RATIONALE:Restrictive allograft syndrome (RAS) is a major cause of mortality following lung transplantation due to progressive fibrosis of the lung allograft with no therapeutic options. Knowledge of the cellular and molecular mechanisms driving fibrosis in RAS remains limited. OBJECTIVE:To characterize the cellular and molecular changes in human RAS lungs through single-cell transcriptomic profiling. METHODS:Single-nucleus RNA-sequencing (snRNA-seq) was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and extended using histologic techniques including immunofluorescence, RNA in situ hybridization, Elastica-van-Gieson immunohistochemistry, quantitative histological analyses, and micro-CT scans. MEASUREMENTS AND MAIN RESULTS:snRNA-seq analysis of RAS lungs revealed previously undescribed aberrant basaloid cells, ectopic COL15A1+ peribronchial vascular endothelial cells (pVECs), and CTHRC1+ fibrotic fibroblasts. Histologic stains disclosed distinctive distribution patterns: aberrant basaloid cells, primarily localized at the fibrotic edge, together with juxtaposed CTHRC1+ fibrotic fibroblasts and ectopic COL15A1+ pVECs form the fibrotic niche of alveolar fibroelastosis (AFE). PRX+ alveolar microvasculature is partially lost in AFE areas. Micro-CT scans revealed changes from pulmonary to systemic perfusion, facilitated by COL15A1+ pVECs. Last, our data reveals potential therapeutic targets in RAS, including integrin αvβ6, activator of TGFβ. CONCLUSION:Considering the multifaceted differences of RAS and idiopathic pulmonary fibrosis, we revealed a surprising general principle of an entity-spanning composition of the fibrotic niche by aberrant basaloid cells localized at the fibrotic edge, ectopic COL15A1+ pVECs and CTHRC1+ fibrotic fibroblasts. This suggests a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.

BACKGROUND:The pulsatile expansion of pulmonary vessels carries dynamic cardiopulmonary information that may reveal disease earlier than structural changes alone. PURPOSE:To test (i) intra- and inter-scan repeatability of dynamic vessel metrics in healthy subjects, and (ii) whether chronic obstructive pulmonary disease (COPD) and postcapillary pulmonary hypertension (PH) exhibit disease-specific differences. STUDY TYPE:Retrospective, multi-cohort feasibility study with repeatability sub-study. SUBJECTS:Healthy: 29 (11 female), COPD: 52 (15 female), PH: 25 (15 female) with isolated postcapillary PH. FIELDSTRENGTH/SEQUENCE:1.5T, free-breathing spoiled gradient-echo sequence, TE = 0.82 ms, TR = 3 ms, flip angle = 5°. ASSESSMENT:2D U-Nets segmented pulmonary vessels throughout each series of 250 images. Dynamic metrics included the coefficient of variation (CV) of vessel area and CV of vessel signal. Delay between vessel signal and vessel expansion as %-of-RR-interval (SE-delay) together with the heart rate and lung area were calculated. STATISTICAL TESTS:Repeatability: Friedman; intraclass correlation coefficient (ICC); standard error of measurement (SEM) and the minimal detectable change (MDC). Group comparisons: Kruskal-Wallis and multiple linear regression. p < 0.05 was considered significant. RESULTS:Only the heart rate and the SE-delay presented significant changes across repetitive scans. Differences in Bland-Altman plots were evenly distributed across the range of measurements and symmetrically scattered around zero within 95% confidence intervals (mean bias 0.08%-5.12%). Across cohorts, CV vessel area and CV lung area differed significantly, showing lowest variability in COPD (2.67%; 7.03%) and highest in PH (4.58%; 16.74%). SE-delay was significantly prolonged in COPD (81.98%) compared to PH (67.20%) and healthy participants (62.97%). Cohort status (Healthy/COPD/PH) remained the strongest significant predictor for all parameters after adjustment (F-values 4.62-34.04), while age and gender had no significant influence (p > 0.391; p > 0.069). DATA CONCLUSION:Free-breathing lung MRI with automated vessel segmentation reveals distinct hemodynamic characteristics in COPD and PH. This method shows potential as a sensitive non-invasive tool for detection, phenotyping, and treatment monitoring in pulmonary pathology. EVIDENCE LEVEL:3. TECHNICAL EFFICACY:2.

Background:Lung hyperinflation in COPD adversely affects cardiac function and may influence autonomic nervous system activity. The onset of cardiac improvement with dual bronchodilator therapy remains uncertain. Methods:In this randomised, placebo-controlled, investigator-blinded, mechanistic, single-dose crossover trial with an open-label 2-week extension we assessed tiotropium/olodaterol (T/O) effects on cardiopulmonary function and muscle sympathetic nerve activity (MSNA) in hyperinflated COPD patients. 32 participants received saline (placebo) and T/O (5 µg/5 µg) in a crossover design, followed by 14 days of open-label T/O. The primary end-point was change in left ventricular end-diastolic volume index (LV-EDVi) measured by magnetic resonance imaging (MRI). Secondary assessments included pulmonary function tests, MSNA measurements and advanced lung imaging including 129Xe-MRI. Findings:Single-dose T/O significantly increased LV-EDVi relative to placebo (3.25 mL·m-2; 95% CI 0.95 to 5.56), driven partly by a decrease under placebo. Compared with baseline, T/O's acute LV-EDVi gain was minimal, yet it reached significance at 14 days (4.70 mL·m-2; 95% CI 1.75 to 7.65). Pulmonary parameters, in contrast, showed immediate improvement, with a marked reduction in residual volume (-0.67 L; 95% CI -0.82 to -0.51) after a single dose. MSNA demonstrated a nonsignificant numerical rise post single-dose T/O. No serious adverse events occurred. Interpretation:T/O rapidly improves pulmonary function in hyperinflated COPD patients, but significant cardiac benefits seem to require sustained therapy. These results underscore the importance of continued dual bronchodilator therapy to achieve cardiovascular improvements. The nonsignificant rise in MSNA after a single dose suggests minimal immediate effect on sympathetic activity; further studies are necessary to evaluate long-term autonomic outcomes.

Understanding intrapulmonary pharmacokinetics (PK) following inhalation remains a significant challenge in drug development and repurposing. Current lung sampling methods include bronchoalveolar lavage (BAL), biopsies, and the more recent bronchosorption technique, which enhances regional specificity while reducing potential quantification errors. This study aimed to develop a pulmonary population physiologically based pharmacokinetic (PBPK) model for inhaled salbutamol by integrating data from all three sampling techniques to improve PK predictions and to compare different sampling strategies to optimize future study designs. A population-based minimal PBPK model was developed using data from a previously published study (NCT03524066) investigating salbutamol's pulmonary and plasma PK in 13 healthy volunteers after inhalation. Simulations assessed the impact of permeability on pulmonary PK profiles and BAL-derived epithelial lining fluid (ELF)-to-plasma ratios using salbutamol as a reference compound. Stochastic simulation-estimation (SSE) methods were employed to assess the feasibility of different sampling strategies for estimating key parameters of the PBPK model. First, we evaluated using one or two sampling techniques within a single bronchoscopy session. Second, we compared uniform and staggered bronchosorption-based sampling strategies for drugs from different permeability categories. The minimal PBPK model described pulmonary PK of salbutamol across the lung and estimated the unbound tissue–plasma partition coefficient for the lung ( K_p,u,lung ) and the effective permeability ( P_eff ) of salbutamol as 11.0 and 0.543 m/h, respectively. Inter-individual variabilities (IIV) were found on plasma clearance and lung deposition fraction. No significant IIV was detected on K_p,u,lung or P_eff . Simulations indicated that low-permeability drugs exhibited higher concentrations in the ELF, while high-permeability drugs accumulated more in lung tissues, after inhalation. Results from SSE showed that bronchosorption plus biopsy were the most informative two-technique combination and bronchosorption alone was the best single-technique option. Additionally, the optimal sampling strategy for both uniform and staggered sampling depended on drug permeability, with early time points favoured for high-permeability drugs and later or broader windows needed for low-permeability drugs. A pulmonary population PBPK model for inhaled salbutamol was developed by integrating detailed intrapulmonary data from bronchoalveolar lavage, biopsy, and bronchosorption. The study revealed that parameter estimates of K_p,u,lung and P_eff were sensitive to the sampling technique. Staggered sampling strategies mitigated the risk of biased estimates, though the ideal sampling windows varied by drug’s permeability. These findings support model-informed, permeability-driven study design in inhaled drug development.

BACKGROUND:Acute respiratory distress syndrome (ARDS) is characterized by increased alveolar-capillary permeability and neutrophil-driven inflammation. Anumigilimab, a novel, fully human, monoclonal antibody, competitively antagonizes the granulocyte colony-stimulating factor (G-CSF) receptor. This trial aimed to assess its potential in modulating pulmonary inflammation induced by segmental lipopolysaccharide (LPS) challenge. METHODS:In this randomized, double-blind, placebo-controlled Phase 1b trial (NCT05653713), healthy adults were randomized 1:1 to receive a single intravenous dose of anumigilimab 0.6 mg/kg or placebo (0.9% saline), followed by LPS challenge on Day 3. Biomarkers were measured in bronchoalveolar lavage (BAL) and blood before and after LPS challenge. The primary endpoint was the percentage reduction from baseline in mean absolute neutrophil cell count in BAL following LPS challenge. Secondary endpoints included additional BAL biomarkers of neutrophilic inflammation and tissue injury. Pharmacokinetics, pharmacodynamics, and safety were also assessed. RESULTS:Participants were randomly assigned to anumigilimab (n = 23) or placebo (n = 22). LPS challenge induced expected increases in BAL neutrophils and inflammatory biomarkers, with no significant difference between groups. Target engagement was confirmed by elevated plasma and BAL G-CSF concentrations and transient reductions in peripheral neutrophil counts following anumigilimab administration. Pharmacokinetic/pharmacodynamic parameters were consistent with prior clinical data. Anumigilimab was well tolerated; mild transient neutropenia occurred in four participants treated, with no serious adverse events. CONCLUSIONS:While anumigilimab had an acceptable safety profile in healthy volunteers, and favorable pharmacokinetic/pharmacodynamic characteristics, it did not attenuate LPS-induced neutrophilic inflammation. Further preclinical investigations are warranted before advancing its clinical development in ARDS.

Dual long-acting bronchodilators are a standard treatment in chronic obstructive pulmonary disease (COPD), aimed at alleviating dyspnea, improving exercise tolerance, and preventing exacerbations. Metronome-paced tachypnea (MPT) offers a feasible alternative to exercise testing for the evaluation of dynamic hyperinflation (DH) in COPD. Because MPT can be performed during MRI, its combination with single-slice phase-resolved functional lung (PREFUL) MRI provides a promising approach to investigate changes in regional ventilation dynamics induced by DH. This approach was evaluated in a randomized, investigator-blinded, placebo-controlled, single-dose (SD) crossover trial with a two-week extension of once daily dual bronchodilator medication, in which patients with stable COPD underwent PREFUL MRI during resting tidal breathing (RTB) and during MPT. During RTB, no significant improvements in MRI-derived parameters were observed after SD treatment compared with placebo. During MPT, however, regional ventilation, flow-volume loop correlation, its defect percentage, and end-expiratory lung area improved significantly after SD treatment compared to the placebo scan (all p < 0.02). After multi-dose treatment, five out of six measured parameters improved during MPT, when compared to the baseline scan without bronchodilator treatment (all p < 0.03). In contrast to RTB, PREFUL MRI during MPT was able to detect changes in COPD patients already after SD treatment. The combination of MPT and PREFUL MRI represents a promising method to evaluate the effects of dual bronchodilators on regional ventilation dynamics and hyperinflation.

Introduction: Climate change may increase the spread of allergenic plants such as ragweed. Ragweed pollen is assumed to be more allergenic than birch pollen. Determining pollen threshold concentrations triggering symptoms is challenging. Technical modifications to the Fraunhofer allergen challenge chamber (ACC) now allow investigation of very low pollen concentrations. Based on Total Nasal Symptom Score (TNSS) data from 15 subjects with birch pollen allergic rhinitis (AR), a birch pollen threshold between 50 and 100 pollen grains/m3 of air was previously derived. This study explored threshold concentrations of ragweed pollen. Methods: Two ragweed pollen AR patients were exposed to varying concentrations of ragweed pollen for 4 h. TNSS was recorded every 20 min, and nasal secretion and peak nasal inspiratory flow hourly. Area under the curve (AUC0-240) values were calculated. Ex vivo basophil activation tests with varying concentrations of ragweed pollen, Amb a 1, birch pollen, and Bet v 1 were performed for both ragweed pollen AR patients and 6 of the 15 birch pollen AR patients. Mean effective concentrations (EC50) for CD63 expression were calculated. Results: Ragweed pollen exposure resulted in the following TNSS AUC0-240 values: 28/23 at 0, 18/17 at 50, 13/27 at 100, 40/40 at 200, 29/16 at 300, and 55/47 at 1,000 pollen grains/m3. For CD63 expression, the mean EC50 was 277 pollen/mL for ragweed pollen, 884 ng/mL for Amb a 1, 11 pollen/mL for birch pollen, and 0.13 ng/mL for Bet v 1. On average, the proportion of Amb a 1 in ragweed pollen was 0.17%, and the proportion of Bet v 1 in birch pollen 1.96%. Conclusion: The modified Fraunhofer ACC can investigate pollen threshold concentrations under defined conditions. The assumed differences in threshold concentrations of ragweed and birch pollen for triggering symptoms were not confirmed. To derive reliable threshold concentrations for ragweed pollen, larger studies have to be followed.

BACKGROUND:Sarcoidosis is a disease of unknown etiology, characterized by the formation of immune cell accumulations (granulomas) in the lung and other tissues. Chronic sarcoidosis may lead to pulmonary fibrosis. AIM:To unravel cellular niches within pulmonary granulomas of patients with chronic sarcoidosis using spatial transcriptomics. METHODS:Spatial transcriptomics using the Visium platform (10x Genomics) was performed on 9 granuloma-containing lung explants from patients with sarcoidosis. Validation of gene expression was performed through immunohistofluorescence protein staining and RNA in situ hybridization. RESULTS:Spatial gene expression covered 30,587 gene expression spots and 173 granulomas. A CD68+ macrophage niche was localized in the center of the granuloma, with a CD3+ T-cell and CD20+ B-cell niche in close proximity, surrounded by a COL3A1+ fibroblast niche. In the Central Granuloma Macrophage niche, expression of the profibrotic macrophage genes SPP1, CHIT1, and CHI3L1 was observed, genes whose expression has recently been described for macrophages in idiopathic pulmonary fibrosis. Additionally, proinflammatory macrophage genes were expressed in the Central Granuloma Macrophage niche, suggesting that macrophages were armed for lysosomal degradation and ready for phagocytosis. Inner granuloma niches showed higher expression of interferon gamma (IFN-γ)-inducible genes. Higher collagen and CTHRC1 expression were observed in fibroblast-containing granuloma niches, characteristics of profibrotic lung remodeling. Ligand-receptor analysis identified proinflammatory and profibrotic interactions between granuloma niches. CONCLUSION:Taken together, macrophages in the center of the sarcoidosis granuloma form an armed-and-ready, hybrid proinflammatory and profibrotic niche, supporting granuloma persistence through continuous IFN-γ stimulation and collagen expression by fibroblasts localized in the periphery of the granuloma.

To evaluate the effects of tiotropium/olodaterol (T/O) on phase-resolved functional lung (PREFUL) MRI parameters in hyperinflated chronic obstructive pulmonary disease (COPD) patients and examine correlations with conventional cardiopulmonary and hyperpolarized 129Xe MRI measures. Retrospective subanalysis of a prospective, randomized, placebo-controlled, crossover trial with open-label extension. Thirty-two patients with moderate-to-severe COPD (61.5 ± 7.7 years; 17 men); 30 completed the MRI extension at 1.5 T. PREFUL analysis yielded regional ventilation (RVent), flow-volume loop correlation metric (FVL-CM), normalized perfusion (QN), ventilation defect percentage (VDP), perfusion defect percentage (QDP), V/Q match metrics (VQM), and pulmonary pulse wave velocity (PWV; post-hoc parameter). Linear mixed-effects models tested treatment effects; correlations were evaluated with Spearman’s rank and bootstrap 95

PURPOSE:To develop a balanced SSFP (bSSFP) sequence for pulmonary 129Xe dissolved-phase imaging based on a previously described double-echo sequence facilitating separation into red blood cell and membrane signals, to test its feasibility and compare its SNR performance and imaging results to an analogous FLASH sequence. METHODS:12 healthy volunteers underwent hyperpolarized 129Xe MRI with both sequences with two different choices of flip angle for FLASH. SNR was quantified using a pseudo multiple replica method, normalized by 129Xe polarization and ventilated volume. Chemical shift separation was performed in k-space taking results from time-domain fits of signal evolution as input, either approximating TE as constant or taking the finite readout duration into account. Results were compared between sequences and methods by linear mixed models, Bland-Altman, and scatter plots. RESULTS:Normalized SNR of the first echo was significantly greater for bSSFP compared to FLASH with the same flip angle, p = 0.003, but not with lower flip angle resulting in equivalent depolarization, p = 0.98. The effective number of signal averages in signal decomposition was significantly higher compared to FLASH with both flip angles and both dissolved resonances, p < 0.008. Neglecting the finite readout duration was found to introduce bias of derived RBC-M ratios but prevent noise amplification. CONCLUSION:Hyperpolarized 129Xe dissolved-phase imaging using dual-echo bSSFP is feasible and provides superior effective number of signal averages resulting in improved SNR efficiency of signal decomposition. Future work should concentrate on the application in patients with lung disease.

EP395, a macrolide with negligible antimicrobial activity but with anti-inflammatory effects in murine lipopolysaccharide (LPS) challenge model, is being developed as a potential treatment to reduce COPD exacerbations. This double-blind, placebo-controlled clinical study evaluated the pharmacodynamics of EP395 in response to inhaled LPS, an established clinical model for assessing anti-inflammatory effects of potential new treatments. Forty-nine healthy, non-smoking participants were randomised to oral 375 mg EP395 or placebo, daily for 3 weeks. An inhaled LPS challenge (2 μg) was then given, followed 6 h later by bronchoscopy for bronchoalveolar lavage fluid (BALF) collection. Blood samples were collected pre, 6 and 24 h after LPS challenge. BALF concentrations of IL-6, TNF-α, MIP-1α, MIP-1β and MCP-1 were lower with EP395 than placebo, while IL-33, IL-8, and IL-1β were higher with EP395 than placebo (not statistically significant). Neutrophil counts were unaffected, but neutrophil elastase and myeloperoxidase were higher with EP395 than placebo (not statistically significant). Serum concentrations of surfactant protein-D significantly increased in the EP395 group in response to LPS at both 6 and 24 h compared with pre-LPS (mean pre-LPS 148.8 ng/mL; mean 24 h post-LPS 183.0 ng/mL) but not in the placebo group (mean pre-LPS 142.4 ng/mL; mean 24 h post-LPS 142.4 ng/mL). The log2 transformed fold difference in the EP395 group, before and 24 h after LPS challenge was 0.33 (95 % CI 0.52, 0.14; p = 0.0007). EP395 treatment increased the host defence response to inhaled LPS, including the epithelial response, whilst reducing inflammatory site pro-inflammatory mediators.

RATIONALE Direct airway mucosal exposure to OM-85 by nasal administration demonstrated very potent and dose-dependent protection toward viral infection and inflammation in vivo in various disease models. This First-in-Human (FIM) clinical trial was designed to evaluate the safety and tolerability of the nasal spray OM-85-IN and to investigate potential immunomodulatory properties based on clinical and biomarker readouts using nasal allergen provocation in patients with mild allergic asthma. METHODS Healthy participants (Phase 1a: 6+3 per Cohort) and patients with mild allergic asthma Step 1 GINA patients (Phase 1b: 9+5) were to be enrolled in parallel groups to receive intranasal treatment with OM-85-IN or placebo as single ascending dose followed by multiple doses for 16 days. Three doses of OM-85-IN were evaluated: Cohort 1 (1.12 mg), Cohort 2 (3.35 mg), and Cohort 3 (6.70 mg). Safety and tolerability were assessed by monitoring of treatment-emergent adverse events. Dose escalations were decided by Safety Review Committee. RESULTS 28 Healthy participants were randomized in Phase 1a, including 13 males and 15 females, with a mean (range) for age of 28.1 (20; 43) years and for BMI of 25.3 (20.1; 30.9) kg/m2. 48 Adverse Events (AEs) were reported during Phase 1a. No AE led to drug discontinuation and 1 AE led to withdrawal from the study (not related to study drug). No Serious AE was reported in the study. Main AEs System Organ Classes (SOCs) were respiratory, thoracic and mediastinal disorders of mild severity (10); Nervous system disorders of mild (6) and moderate (3) severity; Gastrointestinal disorders of mild severity (2) and General disorders and administration site condition of mild (1) and moderate (1) severity. Out of the 48 AEs reported during Phase 1a, 7 were considered possibly related to investigational medicinal product (IMP), 8 were probably/likely related, and 33 were deemed unlikely or unrelated. CONCLUSION FIM administration of OM-85-IN in Healthy Volunteers demonstrated a favorable safety profile of OM-85-IN: low incidence of AEs, all of them assessed with mild or moderate severity, majority of them assessed as not related to the IMP and which resolved spontaneously in the majority of cases. These findings indicate that the direct application of OM-85-IN to the nasal mucosa is safe and well tolerated. Upcoming Phase 1b in patients with mild asthma will study safety and tolerability in diseased conditions including potential protection from nasal allergen challenge.

Rationale: Airway neutrophilia is associated with several lung diseases, including acute lung injury, severe asthma, and chronic obstructive pulmonary disease (COPD). RLS-0071 (Pegtarazimod), a novel dual-targeting anti-inflammatory peptide, has been shown to reduce airway neutrophilia in a human endotoxin challenge model (Cunnion et al., ERJ Open 2024). ChipCytometry allows quantification of airway cells including intracellular staining of effector molecules. This study investigates the effect of RLS-0071 on airway neutrophils following inhaled endotoxin challenge by ChipCytometry. Methods: RLS-0071 at two doses versus placebo was given in a randomized, double-blind, proof-of-mechanism study following inhaled endotoxin (LPS) challenge in 30 healthy participants (NCT05351671). Induced sputum (IS) was collected before, 6 and 24 hours (h) after LPS challenge. IS cells (250,000) were loaded on ChipCytometry-specific slides according to the manufacturer protocol. Cell surface markers (CD45, CD66b, CD14, CD3, CD11a, CD11b, CD89, CD15, CD62L, CD49d, CD35, CD16) and intracellular effector molecules (myeloperoxidase (MPO), neutrophil elastase (NE), eosinophil peroxidase (EPX)) were stained with corresponding antibodies and then analyzed by ChipCytometry. Results: RLS-0071 treatment led to decreased neutrophil numbers in IS at 6h (placebo: 13.0±4.8, low-dose: 4.9±9.6, high-dose: 6.8±3.5 x106/g) and 24h (placebo: 7.9±5.8, low-dose: 3.8±8.9, high-dose: 6.2±9.1 x106/g) compared to placebo. Neutrophils from RLS-0071-treated participants showed a tendency towards reduced expression of neutrophil activation markers CD89, CD11a and CD11b at 6h compared to placebo. Intracellular levels of NE and MPO in neutrophils were decreased at 6h in both treatment groups compared to placebo (NE: p < 0.01 for both doses; MPO: p = 0.06 and 0.01 for low- and high-dose, respectively), and tended to be reduced at 24h. Conclusion: ChipCytometry data confirm and extend previous proof-of-mechanism that RLS-0071 decreases neutrophil numbers in IS and acts on neutrophil activation and adhesion. In line to previous findings of reduced MPO and NE concentrations in sputum supernatant, we found decreased intracellular staining of MPO and NE in neutrophils. Overall, RLS-0071 demonstrates potential as a novel anti-inflammatory treatment for neutrophil-dominated lung diseases by reducing neutrophil activity and infiltration into the lungs.

Granulomas are the hallmark of mycobacterial (MB) infections, forming structured immune environments that contain bacteria but also drive disease persistence. However, their spatial and functional organization remains unclear. Using spatial RNA sequencing on 38 patient samples, we identified five distinct granuloma niches: a necrotic core, an immune-activated inner niche, an inflammatory and an extracellular matrix (ECM)-remodeling middle niche, an outer structural niche, and a tertiary lymphoid structure niche supporting antigen presentation. Immune activity peaks in the inner niche, transitioning to fibrosis at the periphery. Lymph node granulomas display reduced fibroblast involvement but stronger JAK-STAT activation. Mycobacterium tuberculosis (MTB) granulomas exhibit heightened JAK-STAT and IFN-γ signaling, while non-tuberculous mycobacteria (NTM) granulomas show increased hypoxia signatures. Compared to sarcoidosis, MB granulomas feature a structured adaptive immune response, marked by the clustering of plasma cells. Our findings, accessible via , define key disease signatures, guiding biomarker discovery and therapeutic targeting in granuloma-related diseases. ### Competing Interest Statement The authors have declared no competing interest. ERC Starting Grant, 948207 Lower Saxony Center for Artificial Intelligence and Causal Methods in Medicine, ZN4257 Deutsche Forschungsgemeinschaft, 390874280 Lower Saxony MWK Sprung Fund, 19777006 Fritz Thyssen Foundation, 10.21.2.021MN Else Kröner-Fresenius Foundation, 2023_EKCS.18

Introduction: To determine threshold concentrations of pollen inducing symptoms in seasonal allergic rhinitis patients has been a challenge for decades. Allergen challenge chambers (ACC) allow a controlled, reproducible experimental design to address this problem. Hitherto, ACCs were only run with high pollen concentrations. METHODS:The Fraunhofer ACC was technically modified to deploy very low pollen concentrations. Then, adults with birch pollen-induced allergic rhinitis were challenged with varying birch pollen concentrations using a patient-blinded, sham challenge-controlled, part-randomized, titrate-to-effect clinical study setting. Mean increase in Total Nasal Symptom Score (TNSS) ≥0.55 compared to sham challenge was regarded as minimal clinically important difference (MCID). Further endpoints were nasal secretion weight, exhaled nitric oxide (FeNO), and inflammatory cells from nasal lavage. RESULTS:Fifteen participants with mild to moderate allergic rhinitis participated in the experimental study part (mean age 45 years [22-64]; 7 females). Mean TNSS was: 1.08 at 0 pollen/m3; 1.05 at 10 pollen/m3; 1.2 at 50 pollen/m3; 1.74 at 100 pollen/m3; 1.61 at 200 pollen/m3; 2.79 at 1,000 pollen/m3. MCID of TNSS was observed at 100, 200, and 1,000 pollen/m3. More than half of the study population showed a lack of response at 10, 50, and 200 pollen/m3. Nasal secretion increased slightly with concentration. No clinically meaningful results could be derived from FeNO and inflammatory cells. CONCLUSIONS:The applied technical modification of the Fraunhofer ACC produced stable, low pollen concentrations. Based on mean TNSS data, the threshold concentration for inducing symptoms with birch pollen was 50-100 pollen/m3. .

Background: Parenteral COVID-19 vaccines induce strong systemic immunity, but they do not typically trigger pronounced respiratory immunity. In this context, mucosally applied vaccines might help to induce local immune responses for early viral clearance and reduced viral transmission. Methods: In this investigator-initiated, open-label single-dose phase I trial, we analyzed the immunogenicity and safety of the vaccine candidate MVA-SARS-2-ST administered as an inhalation boost in COVID-19-immunized adults (n=23). MVA-SARS-2-ST represents a replication-deficient vector vaccine candidate built on the recombinant Modified Vaccinia virus Ankara (MVA) platform and expresses a prefusion-stabilized version of the full-length spike glycoprotein of SARS-CoV-2. Results: While there was no increase in spike-specific antibodies in the blood, the inhalation of 107 infectious units (IU) MVA-SARS-2-ST led to an increase in IFN-γ release after re-stimulation of whole blood with spike peptides. This enhanced IFN-γ release peaked at day 7 and remained detectable for at least 140 days after vaccination. Notably, selectively individuals with a history of COVID-19 (nucleocapsid protein (NCP)-seropositive study participants), but not individuals without a history of COVID-19 (NCP-seronegative study participants), showed a trend towards increased spike-specific IgA in the lung after inhalation of 107 IU MVA-SARS-2-ST. In contrast, inhaled application of MVA-SARS-2-ST robustly induced spike-specific CD4+ and CD8+ T cell responses in the lung in both NCP-seronegative and NCP-seropositive individuals. Conclusions: Collectively, our study demonstrated that a single booster inhalation of 107 IU MVA-SARS-2-ST did not have a relevant impact on the humoral immune response, but induced specific T cell responses in blood and lung. ### Competing Interest Statement Jens M. Hohlfeld reports grants for clinical trial conduct to his institution from Astellas Pharma GmbH, AstraZeneca, Bayer AG, Boehringer Ingelheim Pharma GmbH & Co. KG, Calibr at Scripps Research, Chiesi, CSL Behring, Desitin Arzneimittel GmbH, EpiEndo, F. Hoffmann-La Roche AG, Genentech, Inc., OM Pharma SA, ReAlta Life Sciences, Sanofi-Aventis Deutschland GmbH, and personal fees from Boehringer Ingelheim Pharma GmbH & Co. KG, Celerion, and Cureteq, all of which are outside the submitted work. ### Clinical Trial NCT05226390 ### Funding Statement This study was funded by MWK Niedersachsen (14-76103-184 CORONA-11/20); DZIF (TTU 01.934 and FF 01.941); DFG (EXC 2155 RESIST); DZL (grant 82DZL002B1). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of Hannover Medical School gave ethical approval for this work (no. 10012\_AMG\_mono_2021). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the corresponding author.

Rationale: Segmental challenge with lipopolysaccharide (LPS) is a well-established method to safely induce transient toll-like receptor 4-driven airway inflammation in human lungs. Molecular and cellular changes are typically captured in bronchoalveolar lavage (BAL), which allows to unravel and investigate pulmonary inflammatory mechanisms as well as the efficacy of investigational drugs. Aim of this work was to investigate the change of human BAL proteome following segmental LPS challenge. Methods: Pre-challenge BAL was collected from ten healthy non-smoking participants, followed by segmental instillation of LPS (40 EU/kg) and contralateral saline as control. BAL was sampled from the challenged lung segments 24 hours after instillation. 1,500 selected proteins were assayed using the SomaScan platform and analyzed when found within limits of detection. Results: As expected, there were no significant differences between pre-challenge BAL and BAL collected after saline challenge. After challenge with LPS compared with saline, 599 proteins were significantly upregulated and 4 proteins (ALDH2, FGFR3, ACP5, ADGRE2) were downregulated. Upregulated proteins were e.g. IL-6, IL-8, MPO, MMP9, VWF, SELL or G-CSF. The three most significant biological processes associated with upregulated proteins were “immune system process”, “response to external stimulus” and “response to chemical”. Conclusion: Segmental LPS challenge in the human lung induced broad upregulation of pro-inflammatory proteins. This study provides a comprehensive analysis of the proteomic profile in BAL following LPS challenge and therefore contributes to a more in-depth characterization and understanding of the LPS challenge model.

Background: House dust mite (HDM) allergies are prevalent, yet current treatments like allergen avoidance, pharmacotherapy, and conventional allergen immunotherapy present limitations. The novel LAMP (lysosomal-associated membrane protein)-based DNA vaccine ASP2390 targets major HDM allergens, potentially shifting immune responses toward nonallergic pathways and minimizing the risk of atopy, with positive safety and efficacy signals in preclinical models. Objective: We evaluated the safety, tolerability, and efficacy of first-in-human intradermal ASP2390 in adults with HDM allergy. Methods: A randomized, double-blind, placebo-controlled phase 1 trial was conducted in adults with HDM-induced allergic rhinitis. Participants received either 1 mg or 4 mg of ASP2390 or placebo intradermally once weekly for 12 weeks, with safety, tolerability, and pharmacodynamic responses assessed over a 63-week period, including early-phase clinical effects assessed via HDM exposure in an allergen challenge chamber. Results: Twenty-eight adults (mean age, 26.9 years; 23 male participants), with 7 receiving 1 mg and 13 receiving 4 mg ASP2390, 8 receiving placebo, showed no serious adverse events or withdrawals due to treatment-emergent adverse events. The most common events were nasopharyngitis, coronavirus disease 2019, headache, fatigue, and diarrhea; fatigue and headache were the most frequent systemic reactions, and injection-site tenderness the most frequent local reaction. There were no substantial changes in allergen-specific immunoglobulin levels, basophil activation, or T helper cell subpopulations, and no difference in allergic clinical responses compared to placebo. Conclusion: Intradermal DNA vaccine ASP2390 is safe and well tolerated but does not show an immunologic or clinical response in a small sample of adults allergic to HDM.

Existing parenteral SARS-CoV-2 vaccines protect against severe disease but do not reliably prevent infection or reinfection. Inhaled vaccines may elicit localized immunity in the respiratory tract, a principal entry site for SARS-CoV-2. In this investigator-initiated, single-center, open-label phase 1 trial ( NCT05226390 , ClinicalTrials.gov ), 23 healthy adults previously immunized with EU-approved SARS-CoV-2 vaccines received a single inhaled dose of Modified Vaccinia virus Ankara-(MVA)-SARS-2-ST (1×10 7 IU), engineered to express a prefusion-stabilized SARS-CoV-2 spike. Participants were followed for 140 days. Primary endpoints included solicited local and systemic reactogenicity through day 7, unsolicited adverse events through day 28, serious adverse events throughout, and changes in spirometry and laboratory parameters. Secondary endpoints were changes in SARS-CoV-2 S1-specific IgG in serum and bronchoalveolar lavage; exploratory endpoints included changes in S1-specific IgA in serum and bronchoalveolar lavage, and methacholine responsiveness. No serious adverse events were observed. Over 28 days, mild or moderate adverse events occurred in 87% of participants, predominantly cough, headache, and fatigue, all resolved. Pulmonary function and methacholine responsiveness were stable, except for one transient 20% decrease in FEV1 on Day 14 that normalized subsequently. Serum IgG responses remained minimal, whereas a subset displayed increased bronchoalveolar IgA. A single inhaled booster dose of MVA-SARS-2-ST was safe and generally well tolerated. While systemic antibody levels did not rise substantially, the observed mucosal IgA response in some participants points to a localized mucosal effect. Further studies are warranted to clarify underlying mechanisms and the significance of this response in diverse populations.