17q21 Variants Disturb Mucosal Host Defense in Childhood Asthma.

Rationale: The strongest genetic risk factor for childhood-onset asthma, the 17q21 locus, is associated with increased viral susceptibility and disease-promoting processes.Objectives: To identify biological targets underlying the escalated viral susceptibility associated with the clinical phenotype mediated by the 17q21 locus.Methods: Genome-wide transcriptome analysis of nasal brush samples from 261 children (78 healthy, 79 with wheezing at preschool age, 104 asthmatic) within the ALLIANCE (All-Age-Asthma) cohort, with a median age of 10.0 (range, 1.0-20.0) years, was conducted to explore the impact of their 17q21 genotype (SNP rs72163891). Concurrently, nasal secretions from the same patients and visits were collected, and high-sensitivity mesoscale technology was employed to measure IFN protein levels.Measurements and Main Results: This study revealed that the 17q21 risk allele induces a genotype- and asthma/wheeze phenotype-dependent enhancement of mucosal GSDMB expression as the only relevant 17q21-encoded gene in children with preschool wheeze. Increased GSDMB expression correlated with the activation of a type-1 proinflammatory, cell-lytic immune, and natural killer signature, encompassing key genes linked to an IFN type-2-signature (IFNG, CXCL9, CXCL10, KLRC1, CD8A, GZMA). Conversely, there was a reduction in IFN type 1 and type 3 expression signatures at the mRNA and protein levels.Conclusions: This study demonstrates a novel disease-driving mechanism induced by the 17q21 risk allele. Increased mucosal GSDMB expression is associated with a cell-lytic immune response coupled with compromised airway immunocompetence. These findings suggest that GSDMB-related airway cell death and perturbations in the mucosal IFN signature account for the increased vulnerability of 17q21 risk allele carriers to respiratory viral infections during early life, opening new options for future biological interventions.The All-Age-Asthma (ALLIANCE) cohort is registered at www.clinicaltrials.gov (pediatric arm, NCT02496468).

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Impact of climate change on immune responses and barrier defense.

Climate change is not just jeopardizing the health of our planet but is also increasingly affecting our immune health. There is an expanding body of evidence that climate-related exposures such as air pollution, heat, wildfires, extreme weather events, and biodiversity loss significantly disrupt the functioning of the human immune system. These exposures manifest in a broad range of stimuli, including antigens, allergens, heat stress, pollutants, microbiota changes, and other toxic substances. Such exposures pose a direct and indirect threat to our body's primary line of defense, the epithelial barrier, affecting its physical integrity and functional efficacy. Furthermore, these climate-related environmental stressors can hyperstimulate the innate immune system and influence adaptive immunity-notably, in terms of developing and preserving immune tolerance. The loss or failure of immune tolerance can instigate a wide spectrum of noncommunicable diseases such as autoimmune conditions, allergy, respiratory illnesses, metabolic diseases, obesity, and others. As new evidence unfolds, there is a need for additional research in climate change and immunology that covers diverse environments in different global settings and uses modern biologic and epidemiologic tools.

Urinary eosinophil-derived neurotoxin is associated with reduced lung function in pediatric asthma.

INTRODUCTION: Eosinophil-derived neurotoxin (EDN) is a biomarker for eosinophilic activation. Urinary (u) EDN may allow non-invasive monitoring of asthma, but clinical recommendations are lacking. We assessed the potential of uEDN as a marker of disease activity in pediatric asthma. METHODS: We assessed urine samples of 371 children from the German ALLIANCE study cohort, from which we had: 169 preschool wheezers (<6 years), 80 asthmatics (≥6 years), and 122 healthy controls using the ImmunoCAP™ EDN Assay. Creatinine (Cr)-adjusted uEDN values were analyzed using correlations, association tests, (non) parametric statistics, multiple linear, and multivariable regression. RESULTS: uEDN/uCr values were higher in atopic versus non-atopic preschool-aged subjects (p = .035) and associated with the sum of allergen-specific IgE in younger (r = 0.24, p = .003), and older subjects (r = 0.23, p = .043). uEDN/uCr was marginally a good determinant for atopy (p = .078, for subjects aged <6 years, and p = .058 for subjects ≥6 years). Children with the T2-high phenotype had higher uEDN/uCr (p < .001) versus T2-low-irrespective of using uEDN/uCr or blood eosinophils in combination to allergen sIgE for disease phenotyping. uEDN/uCr significantly correlated with reduced lung function among asthmatics (FEV1 z-scores: r = -0.30, p = .007, and FEV1/FVC z-scores: r = -0.24, p = .038). Using multivariable modeling, uEDN/uCr was an independent determinant of FEV1 (p = .038), and to a lesser extent, FEV1/FVC (p = .080). CONCLUSIONS: uEDN/uCr may serve as a non-invasive biomarker for clinical features such as lung function in pediatric asthma. We highlight the utility of uEDN/uCr as a biomarker that can be easily assessed using widely available robust diagnostic immunoassays.

Respiratory viral co-infections in patients with COVID-19 and associated outcomes: A systematic review and meta-analysis.

The aim of this systematic review and meta-analysis was to critically assess the published literature related to community-acquired viral co-infections and COVID-19 and to evaluate the prevalence, most identified co-pathogens, and relevant risk factors. Furthermore, we aimed to examine the clinical features and outcomes of co-infected compared to mono-infected COVID-19 patients. We systematically searched PubMed, Web of Science, Embase, Scopus, and The Cochrane Library for studies published from 1 November 2019 to 13 August 2021. We included patients of all ages and any COVID-19 severity who were screened for respiratory viral co-infection within 48 h of COVID-19 diagnosis. The main outcome was the proportion of patients with a respiratory viral co-infection. The systematic review was registered to PROSPERO (CRD42021272235). Out of 6053 initially retrieved studies, 59 studies with a total of 16,643 SARS-CoV-2 positive patients were included. The global pooled prevalence was 5.01% (95% CI 3.34%-7.27%; I2  = 95%) based on a random-effects model, with Influenza Viruses (1.54%) and Enteroviruses (1.32%) being the most prevalent pathogens. Subgroup analyses showed that co-infection was significantly higher in paediatric (9.39%) than adult (3.51%) patients (p-value = 0.02). Furthermore, co-infected patients were more likely to be dyspnoeic and the odds of fatality (OR = 1.66) were increased. Although a relatively low proportion of COVID-19 patients have a respiratory viral co-infection, our findings show that multiplex viral panel testing may be advisable in patients with compatible symptoms. Indeed, respiratory virus co-infections may be associated with adverse clinical outcomes and therefore have therapeutic and prognostic implications.

Comorbidity defines asthmatic patients' risk of COVID-19 hospitalization: A global perspective.

BACKGROUND: The global epidemiology of asthma among patients with coronavirus disease 2019 (COVID-19) presents striking geographic differences, defining prevalence zones of high and low co-occurrence of asthma and COVID-19. OBJECTIVE: We aimed to compare asthma prevalence among hospitalized patients with COVID-19 in major global hubs across the world by applying common inclusion criteria and definitions. METHODS: We built a network of 6 academic hospitals in Stanford (Stanford University)/the United States; Frankfurt (Goethe University), Giessen (Justus Liebig University), and Marburg (Philipps University)/Germany; and Moscow (Clinical Hospital 52 in collaboration with Sechenov University)/Russia. We collected clinical and laboratory data for patients hospitalized due to COVID-19. RESULTS: Asthmatic individuals were overrepresented among hospitalized patients with COVID-19 in Stanford and underrepresented in Moscow and Germany as compared with their prevalence among adults in the local community. Asthma prevalence was similar among patients hospitalized in an intensive care unit and patients hospitalized in other than an intensive care unit, which implied that the risk for development of severe COVID-19 was not higher among asthmatic patients. The numbers of males and comorbidities were higher among patients with COVID-19 in the Stanford cohort, and the most frequent comorbidities among these patients with asthma were other chronic inflammatory airway disorders such as chronic obstructive pulmonary disease. CONCLUSION: The observed disparity in COVID-19-associated risk among asthmatic patients across countries and continents is connected to the varying prevalence of underlying comorbidities, particularly chronic obstructive pulmonary disease.

Impaired systemic nucleocapsid antigen clearance in severe COVID-19.

The circulating nucleocapsid (NCP) antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detectable in coronavirus disease-2019 (COVID-19) patients. To better understand the biology of disease severity, we investigated NCP clearance kinetics in hospitalized COVID-19 patients. Serum NCP was quantified using a commercial NCP-specific enzyme-linked immunoassay in hospitalized COVID-19 patients (n = 63) during their hospital stay. Results were correlated to COVID-19 disease severity, inflammation parameters, antibody response, and results of SARS-CoV-2 PCR from nasopharyngeal swabs. We demonstrate that NCP antigen levels in serum remained elevated in 21/45 (46.7%) samples from patients in intensive care units (ICU) after >8 days postdiagnosis. The proportion of ICU patients with detectable antigenemia declined only gradually from 84.6% to 25.0% over several weeks. This was in contrast to complete NCP clearance in all non-ICU patients after 8 days, and also in contrast to mucosal clearance of the virus as measured by PCR. Antigen clearance was associated with higher IgG against S1 but not NCP. Clearance of NCP antigenemia is delayed in >40% of severely ill COVID-19 patients. Thus, NCP antigenemia detected after 8 days post COVID-19 diagnosis is a characteristic of patients requiring intensive care. Prospective trials should further investigate NCP antigen clearance kinetics as a mechanistic biomarker.

Acute and chronic impacts of heat stress on planetary health.

Heat waves are increasing in intensity, frequency, and duration causing significant heat stress in all living organisms. Heat stress has multiple negative effects on plants affecting photosynthesis, respiration, growth, development, and reproduction. It also impacts animals leading to physiological and behavioral alterations, such as reduced caloric intake, increased water intake, and decreased reproduction and growth. In humans, epidemiological studies have shown that heat waves are associated with increased morbidity and mortality. There are many biological effects of heat stress (structural changes, enzyme function disruption, damage through reactive oxygen or nitrogen species). While plants and animals can mitigate some of these effects through adaptive mechanisms such as the generation of heat shock proteins, antioxidants, stress granules, and others, these mechanisms may likely be inadequate with further global warming. This review summarizes the effects of heat stress on plants and animals and the adaptative mechanisms that have evolved to counteract this stress.

Deep phenotypical characterization of human CD3+ CD56+ T cells by mass cytometry.

CD56+ T cells are a group of pro-inflammatory CD3+ lymphocytes with characteristics of natural killer cells, being involved in antimicrobial immune defense. Here, we performed deep phenotypic profiling of CD3+ CD56+ cells in peripheral blood of normal human donors and individuals sensitized to birch-pollen or/and house dust mite by high-dimensional mass cytometry combined with manual and computational data analysis. A co-regulation between major conventional T-cell subsets and their respective CD3+ CD56+ cell counterparts appeared restricted to CD8+ , MAIT, and TCRγδ+ T-cell compartments. Interestingly, we find a co-regulation of several CD3+ CD56+ cell subsets in allergic but not in healthy individuals. Moreover, using FlowSOM, we distinguished a variety of CD56+ T-cell phenotypes demonstrating a hitherto underestimated heterogeneity among these cells. The novel CD3+ CD56+ subset description comprises phenotypes superimposed with naive, memory, type 1, 2, and 17 differentiation stages, in part represented by a phenotypical continuum. Frequencies of two out of 19 CD3+ CD56+ FlowSOM clusters were significantly diminished in allergic individuals, demonstrating less frequent presence of cells with cytolytic, presumably protective, capacity in these donors consistent with defective expansion or their recruitment to the affected tissue. Our results contribute to defining specific cell populations to be targeted during therapy for allergic conditions.

T2-high asthma phenotypes across lifespan.

RATIONALE: In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. OBJECTIVES: To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. METHODS: In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. MEASUREMENTS AND MAIN RESULTS: Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. CONCLUSIONS: Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Epidemiology and management of asthma and atopic dermatitis in Sub-Saharan Africa.

Sub-Saharan Africa (SSA) is currently undergoing a transformation process of unprecedented magnitude owing to economic development and urbanization. This process is paralleled by a dramatic increase in prevalence and incidence of noncommunicable diseases. In this article we analyze the current situation with regard to 1 group of the earliest noncommunicable diseases in a person's life, namely, allergies and asthma. This article provides an update on the epidemiology, availability, and access to management strategies by patients experiencing bronchial asthma or atopic dermatitis in SSA. Despite all of the progress, there is still a tremendous need to support education and training, transfer of resources, and cooperation with pharmaceutical and diagnostic companies to achieve adequate treatment and sustainability in SSA with regard to allergy, asthma, and eczema management.

Validation study of a new chemiluminescent singleplex IgE assay in a set of Italian allergic rhinitis patients.

BACKGROUND: The measurement of specific IgE to allergenic extracts and molecules in patients with allergic rhinitis (AR) is crucial for a precise diagnosis and further immunotherapy. Companies providing in vitro diagnostic methods in allergology continuously strive for the optimization and modernization of such methods. A new generation of automated allergy tests based on chemiluminescence detection and paramagnetic microparticles is now available, with possible advantages in sample volume, cost-effectiveness and avoidance of sample-related interference. OBJECTIVES: To test whether sIgE antibody levels obtained with a new singleplex chemiluminescent method have a good agreement with the corresponding results obtained with a "gold standard" test. METHODS: We tested sera from 368 AR patients. Specific IgE sera levels (kU/L) to a comprehensive panel of 15 allergen extracts and 6 molecules were tested with ImmunoCAP® (Thermo Fisher Scientific Inc, Phadia AB, Uppsala, Sweden) and NOVEOS™ (HYCOR® Biomedical, Garden Grove, CA, USA). We evaluated the qualitative and quantitative performance of the new NOVEOS system in matching the outcome of ImmunoCAP to each of the examined allergens. RESULTS: In relation to ImmunoCAP, the overall diagnostic sensitivity and specificity of sIgE tests with NOVEOS were 90.8% (95% CI = 88.6-92.7) and 96.2% (95% CI = 93.9-97.8), respectively. These values were higher when only molecules were considered (sensitivity = 98.7% [95% CI = 96.4%-99.7%]; specificity = 94.2% [95% CI = 88.4%-97.6%]) and lower when only extracts were considered (sensitivity = 87.6% [95% CI = 84.7%-90.2%]; specificity = 97% [95% CI = 94.4%-98.6%]). Spearman's correlation between the data set of both methods for a ≥ 0.1 kU/L cut-off was 0.84 (p < .001). CONCLUSIONS: The new singleplex NOVEOS system presented good results for qualitative and quantitative comparisons when testing specific serum IgE antibodies against a range of 21 allergens. This novel immunoassay system using only 4 µl of sample per test appears to be robust and reliable and can, therefore, be used as an aid in allergy diagnosis.

Allergen extract- and component-based diagnostics in children of the ALLIANCE asthma cohort.

BACKGROUND: Current in vitro allergen-specific IgE (sIgE) detection assays measure IgE against allergen extracts or molecules in a single- or multiplex approach. Direct comparisons of the performance of such assays among young children with common presentations of allergic diseases regardless of sensitization status are largely missing. OBJECTIVES: The aim of this study was a comparison of the analytical and diagnostic performance for common clinical questions of three commonly used technologies which rely upon different laboratory methodologies among children of the All Age Asthma (ALLIANCE) cohort (clinicaltrials.gov: NCT02496468). METHODS: Sera from 106 paediatric study participants (mean age 4 years) were assessed for the presence of sIgE by means of the ImmunoCAP™ sx1 and fx5 mixes, the ImmunoCAP ISAC™ 112 microarray and a Euroline™ panel. RESULTS: Total and negative concordance was high (>82%->89%), while positive concordance varied considerably (0%-100%) but was also >50% for the most common sensitizations analysed (house dust mite and birch). All three test systems showed good sensitivity and specificity (AUC consistently > 0.7). However, no significant differences with regard to identifying sIgE sensitizations associated with symptoms in children with suspected pollen- or dust-triggered wheeze or presenting with symptoms of allergic rhinoconjunctivitis or food allergy were detected. Extending the number of allergens did not change the similar performance of the three assay systems. CONCLUSION AND CLINICAL RELEVANCE: Among young children, the three sIgE assays showed good analytical and diagnostic concordance. Our results caution that the identification of larger numbers of sensitizations by more comprehensive multiplex approaches may not improve the clinical utility of sIgE testing in this age group.

IL-1ß augments TGF-ß inducing epithelial-mesenchymal transition of epithelial cells and associates with poor pulmonary function improvement in neutrophilic asthmatics.

BACKGROUND: Neutrophilic asthmatics (NA) have less response to inhaled corticosteroids. We aimed to find out the predictor of treatment response in NA. METHODS: Asthmatics (n = 115) and healthy controls (n = 28) underwent clinical assessment during 6-month follow-up with standardized therapy. Asthmatics were categorized by sputum differential cell count. The mRNA expressions were measured by RT-qPCR for sputum cytokines (IFN-γ, IL-1ß, IL-27, FOXP3, IL-17A, and IL-5). The protein of IL-1ß in sputum supernatant was detected by ELISA. Reticular basement membranes (RBM) were measured in the biopsy samples. The role and signaling pathways of IL-1ß mediating the epithelial-mesenchymal transition (EMT) process were explored through A549 cells. RESULTS: NA had increased baseline sputum cell IL-1ß expression compared to eosinophilic asthmatics (EA). After follow-up, NA had less improvement in FEV1 compared to EA. For all asthmatics, sputum IL-1ß mRNA was positively correlated with protein expression. Sputum IL-1ß mRNA and protein levels were negatively correlated to FEV1 improvement. After subgrouping, the correlation between IL-1ß mRNA and FEV1 improvement was significant in NA but not in EA. Thickness of RBM in asthmatics was greater than that of healthy controls and positively correlated with neutrophil percentage in bronchoalveolar lavage fluid. In vitro experiments, the process of IL-1ß augmenting TGF-ß1-induced EMT cannot be abrogated by glucocorticoid or montelukast sodium, but can be reversed by MAPK inhibitors. CONCLUSIONS: IL-1ß level in baseline sputum predicts the poor lung function improvement in NA. The potential mechanism may be related to IL-1ß augmenting TGF-ß1-induced steroid-resistant EMT through MAPK signaling pathways. TRIAL REGISTRATION: This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (IRB ID: 20150406).

Asthma-associated risk for COVID-19 development.

The newly described severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a pandemic (coronavirus disease 2019 [COVID-19]). It is now well established that certain comorbidities define high-risk patients. They include hypertension, diabetes, and coronary artery disease. In contrast, the context with bronchial asthma is controversial and shows marked regional differences. Because asthma is the most prevalent chronic inflammatory lung disease worldwide and SARS-CoV-2 primarily affects the upper and lower airways leading to marked inflammation, the question arises about the possible clinical and pathophysiological association between asthma and SARS-CoV-2/COVID-19. Here, we analyze the global epidemiology of asthma among patients with COVID-19 and propose the concept that patients suffering from different asthma endotypes (type 2 asthma vs non-type 2 asthma) present with a different risk profile in terms of SARS-CoV-2 infection, development of COVID-19, and progression to severe COVID-19 outcomes. This concept may have important implications for future COVID-19 diagnostics and immune-based therapy developments.

Validation of the analytical performance of the NOVEOS™ System, a system which improves upon the third-generation in vitro allergy testing technology.

Objectives Detection of allergen-specific immunoglobulin E (sIgE) is important for the diagnosis of allergy. IgE sensitization is commonly demonstrated in vivo by skin prick testing (SPT), or in vitro utilizing automated systems. Recently, HYCOR® Biomedical launched its new system for allergen sIgE testing called the NOVEOS™ Immunoanalyzer. This study aims to evaluate the analytical performance of the NOVEOS system in a bi-center study at Philipps-University Marburg (Site-1) and Charité Medical University Berlin (Site-2), respectively. Methods The analytical performance was evaluated based on the guidelines I/LA20-A3, EP5-A3, EP17-A2, EP6-A, EP7-A3, and EP9-A3 of the Clinical and Laboratory Standards Institute (CLSI). Results The conducted repeatability and within-laboratory precision tests provided acceptable performance with 3.0%-11.9% coefficient of variation across both sites. The limit of blank (LoB) and limit of detection (LoD) were <0.1 kU/L at both centers. A within-parameter linearity for all tested allergens was reported at both sites. Of note, no significant interference was observed for high levels of biotin, methylprednisolone, diphenhydramine, omalizumab, or ranitidine. Method comparison between the NOVEOS calibration and the latest World Health Organization (WHO) reference standard showed good agreement at both sites. Conclusions The results from the analytical performance of the NOVEOS allergen sIgE assay and instrument testing at both sites were comparable. Overall, a good precision and linearity as well as a detection limit <0.1 kU/L were observed, with minimal impact of common interfering substances on patient recoveries. The NOVEOS is calibrated to the latest WHO reference standard and adds benefits like a small sample size and para-magnetic microparticles that improve upon third-generation allergen sIgE assays' design and performance.

Molecular diagnosis for allergen immunotherapy.

The extensive use of allergen molecules in birth cohort studies revealed that atopic sensitization is a sequential IgE response to distinct non-cross-reacting molecules from the same allergenic source (ie, molecular spreading), starting with an initiator molecule. This phenomenon reaches different degrees of progression (monomolecular, oligomolecular, and polymolecular) according to the individual atopic propensity and allergen exposure, thus producing an extreme heterogeneity of IgE sensitization profiles in patient populations. In patients with allergic rhinitis, the broader the IgE molecular sensitization profile, the greater is the risk of asthma and other allergic comorbidities, such as oral allergy syndrome. Hence it has been proposed to anticipate immunologic intervention at disease onset (early allergen immunotherapy) or even earlier during the preclinical sensitization stage (allergen immunoprophylaxis). Diagnostic algorithms based on singleplex or multiplex molecular IgE tests allow the discrimination of genuine from cross-reacting sensitization and the selection of the right extracts for allergen immunotherapy composition. Patients with extreme molecular poly-sensitization and greater risk of asthma or other IgE-mediated comorbidities, can be easily identified by means of allergen microarray or macroarray procedures and might benefit from anti-IgE treatment. IgE molecular tests have opened the era of precision allergology, and their routine use should aim at cost-effectiveness, according to the principles of the Choosing Wisely initiative.

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses.

BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.

Postpartum human breast milk levels of neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9)/NGAL complex in normal and pregnancies complicated with insulin-dependent gestational diabetes mellitus. A prospective pilot case-control study.

Neutrophil gelatinase-associated lipocalin (NGAL) and its complex with matrix metalloproteinase-9 (MMP-9) are present in a variety of human tissues and extracellular fluids. The aim of this pilot prospective case-control study was to detect NGAL and MMP-9/NGAL complex in human breast milk postpartum in women with normal and pregnancies that developed insulin-depended gestational diabetes mellitus (iGDM). We detected both biomarkers in human breast milk and concentrations were determined at the first day of colostrum secretion and two days after, in 22 normal pregnancies and 13 pregnancies with iGDM. Mean NGAL concentration decreased significantly from the first to the second sample, in both groups. Mean MMP-9/NGAL complex concentration decreased also significantly from the first to the second sample in normal pregnancies. Mean complex concentration was significantly higher in diabetic pregnancies compared to normal ones in the second sample.IMPACT STATEMENTWhat is already known on this subject? There is limited information on the presence of Neutrophil gelatinase-associated lipocalin (NGAL) in human milk and its physiological role.What the results of this study add? It is the first time that MMP-9/NGAL complex is detected in human milk in both normal and pregnancies complicated with insulin-depended gestational diabetes mellitus (iGDM). We confirm the presence of NGAL in colostrum of normal pregnancies and for the first time we detected NGAL in milk of pregnancies with iGDM. Concentrations of NGAL and MMP-9/NGAL complex tend to lessen postpartum in both groups. Pregnancies with iGDM compared to normal ones showed significantly higher concentration of MMP-9/NGAL complex two days after the beginning of lactation.What the implications are of these findings for clinical practice and/or further research? Further studies are necessary to determine the levels of NGAL and MMP-9/NGAL complex in human milk postpartum in normal and pathological pregnancies. Taking into consideration the well-established NGAL's ability to act as a bacteriostatic agent and its mucosal healing activity in gastrointestinal track, early breastfeeding of neonates is a logical recommendation. Finally, new studies on the actual physiological role of milk NGAL in neonates are necessary.

Bronchiolitis needs a revisit: Distinguishing between virus entities and their treatments.

Current data indicate that the "bronchiolitis" diagnosis comprises more than one condition. Clinically, pathophysiologically, and even genetically three main clusters of patients can be identified among children suffering from severe bronchiolitis (or first wheezing episode): (a) respiratory syncytial virus (RSV)-induced bronchiolitis, characterized by young age of the patient, mechanical obstruction of the airways due to mucus and cell debris, and increased risk of recurrent wheezing. For this illness, an effective prophylactic RSV-specific monoclonal antibody is available; (b) rhinovirus-induced wheezing, associated with atopic predisposition of the patient and high risk of subsequent asthma development, which may, however, be reversed with systemic corticosteroids in those with severe illness; and (c) wheeze due to other viruses, characteristically likely to be less frequent and severe. Clinically, it is important to distinguish between these partially overlapping patient groups as they are likely to respond to different treatments. It appears that the first episode of severe bronchiolitis in under 2-year-old children is a critical event and an important opportunity for designing secondary prevention strategies for asthma. As data have shown bronchiolitis cannot simply be diagnosed using a certain cutoff age, but instead, as we suggest, using the viral etiology as the differentiating factor.