Poster Tour: Thursday

Aim: Disruptions in neonatal lung microbiota are associated with asthma susceptibility, yet their impact on immune priming and airway barrier function remains unclear. We aimed to investigate whether early-life microbial perturbations predispose neonatal lungs to heightened allergic airway inflammation.

Methods: Neonatal (14- and 21-day-old) BALB/c mice and adult (8-week-old) mice were exposed intranasally to Streptococcus pneumoniae (10³–10⁵ CFU) three times over five days; then received house dust mite extract (HDM) instillations every 48 h for 14 days. Microbial shifts were confirmed by 16S rRNA gene amplicon sequencing; immune cell composition and priming were characterized by flow cytometry.

Results: Early-life exposure to S. pneumoniae and HDM led to lung dysbiosis, increased type 2 and type 3 innate lymphoid cells (ILC2/3) and elevated memory T-cell frequencies in the lung, and enhanced eosinophilia in bronchoalveolar lavage fluid (BALF). These effects were most pronounced in 14-day-old compared to 21-day-old mice. Adult mice showed fewer innate changes but stronger Th2 cytokine responses. The observed effects were sex-specific with female mice exhibiting enhanced BALF eosinophilia and increased DC activation (CD80).

Conclusions: Early-life S. pneumoniae exposure profoundly alters neonatal lung microbiota and allergic airway inflammation compared with adult exposure, highlighting a critical window of susceptibility. These results represent an initial step toward delineating the mechanisms underlying early-life lung dysbiosis-mediated allergic susceptibility.

Introduction & Aim:
Cat allergy, mainly triggered by the major allergen Fel d 1, can cause allergic rhinitis and asthma. A novel vaccine was developed using Eggplant Mosaic Virus (EMV) virus-like particles (VLPs) displaying Fel d 1 to induce a protective immune response. This study evaluated the immunogenicity of alternative vaccine formulations and investigated different delivery methods, including subcutaneous and intranasal administration, in a mouse model.

Design & Methods:
Fel d 1 was genetically fused to the EMV VLP capsid protein, expressed in bacteria, and purified. Mice were vaccinated with VLP-Fel d 1, VLP, or Fel d 1 protein, and received homologous or heterologous immunizations. Specific antibody responses (IgG, IgG subclasses, IgA) were measured in serum and bronchoalveolar lavage fluid (BALF) by ELISA.

Results:
Both homologous and heterologous vaccination strategies induced strong Fel d 1-specific IgG responses, with levels rising after booster immunization. At Day 28, IgG1, IgG2b, and IgG3 subclasses were highly elevated, whereas IgG2a responses were lower. IgA levels remained low in both serum and BALF. IgG avidity improved after boosting, especially in groups vaccinated with EMV-Fel d 1 VLPs.

Conclusion:
The EMV-Fel d 1 VLP vaccine induced a strong and specific IgG response through both homologous and heterologous strategies, supporting its potential as a safe and effective immunotherapy for cat allergy. Ongoing studies will further assess the impact of vaccination route, long-term immunity, and lung histopathology.

Eicosanoids regulate chronic inflammation, particularly in therapy-resistant airway disease. Helminth products can act as potent natural immunoregulators. We identified glutamate dehydrogenase (GDH) from the larval extract of Heligmosomoides polygyrus bakeri (Hpb) as an immune-modulatory factor that regulates type 2 inflammation by modulating eicosanoid metabolism.

Mice subjected to house dust mite–induced airway inflammation were intranasally treated with Hpb extract (HpbE) or helminthic GDH (heGDH). Human monocyte-derived macrophages, granulocytes, and nasal polyp tissue were treated ex vivo with HpbE or heGDH. Eicosanoids and cytokines were analyzed by LC-MS/MS or multiplex assays, and lung inflammation was assessed via histology, flow cytometry, and chemotaxis assays.

In macrophages, HpbE and heGDH induced an anti-inflammatory eicosanoid shift from 5-lipoxygenase to cyclooxygenase (COX) metabolites, resulting in reduced granulocyte recruitment. Mechanistically, PGE₂ induction required the N-Terminus of heGDH, which induced p300-mediated histone acetylation. In contrast, the suppression of leukotriene (LT) synthesis depended on heGDH’s catalytic activity. Treatment with HpbE or heGDH or adoptive transfer of HpbE-conditioned macrophages attenuated allergic inflammation in mice via COX-2. Consistent with its anti-inflammatory effects in vivo and human leukocytes, heGDH decreased LTs while increased PGE₂ in human nasal polyp tissue.

These findings highlight HpbE and its immunoregulatory component -heGDH- as a promising immunomodulatory therapy for type 2 inflammatory diseases.

Aim: Extended serological profiling may improve autoimmune connective tissue diseases (CTD) diagnosis. We evaluated the diagnostic utility of machine learning (ML) classifiers based on the 15-antibody profile by a novel, single-use, multiplexed microarray immunoassay, using its fully automated high-throughput proprietary system for simultaneous the detection of IgG antibodies directed to dsDNA, SS-A 60, TRIM21, SS-B, Sm, Sm/RNP, U1RNP, Jo-1, Scl-70, CENP-B, Chromatin, Ribosomal P, DFS70, RNAP III and CCP2.

Methods: De-identified sera from 475 patients with autoimmune CTD [127 systemic lupus erythematosus (SLE), 74 systemic sclerosis, 76 Sjögren’s syndrome (SjS), 71 idiopathic inflammatory myopathies, 54 mixed CTD, 73 rheumatoid arthritis] and 652 disease controls were tested with the MosaiQ AiPlex^® CTDplus (AliveDx, Switzerland) assay. Classification models were developed with the RandomForest (RF) algorithm, using all 15 antibodies or a subset. Diagnostic performance was assessed by receiver operating characteristic curve analysis.

Results: For SLE, the 15-plex RF classifier (RFC) achieved an area under the curve (AUC) of 0.92, outperforming individual markers dsDNA (0.68) and Sm (0.60). For SjS, the 15-plex RFC achieved an AUC of 0.83, versus a 3-plex RFC based on antibodies to SS-A 60, TRIM21, and SS-B (0.62). The AUCs for these markers were 0.63, 0.59, and 0.58, respectively. Similarly, 15-plex classifiers outperformed individual disease-specific markers.

Conclusions: Multiplex autoantibody testing combined with ML algorithms has the potential to improve CTD diagnosis.

Background: The lymphocyte transformation test (LTT) is an in vitro test to detect T cell reactivity to a drug in suspected drug hypersensitivity (DH) by measuring T cell proliferation. We adapted this test by measuring 5 cytokines (IL-5, IL-13, IFNγ, granzyme B, granulysin) with a multiplex bead-assay instead of proliferation (Cyto-LTT). Previous studies have shown that this test modification led to an improved sensitivity with preserved specificity [Lochmatter P, 2008].

Methods: This retrospective analysis of 851 positive Cyto-LTT results (97 DRESS, 754 exanthems) assessed responses to amoxicillin, vancomycin, and aromatic sulfonamides. Lymphocytes were cultured with drugs for six days, cytokine secretion was quantified, with a positive response defined as a stimulation index (SI) >2 in at least two drug concentrations and two cytokines.

Results: Our key findings were (1) dose dependency: Cytokine secretion increased significantly with drug concentration (p<0.001). (2) reaction strength: DRESS cases showed 3-6× higher IL-5/IL-13 and 2× higher cytotoxic mediators than exanthems. 65% of DRESS vs 34% of exanthems had strong reactions (SI>75th percentile in ≥2 cytokines). (3) Drug-specific patterns: amoxicillin/vancomycin induced Th2 cytokines (IL-5/IL-13), aromatic sulfonamides triggered Th1/cytotoxic responses (IFNγ/GzB).

Conclusion: The Cyto-LTT, utilizing five selected cytokines, identifies culprit drugs, quantifies T-cell reactivity, can distinguish severe DH like DRESS from exanthems and reveals drug-specific immune patterns, improving DH diagnosis and risk stratification.