An optimized, robust and reproducible protocol to generate well-differentiated primary nasal epithelial models from extremely premature infants

Extremely premature infants are prone to severe respiratory infections, and the mechanisms underlying this exceptional susceptibility are largely unknown. Nasal epithelial cells (NEC) represent the first-line of defense and adult-derived ALI cell culture models show promising results in mimicking in vivo physiology. Therefore, the aim of this study was to develop a robust and reliable protocol for generating well-differentiated cell culture models from NECs of extremely premature infants. Nasal brushing was performed in 13 extremely premature infants at term corrected age and in 11 healthy adult controls to obtain NECs for differentiation at air-liquid interface (ALI). Differentiation was verified using imaging and functional analysis. Successful isolation and differentiation was achieved for 5 (38.5%) preterm and 5 (45.5%) adult samples. Preterm and adult ALI-cultures both showed well-differentiated morphology and ciliary function, however, preterm cultures required significantly longer cultivation times for acquiring full differentiation (44 +/- 3.92 vs. 23 +/- 1.83 days; p <0.0001). Moreover, we observed that recent respiratory support may impair successful NECs isolation. Herewithin, we describe a safe, reliable and reproducible method to generate well-differentiated ALI-models from NECs of extremely premature infants. These models provide a valuable foundation for further studies regarding immunological and inflammatory responses and respiratory disorders in extremely premature infants.Peer reviewe

Acute urticaria caused by the injection of goat-derived hyaluronidase

Hyaluronidase is a goat testicular protein that hydrolyzes hyaluronic acid, a structural component of the intercellular matrix. It is commonly used as a spreading factor to improve the diffusion of drugs, including local anesthetics and chemotherapeutics. We experienced a 55-yr-old female with generalized urticaria that developed within 1 hr after the epidural injection of hyaluronidase. She had a history of allergic rhinitis, and had suffered from post-herpetic neuralgia and a herniated disc for several years. To relieve her pain, she had been given epidural injections consisting of mepivacaine hydrochloride, triamcinolone acetonide, and morphine sulfate biweekly for one year. Hyaluronidase had been administered several times with these drugs before this episode of generalized urticaria. Skin prick testing showed a positive response to 1,500 IU/mL of hyaluronidase extract, as compared to histamine. The patient's serum hyaluronidase-specific IgE level, determined using an enzyme-linked immunosorbent assay (ELISA), was markedly elevated, as compared to unexposed healthy controls. An IgE immunoblot analysis using hyaluronidase extract and the patient's serum showed IgE binding components at 31 and 21 kDa, whereas no corresponding IgE binding component was found in healthy controls. An ELISA inhibition test showed significant, dose-dependent inhibition with the serial addition of hyaluronidase extract. This is the first case of an IgE-medicated allergic reaction to goat (Naemorhedus goral raddenus) hyaluronidase, demonstrated by skin testing and a specific IgE and immunoblot assay

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.Fil: Christiansen, Anders. Technical University of Denmark; DinamarcaFil: Kringelum, Jens V.. Technical University of Denmark; DinamarcaFil: Hansen, Christian S.. Technical University of Denmark; DinamarcaFil: Bøgh, Katrine L.. Technical University of Denmark; DinamarcaFil: Sullivan, Eric. Roche Nimble Gen; Estados UnidosFil: Patel, Jigar. Roche Nimble Gen; Estados UnidosFil: Rigby, Neil M.. Institute of Food Research; Reino UnidoFil: Eiwegger, Thomas. Medical University of Vienna; AustriaFil: Szépfalusi, Zsolt. Medical University of Vienna; AustriaFil: Masi, Federico De. Technical University of Denmark; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Lund, Ole. Technical University of Denmark; DinamarcaFil: Dufva, Martin. Technical University of Denmark; Dinamarc

The development of TH2 responses from infancy to 4 years of age and atopic sensitization in areas endemic for helminth infections

BACKGROUND: Helminth infections and allergies are associated with TH(2) responses. Whereas the development of TH(2) responses and allergic disorders in pediatric populations has been examined in affluent countries, no or little data exist from low income regions of the world. The aim of this study is to examine factors influencing the development of TH(2) responses of children born in areas endemic for helminth infections and to relate these factors to atopic sensitization at 4 years of age. METHODS: Data were collected from pregnant mothers on helminth infections, education and socioeconomic status (SES). Total IgE, IL-5 in response to mitogen, and helminth antigens were measured in children at 2, 5, 12, 24 and 48 months of age. Skin prick testing (SPT) and allergen-specific IgE were determined at 4 years of age. RESULTS: Strong TH(2) responses were seen at 5 months of age and increased with time. Although maternal filarial infection was associated with helminth-antigen specific TH(2) responses, it was low maternal education or SES but not helminth infection, which was associated with the development of high total IgE and PHA-induced IL-5. At 4 years of age when allergen reactivity was assessed by SPT, the high general TH(2) responses did not translate into higher prevalence of SPT. The risk factor for SPT reactivity was low maternal education which decreased the risk of SPT positivity to allergens (adjusted OR, 0.32; 95% CI, 0.12 – 0.87) independently of maternal filarial infection which tended to reduce the child’s risk for being SPT positive (adjusted OR, 0.35; 95% CI, 0.07 – 1.70). CONCLUSIONS: In areas endemic for helminths, potent TH(2) responses were seen early in life, but did not translate into a higher SPT reactivity to allergens. Therefore, in many parts of the world TH(2) responses in general and IgE in particular cannot be used for diagnosis of allergic diseases

Is Aboriginal Food Less Allergenic? Comparing IgE-Reactivity of Eggs from Modern and Ancient Chicken Breeds in a Cohort of Allergic Children

BACKGROUND: Hen's egg allergy ranks among the most frequent primary food allergies in children. We aimed to investigate sensitization profiles of egg allergic patients and compare in vitro IgE reactivities of eggs from ancient chicken breeds (Araucana and Maran) with those from conventional laying hen hybrids. METHODOLOGY: Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1-5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays. PRINCIPAL FINDINGS: We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3-5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken. CONCLUSION/SIGNIFICANCE: Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen's egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family