Yael Gernez

A contributive result of open-lung biopsy improves survival in acute respiratory distress syndrome patients.

Objective:The impact of a contributive result of open-lung biopsy on the outcome of patients with acute respiratory distress syndrome (ARDS) has not been extensively investigated. The aim of this study was therefore to determine the rate of contributive open-lung biopsy and whether it improved the prognosis of ARDS patients. Design:Prospective study conducted during an 8-yr period. Setting:A 14-bed medico-surgical intensive care unit and a 12-bed medical intensive care unit from the same hospital. Patients:One hundred open-lung biopsies were performed in 100 patients presenting ARDS. Interventions:Open-lung biopsy was performed after ≥5 days of evolution of ARDS when there was no improvement in the respiratory status despite negative microbiological samples cultures and potential indication for corticosteroid treatment. Measurements and Main Results:Ten patients presented a mechanical complication following open-lung biopsy (two pneumothoraces and eight moderate air leaks). The unique independent factor associated with this complication was the minute ventilation when open-lung biopsy was performed (odds ratio, 1.20; 95% confidence interval, 1.03–1.41; p = .02). Fibrosis was noted in 53 patients but was associated with an infection in 29 of these 53 patients (55%). A contributive result of open-lung biopsy (defined as the addition of a new drug) was noted in 78 patients. Simplified Acute Physiology Score II was the only independent predictive factor of a contributive open-lung biopsy (odds ratio, 0.96; 95% confidence interval, 0.92–0.99; p = .04). Survival was higher in patients with a contributive open-lung biopsy (67%) than in patients in whom open-lung biopsy results did not modify the treatment (14%) (p < .001). The factors predicting survival were a contributive result of open-lung biopsy, female gender, and the Organ System Failures score the day of open-lung biopsy. Conclusions:The present study shows that open-lung biopsy provided a contributive result in 78% of ARDS patients with a negative bronchoalveolar lavage. Survival of ARDS patients improved when open-lung biopsy was contributive.

Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways

Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b+ and CD66b+ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63+ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.

Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs

Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor- and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa- and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTOR-inducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.

Basophil CD203c Levels Are Increased at Baseline and Can Be Used to Monitor Omalizumab Treatment in Subjects with Nut Allergy

Rationale: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. Methods: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. Results: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. Conclusions: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

Immunotherapy for Food Allergy: Are We There Yet?

Current clinical research focuses on food allergen-specific immunotherapy through oral (OIT), sublingual (SLIT), or epicutaneous (EPIT) routes. Immunotherapy relies on the delivery of gradually increasing doses of specific allergens to induce desensitization (defined as temporary antigen hyporesponsiveness that depends on regular food ingestion) and, ultimately, tolerance (defined as the ability to ingest food without symptoms despite prolonged periods of avoidance or irregular intake). Although the majority of the patients treated with OIT achieve desensitization, only a minority achieves tolerance. OIT involves higher maintenance doses of food protein (300 mg-4g) compared with SLIT (2.5-7.5 mg) and EPIT (250-500 mcg). OIT efficacy is higher compared with SLIT, but OIT is associated with higher rate of systemic adverse events compared with SLIT and EPIT. OIT is also associated with a minor risk of eosinophilic esophagitis. Combined treatment of OIT and anti-IgE monoclonal antibody has improved safety but not efficacy compared with OIT alone. Early initiation of peanut OIT in peanut-allergic infants and young children may afford superior efficacy and safety. In this review, we discuss the allergen-specific strategies currently explored for the treatment of food allergy, including immunotherapy with native and heat-modified food proteins. Additional research employs strategies to improve the safety and efficacy of allergen immunotherapy through modifications of allergen structure and addition of immunomodulatory adjuvants.

Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens

INTRODUCTION Fifteen to sixty percent of cystic fibrosis patients harbor Aspergillus fumigatus (Af) in their airways (CF-AC) and some will develop allergic bronchopulmonary aspergillosis (CF-ABPA). Since basophils play a key role in allergy, we hypothesized that they would display alterations in CF-ABPA patients compared to CF-AC or patients without Af colonization (CF). METHODS Using flow cytometry, we measured CD203c, CD63 and CD123 levels on basophils from CF-ABPA (N=11), CF-AC (N=14), and CF (N=12) patients before and after ex vivo stimulation with Af allergens. RESULTS Baseline CD203c was increased in basophils from CF-ABPA compared to CF-AC and CF patients. Af extract and recombinant Aspf1 stimulated basophils from CF-ABPA patients to markedly upregulate CD203c, along with modest upregulation of CD63 and a CD123 downward trend. Plasma TARC/CCL17 at baseline and post-stimulation cell supernatant histamine levels were similar in the three groups. CONCLUSIONS In CF-ABPA, blood basophils are primed and hyperresponsive to Af allergen stimulation.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis

BACKGROUND Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

Blood basophil activation is a reliable biomarker of allergic bronchopulmonary aspergillosis in cystic fibrosis

The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA. We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6 months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort). Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus. Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF. Blood basophil surface CD203c level is a method to diagnose ABPA in CF, and study phenotypes, therapy and management http://ow.ly/Sc8zr

Increased HLA-DR Expression on Tissue Eosinophils in Eosinophilic Esophagitis

Objective: The aim of the study was to investigate whether eosinophils have increased human leukocyte antigen (HLA)-DR expression in subjects with eosinophilic esophagitis (EoE) compared with controls. Patients and Methods: Patients who were undergoing an upper endoscopy with biopsies for suspected gastroesophageal reflux disease (GERD) or EoE at Lucile Packard Childrens Hospital were enrolled. In total, the blood and tissue samples of 10 healthy controls (HC), 11 subjects with GERD, and 10 with EoE were studied. Multiple tissue staining to identify eosinophils (via eosinophil cationic protein-clone EG2) and major histocompatibility complex class II cell surface receptors (via HLA-DR) was performed via immunohistochemistry. The peripheral blood was analyzed using flow cytometry to detect eosinophil HLA-DR expression among these subjects. Results: In the tissue, a greater proportion of eosinophils expressed HLA-DR among the subjects with EoE (mean 0.83 ± 0.14, n = 9) relative to those with GERD (mean 0.18 ± 0.19, n = 8, P < 0.01) and HC (mean 0.18 ± 0.13, n = 6, P < 0.01). In total, 6 participants (4 HC subjects and 2 subjects with GERD) did not have any eosinophils identified on tissue staining and were unable to be included in the present statistical analysis. In the blood, there was no statistically significant difference in eosinophil HLA-DR expression among HC subjects (mean 415 ± 217, n = 6), subjects with GERD (mean 507 ± 429, n = 2), and those with EoE (mean 334 ± 181, n = 6). Conclusions: These data demonstrate that the eosinophils from the esophagus of subjects with EoE have increased HLA-DR expression within this tissue.

Immunophenotyping of Peripheral Eosinophils Demonstrates Activation in Eosinophilic Esophagitis

Background and Aim: Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder characterized by upper gastrointestinal symptoms and the presence of high numbers of eosinophils in the esophagus. Although eosinophils in the esophagus have been found to be activated in subjects with EoE, detailed studies of intracellular signaling pathways involved in the mechanism of activation of eosinophils in EoE have heretofore been limited. The aim of the study was to assess whether any surface molecules or transcription factors are activated in peripheral eosinophils in subjects with EoE. Methods: Eosinophils and CD3+ lymphocytes were identified directly from 50 μL of whole blood of EoE and control subjects. Using Hi-FACS, levels of surface activation markers, including CD66b, and intracellular phosphoepitopes, including phosphorylated forms of signal transducer and activator of transcription (phospho-STAT) 1 and 6, were measured within each cell subset. Results: Levels of surface CD66b as well as levels of intracellular phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were significantly higher for untreated subjects with EoE vs healthy controls (P < 0.05). Levels of phospho-STAT1 and phospho-STAT6 in peripheral blood eosinophils were lower in subjects with EoE on therapy versus untreated subjects with EoE (P < 0.05). Conclusions: Levels of phospho-STAT1 and phospho-STAT6, transcription factors involved in inflammatory processes, were both significantly higher in peripheral eosinophils from untreated (ie, newly diagnosed) subjects with EoE versus subjects with EoE on therapy, healthy controls. Blood-based measurements of CD66b and phospho-STAT levels in peripheral eosinophils may be beneficial for identifying EoE.