV-2 vaccines or in COVID-19 convalescent subjects. CONCLUSION: The PreS-RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS-CoV-2 and HBV by stopping viral replication through the inhibition of cellular virus entry.

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes.

BACKGROUND: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. METHODS: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus. RESULTS: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. CONCLUSION: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.

Microarray Technology May Reveal the Contribution of Allergen Exposure and Rhinovirus Infections as Possible Triggers for Acute Wheezing Attacks in Preschool Children.

Allergen exposure and rhinovirus (RV) infections are common triggers of acute wheezing exacerbations in early childhood. The identification of such trigger factors is difficult but may have therapeutic implications. Increases of IgE and IgG in sera, were shown against allergens and the N-terminal portion of the VP1 proteins of RV species, respectively, several weeks after allergen exposure or RV infection. Hence, increases in VP1-specific IgG and in allergen-specific IgE may serve as biomarkers for RV infections or allergen exposure. The MeDALL-allergen chip containing comprehensive panels of allergens and the PreDicta RV chip equipped with VP1-derived peptides, representative of three genetic RV species, were used to measure allergen-specific IgE levels and RV-species-specific IgG levels in sera obtained from 120 preschool children at the time of an acute wheezing attack and convalescence. Nearly 20% of the children (22/120) showed specific IgE sensitizations to at least one of the allergen molecules on the MeDALL chip. For 87% of the children, increases in RV-specific IgG could be detected in the follow-up sera. This percentage of RV-specific IgG increases was equal in IgE-positive and -negative children. In 10% of the children, increases or de novo appearances of IgE sensitizations indicative of allergen exposure could be detected. Our results suggest that, in the majority of preschool children, RV infections trigger wheezing attacks, but, in addition, allergen exposure seems to play a role as a trigger factor. RV-induced wheezing attacks occur in IgE-sensitized and non-IgE-sensitized children, indicating that allergic sensitization is not a prerequisite for RV-induced wheeze.

Dissociation of the respiratory syncytial virus F protein-specific human IgG, IgA and IgM response.

Human respiratory syncytial virus (RSV) is one of the most important causes of severe respiratory tract infections in early childhood. The only prophylactic protection is the neutralizing antibody, palivizumab, which targets a conformational epitope of the RSV fusion (F) protein. The F protein is generated as a F0 precursor containing two furin cleavage sites allowing excision of the P27 fragment and then gives rise to a fusion-competent version consisting of the N-terminal F2 subunit and the a C-terminal F1 subunits linked by two disulphide bonds. To investigate natural human F-specific antibody responses, F2 conferring the species-specificity of RSV, was expressed in Escherichia coli. Furthermore, the F0 protein, comprising both subunits F2 and F1, was expressed as palivizumab-reactive glycoprotein in baculovirus-infected insect cells. Six overlapping F2-derived peptides lacking secondary structure were synthesized. The analysis of IgG, IgA and IgM responses of adult subjects to native versions and denatured forms of F2 and F0 and to unfolded F2-derived peptides revealed that mainly non-conformational F epitopes, some of which represented cryptic epitopes which are not exposed on the proteins were recognized. Furthermore, we found a dissociation of IgG, IgA and IgM antibody responses to F epitopes with F2 being a major target for the F-specific IgM response. The scattered and dissociated immune response to F may explain why the natural RSV-specific antibody response is only partially protective underlining the need for vaccines focusing human antibody responses towards neutralizing RSV epitopes.

Features of the Human Antibody Response against the Respiratory Syncytial Virus Surface Glycoprotein G.

Respiratory syncytial virus (RSV) infections are a major cause of serious respiratory disease in infants. RSV occurs as two major subgroups A and B, which mainly differ regarding the surface glycoprotein G. The G protein is important for virus attachment and G-specific antibodies can protect against infection. We expressed the surface-exposed part of A2 strain-derived G (A2-G) in baculovirus-infected insect cells and synthesized overlapping peptides spanning complete A2-G. The investigation of the natural IgG response of adult subjects during a period of one year showed that IgG antibodies (i) recognize G significantly stronger than the fusion protein F0, (ii) target mainly non-conformational, sequential peptide epitopes from the exposed conserved region but also buried peptides, and (iii) exhibit a scattered but constant recognition profile during the observation period. The IgG subclass reactivity profile (IgG1 > IgG2 > IgG4 = IgG3) was indicative of a mixed Th1/Th2 response. Two strongly RSV-neutralizing sera including the 1st WHO standard contained high IgG anti-G levels. G-specific IgG increased strongly in children after wheezing attacks suggesting RSV as trigger factor. Our study shows that RSV G and G-derived peptides are useful for serological diagnosis of RSV-triggered exacerbations of respiratory diseases and underlines the importance of G for development of RSV-neutralizing vaccines.

ELISA-Based Assay for Studying Major and Minor Group Rhinovirus-Receptor Interactions.

Rhinovirus (RV) infections are a major cause of recurrent common colds and trigger severe exacerbations of chronic respiratory diseases. Major challenges for the development of vaccines for RV include the virus occurring in the form of approximately 160 different serotypes, using different receptors, and the need for preclinical models for the screening of vaccine candidates and antiviral compounds. We report the establishment and characterization of an ELISA-based assay for studying major and minor group RV-receptor interactions. This assay is based on the interaction of purified virus with plate-bound human receptor proteins, intercellular adhesion molecule 1 (ICAM-1), and low density lipoprotein receptor (LDLR). Using RV strain-specific antibodies, we demonstrate the specific binding of a panel of major and minor RV group types including RV-A and RV-B strains to ICAM-1 and LDLR, respectively. We show that the RV-receptor interaction can be blocked with receptor-specific antibodies as well as with soluble receptors and neutralizing RV-specific antibodies. The assay is more sensitive than a cell culture-based virus neutralization test. The ELISA assay will therefore be useful for the preclinical evaluation for preventive and therapeutic strategies targeting the RV-receptor interaction, such as vaccines, antibodies, and anti-viral compounds.

Toward personalization of asthma treatment according to trigger factors.

Asthma is a severe and chronic disabling disease affecting more than 300 million people worldwide. Although in the past few drugs for the treatment of asthma were available, new treatment options are currently emerging, which appear to be highly effective in certain subgroups of patients. Accordingly, there is a need for biomarkers that allow selection of patients for refined and personalized treatment strategies. Recently, serological chip tests based on microarrayed allergen molecules and peptides derived from the most common rhinovirus strains have been developed, which may discriminate 2 of the most common forms of asthma, that is, allergen- and virus-triggered asthma. In this perspective, we argue that classification of patients with asthma according to these common trigger factors may open new possibilities for personalized management of asthma.

Microarray-Based Allergy Diagnosis: Quo Vadis?

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

Epitope mapping of antibodies induced with a conserved rhinovirus protein generating protective anti-rhinovirus immunity.

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and chronic obstructive pulmonary disease (COPD) exacerbations. Currently there is no vaccine for RV which is largely due to the existence of ∼160 serotypes/strains. We demonstrated previously that immunising mice with highly conserved VP4 and VP2 regions of the RV polyprotein (RV-A16 VP0) generated cross-reactive immunity to RV in vivo. The current study investigated and mapped the epitopes of RV-A16 VP0 that are targets for antibodies in serum samples from VP0 immunisation and RV challenge studies in mice. Recombinant capsid proteins, peptide pools and individual peptides spanning the immunogen sequence (RV-A16 VP0) were assessed for IgG binding sites to identify epitopes. We found that peptide pools covering the C-terminus of VP4, the N-terminus of VP2 and the neutralising NIm-II site within VP2 were bound by serum IgG from immunised mice. The NIm-II site peptide pool blocked IgG binding to the immunogen RV-A16 VP0 and individual peptides within the pool binding IgG were further mapped. Thus, we have identified immunodominant epitopes of RV vaccine candidate RV-A16 VP0, noting that strong IgG binding antibodies were observed that target a key neutralising epitope that is highly variable amongst RV serotypes.

Rhinovirus Species-Specific Antibodies Differentially Reflect Clinical Outcomes in Health and Asthma.

Rationale: Rhinoviruses (RVs) are major triggers of common cold and acute asthma exacerbations. RV species A, B, and C may have distinct clinical impact; however, little is known regarding RV species-specific antibody responses in health and asthma.Objectives: To describe and compare total and RV species-specific antibody levels in healthy children and children with asthma, away from an acute event.Methods: Serum samples from 163 preschool children with mild to moderate asthma and 72 healthy control subjects from the multinational Predicta cohort were analyzed using the recently developed PreDicta RV antibody chip.Measurements and Main Results: RV antibody levels varied, with RV-C and RV-A being higher than RV-B in both groups. Compared with control subjects, asthma was characterized by significantly higher levels of antibodies to RV-A and RV-C, but not RV-B. RV antibody levels positively correlated with the number of common colds over the previous year in healthy children, and wheeze episodes in children with asthma. Antibody levels also positively correlated with asthma severity but not with current asthma control.Conclusions: The variable humoral response to RV species in both groups suggests a differential infectivity pattern between RV species. In healthy preschoolers, RV antibodies accumulate with colds. In asthma, RV-A and RV-C antibodies are much higher and further increase with disease severity and wheeze episodes. Higher antibody levels in asthma may be caused by a compromised innate immune response, leading to increased exposure of the adaptive immune response to the virus. Importantly, there is no apparent protection with increasing levels of antibodies.

PreDicta chip-based high resolution diagnosis of rhinovirus-induced wheeze.

Rhinovirus (RV) infections are major triggers of acute exacerbations of severe respiratory diseases such as pre-school wheeze, asthma and chronic obstructive pulmonary disease (COPD). The occurrence of numerous RV types is a major challenge for the identification of the culprit virus types and for the improvement of virus type-specific treatment strategies. Here, we develop a chip containing 130 different micro-arrayed RV proteins and peptides and demonstrate in a cohort of 120 pre-school children, most of whom had been hospitalized due to acute wheeze, that it is possible to determine the culprit RV species with a minute blood sample by serology. Importantly, we identify RV-A and RV-C species as giving rise to most severe respiratory symptoms. Thus, we have generated a chip for the serological identification of RV-induced respiratory illness which should be useful for the rational development of preventive and therapeutic strategies targeting the most important RV types.

Betamethasone prevents human rhinovirus- and cigarette smoke- induced loss of respiratory epithelial barrier function.

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

Reduced CDHR3 expression in children wheezing with rhinovirus.

BACKGROUND: Rhinovirus-induced wheezing in young children has been associated with increased asthma risk at school age. Recently, the transmembrane protein cadherin-related family member 3 (CDHR3) was identified as the RV-C receptor and the genetic variant rs6967330 (p.Cys529Tyr) was reported to be associated with enhanced RV-C binding and increased replication in vitro. The aim of this study was to examine rs6967330 genotypes and mRNA expression of CDHR3 in relation to presence of rhinovirus and clinical symptoms in children with acute wheezing and compare to a group of age-matched healthy children. METHODS: rs6967330;G>A was genotyped (n = 216), and CDHR3 mRNA expression was measured in peripheral blood leukocytes (n = 69) from a subgroup of children wheezing with RV infection acute and at a follow-up visit 2-3 months later, and in healthy controls. Standardized TaqMan assays were used. RESULTS: The risk allele rs6967330-A was over-represented in the wheezing group (P < .001). Reduced mRNA levels of CDHR3 were found in children with acute wheezing as compared to the control group (P = .001). Children with the rs6967330 genotypes AA/AG showed the largest differences in CDHR3 expression between acute and follow-up visit (P < .04). CONCLUSIONS: Preschool children with RV-induced wheezing were shown to have reduced CDHR3 mRNA levels, which might result in an increased permeability of the epithelial layers of the airways and thereby an increased vulnerability. Thus, measuring CDHR3 mRNA levels might help to identify a more severe phenotype of wheezing preschool children.