Background Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients. Methods A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival. Results In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6–24.0, P = 0.52) and 22.4% (97.5% CI: 17.2–28.3, P < 0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline. Conclusions Tocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline. Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092).

Asthma is a chronic inflammatory disease that is associated with airway remodeling, including hyperplasia of airway epithelial cells and airway smooth muscle cells, and goblet cell differentiation. We wished to address the potential role of histamine, a key biogenic amine involved in allergic reactions, in airway remodeling through the epidermal growth factor receptor (EGFR) pathway. Here, we demonstrate that histamine releases 2 EGFR ligands, amphiregulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF), from airway epithelial cells. Amphiregulin and HB-EGF were expressed in airway epithelium of patients with asthma. Histamine up-regulated their mRNA expression (amphiregulin 3.2-fold, P<0.001; HB-EGF 2.3-fold, P<0.05) and triggered their release (amphiregulin EC(50) 0.50 μM, 31.2 ± 2.7 pg/ml with 10 μM histamine, P<0.01; HB-EGF EC(50) 0.54 μM, 78.5 ± 1.8 pg/ml with 10 μM histamine, P<0.001) compared to vehicle control (amphiregulin 19.3 ± 0.9 pg/ml; HB-EGF 60.2 ± 1.0 pg/ml), in airway epithelial cells. Histamine increased EGFR phosphorylation (2.1-fold by Western blot analysis) and induced goblet cell differentiation (CLCA1 up-regulation by real-time qPCR) in normal human bronchial epithelial (NHBE) cells. Moreover, amphiregulin and HB-EGF caused proliferation and migration of both NHBE cells and human airway smooth muscle cells. These results suggest that histamine may induce airway remodeling via the epithelial-derived EGFR ligands amphiregulin and HB-EGF.

Background: Despite bronchoscopic lung volume reduction (BLVR) with valves is a minimally invasive treatment for emphysema, it can associate with some complications. We aimed at evaluating the rate and type of complications related to valve treatment and their impact on clinical outcomes. Methods: It is a retrospective multicenter study including all consecutive patients with severe heterogeneous emphysema undergoing BLVR with endobronchial valve treatment and developed any complications related to this procedure. The type of complication, the time of onset, the treatment required and the outcome were evaluated. Response to treatment was assessed according to the minimal clinically important difference (MCID) as follows: an improvement of ≥15% in forced expiratory volume in one second (FEV 1); of −8% in residual volume (RV); of ≥26 m in 6-minnute walking distance (6MWD); and of ≥4 points on the St. George's Respiratory Questionnaire (SGRQ). Target lobe volume reduction (TLVR) ≥350 mL was considered significant. Results: One hundred and seven out of 423 (25.3%) treated patients had complications related to valve treatment including pneumothorax (17.3%); pneumonia (1.7%), chronic obstructive pulmonary disease (COPD) exacerbation (0.9%), respiratory failure (1.4%), valve migration (2.1%), and hemoptysis (1.9%). In all cases complications resolved with appropriate treatment including removal of valves in 21/107 cases (19.6%). Patients with TLVR ≥350 mL (n=64) vs. those <350 mL (n=43) had a statistically significant higher improvement in FEV 1 (19.0%±3.9% vs. 3.0%±0.9%; P=0.0003); in RV (−10.0%±4.8% vs. −4.0%±2.9%; P=0.002); in 6MWD (33.0±19.0 vs. 12.0±6.3 metres; P=0.001); and in SGRQ (−15.0±2.9 vs. −8.0±3.5 points; P=0.01). Only patients with TLVR ≥350 mL met or exceeded the MCID cutoff criteria for FEV 1 (19.0%±3.9%), RV (−10.0%±4.8%), 6MWT (33.0±19.0 metres), and SGQR (−15.0±2.9 points). Five patients (1.2%) died during follow-up for causes not related to valves treatment neither to any of the complications described.

Contact between airway smooth muscle (ASM) cells and activated CD4(+) T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4(+) T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4(+) T cells. CD4(+) T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4(+) T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD(+) T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4(+) T cells that is dependent on interaction with a structural cell.

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-β was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.

New formulations of extrafine particles of long-acting beta-2 agonists plus inhaled corticosteroids (LABA + ICS) have been shown to reach peripheral regions of the lung. The aim of the study was to assess the effect on small airway obstruction of long-term treatments with two different LABA + ICS formulations in asthma. Ten subjects with moderate persistent asthma were enrolled. After a 4-week washout period they were treated in a randomized crossover design for 24 weeks with formoterol, 12 micrograms, and beclomethasone, 200 micrograms, hydrofluoroalkane (HFA; by metered-dose inhaler) b.i.d. (FB) or salmeterol, 50 micrograms, and fluticasone, 250 micrograms (by dry-powder inhaler), b.i.d. (SF). At baseline and at the end of each period subjects underwent an Asthma Control Test (ACT) and Pulmonary Function Testing. The N(2) phase III slope and closing volume (CV) during single-breath washout test and difference between the maximal expiratory flow rates with air and heliox at isovolume corresponding to 50% [Delta(heliox-air)MEF(50%)] were measured to assess changes on peripheral airways function. Two subjects dropped out and eight completed the study. After SF and FB, forced expiratory volume at 1 second (FEV(1); p < 0.01) and FEV(1)/forced vital capacity (FVC; p < 0.01 for SF and p < 0.05 for FB) increased when compared with baseline. Although both FB and SF treatments slightly increased delta(heliox-air)MEF(50% isovolume) versus baseline, only after FB the N(2) phase III slope and CV decreased from 1.61 ± 0.61%/L to 1.35 ± 0.49 N(2)%/L (p = 0.054) and from 0.98 ± 0.56 L to 0.88 ± 0.58 L (p < 0.05), respectively. ACT score raised from 19 ± 5 (baseline) to 23 ± 1 after FB (p < 0.02) and 23 ± 2 after SF (p < 0.05). When compared with baseline and in contrast to SF (50/250 micrograms b.i.d.), FB HFA (12/200 micrograms b.i.d.) significantly improved functional parameters reflecting small airway obstruction in asthmatic patients. Registered in the public trial registry at www.ClinicalTrials.gov identifier: NCT01255579.

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.

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