Deep immune profiling of chronic rhinosinusitis in allergic and non-allergic cohorts using mass cytometry.

Chronic rhinosinusitis (CRS) is a persistent nasal and paranasal sinus mucosa inflammation comprising two phenotypes, namely CRS with nasal polyps (CRSwNP) and without (CRSsNP). CRSwNP can be associated with asthma and hypersensitivity to non-steroidal anti-inflammatory drug (NSAID) in a syndrome known as NSAID-exacerbated respiratory disease (N-ERD). Furthermore, CRS frequently intertwines with respiratory allergies. This study investigated levels of 33 different nasal and serum cytokines and phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) within cohorts of CRS patients (n = 24), additionally examining the influence of comorbid respiratory allergies by mass cytometry. N-ERD patients showed heightened type 2 nasal cytokine levels. Mass cytometry revealed increased activated naive B cell levels in CRSwNP and N-ERD, while resting naive B cells were higher in CRSsNP. Th2a cell levels were significantly elevated in allergic subjects, but not in CRS groups. In conclusion, there are distinct immunological features in PBMCs of CRS phenotypes and allergy.

Dupilumab increases aspirin tolerance in NSAID-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (N-ERD) comprises the triad of chronic rhinosinusitis with nasal polyps, asthma and intolerance to NSAIDs. Dupilumab treatment, targeting the interleukin-4 (IL-4) receptor α, significantly reduces polyp burden as well as asthma symptoms. Here we aimed to investigate the effect of dupilumab on aspirin intolerance, burden of disease and nasal cytokine profiles in patients with N-ERD. METHODS: In this open-label trial, adult patients with confirmed N-ERD were treated with dupilumab for 6 months. Clinical parameters (e.g. total polyp scores, quality of life questionnaires, smell test, spirometry), oral aspirin provocation testing and blood, nasal and urine sampling were monitored at regular intervals for up to 6 months after starting dupilumab therapy. RESULTS: Of the 31 patients included in the study, 30 completed both aspirin provocation tests. After 6 months of treatment with dupilumab, 23% of patients (n=7 of 30) developed complete aspirin tolerance and an additional 33% of patients (n=10 of 30) tolerated higher doses. Polyp burden was significantly reduced (total polyp score: -2.68±1.84, p<0.001), while pulmonary symptoms (asthma control test: +2.34±3.67, p<0.001) and olfactory performance improved (University of Pennsylvania Smell Identification Test: +11.16±9.54, p<0.001) in all patients after therapy. Patients with increased aspirin tolerance showed a significant decrease in urinary leukotriene E4 levels and their improvement in clinical parameters was associated with a reduction of eotaxin-1, C-C motif chemokine ligand 17, IL-5, IL-17A and IL-6. CONCLUSION: In this study, 57% of N-ERD patients tolerated higher doses of aspirin under dupilumab therapy.

Using mass cytometry for the analysis of samples of the human airways.

Mass cytometry (MC) is a powerful method for mapping complex cellular systems at single-cell levels, based on the detection of cellular proteins. Numerous studies have been performed using human blood, but there is a lack of protocols describing the processing and labeling of bronchoalveolar lavage fluid (BALF) and nasal polyps (NP) for acquisition by MC. These specimens are essential in the investigation of immune cell characteristics in airway diseases such as asthma and chronic rhinosinusitis with NP (CRSwNP). Here we optimized a workflow for processing, labeling, and acquisition of BALF and NP cells by MC. Among three methods tested for NP digestion, combined enzymatic/mechanical processing yielded maximum cell recovery, viability and labeling patterns compared to the other methods. Treatment with DNAse improved sample acquisition by MC. In a final step, we performed a comparison of blood, BALF and NP cell composition using a 31-marker MC antibody panel, revealing expected differences between the different tissue but also heterogeneity among the BALF and NP samples. We here introduce an optimized workflow for the MC analysis of human NP and BALF, which enables comparative analysis of different samples in larger cohorts. A deeper understanding of immune cell characteristics in these samples may guide future researchers and clinicians to a better disease management.

Combination With Tomatidine Improves the Potency of Posaconazole Against Trypanosoma cruzi.

Azoles such as posaconazole (Posa) are highly potent against Trypanosoma cruzi. However, when tested in chronic Chagas disease patients, a high rate of relapse after Posa treatment was observed. It appears that inhibition of T. cruzi cytochrome CYP51, the target of azoles, does not deliver sterile cure in monotherapy. Looking for suitable combination partners of azoles, we have selected a set of inhibitors of sterol and sphingolipid biosynthetic enzymes. A small-scale phenotypic screening was conducted in vitro against the proliferative forms of T. cruzi, extracellular epimastigotes and intracellular amastigotes. Against the intracellular, clinically relevant forms, four out of 15 tested compounds presented higher or equal activity as benznidazole (Bz), with EC50 values ≤2.2 µM. Ro48-8071, an inhibitor of lanosterol synthase (ERG7), and the steroidal alkaloid tomatidine (TH), an inhibitor of C-24 sterol methyltransferase (ERG6), exhibited the highest potency and selectivity indices (SI = 12 and 115, respectively). Both were directed to combinatory assays using fixed-ratio protocols with Posa, Bz, and fexinidazole. The combination of TH with Posa displayed a synergistic profile against amastigotes, with a mean ΣFICI value of 0.2. In vivo assays using an acute mouse model of T. cruzi infection demonstrated lack of antiparasitic activity of TH alone in doses ranging from 0.5 to 5 mg/kg. As observed in vitro, the best combo proportion in vivo was the ratio 3 TH:1 Posa. The combination of Posa at 1.25 mpk plus TH at 3.75 mpk displayed suppression of peak parasitemia of 80% and a survival rate of 60% in the acute infection model, as compared to 20% survival for Posa at 1.25 mpk alone and 40% for Posa at 10 mpk alone. These initial results indicate a potential for the combination of posaconazole with tomatidine against T. cruzi.