Monica Boita

Clonal CD8+ TCR-Vβ expanded populations with effector memory phenotype in Churg Strauss Syndrome

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

The Expression of TSLP Receptor in Chronic Rhinosinusitis with and without Nasal Polyps

Chronic Rhinosinusitis with or without Nasal Polyps (CRSwNP and CRSsNP) may be characterized by different cytokine profiles. Generally, Th2 cytokines and eosinophilic infiltration have been reported to be more specific of CRSwNP compared to CRSsNP, where neutrophils seem to play a major role. The epithelial cell-derived thymic stromal lymphopoietin (TSLP) has been recently identified as a key factor in Th2-inflammatory response. The aim of this study is to investigate the expression of TSLP Receptor (TSLP R) in surgical specimens obtained from patients affected by CRSwNP (n=10) and CRSsNP (n = 5) by immunohistochemical techniques (immunostaining score, IS). TSLP R expression was significantly higher in the inflammatory infiltrate and in the epithelial cells of CRSwNP, CRSsNP patients compared to the control group (IS 4.5±0.68, 4.4+1.44 and 0.4310.3 respectively, p=0.0024 for inflammatory infiltrate and IS 5.8±0.92, 7.8±2.06 and 0.86±0.55 respectively, p=0.0018 for epithelial cells). No significant difference was observed in IS of inflammatory infiltrate and epithelial cells in CRSwNP compared to CRSsNP. Very low IS for TSLP R was found in connective tissue of all the samples, with no difference among the groups. TSLP receptor is highly expressed in CRS compared to controls and independently from the polyps suggesting an early common inflammatory pathway in the two CRS phenotypes.

Inflammatory cytokines and VEGF measured in exhaled breath condensate are correlated with tumor mass in non-small cell lung cancer

Inflammation mediated by the immune system is known to be important in carcinogenesis and, specifically, T helper 17 cells have been reported to play a role in tumor progression by promoting neo-angiogenesis. The aim of this study was to investigate whether inflammatory cytokines and vascular endothelial growth factor (VEGF) levels in exhaled breath condensate (EBC) and in serum were related to tumor size in patients with non-small cell lung cancer (NSCLC). Il-6, IL-17, TNF-α and VEGF levels were measured in EBC and serum of 15 patients with stage I-IIA NSCLC and in 30 healthy controls by immunoassay. The tumor size was measured by a CT scan. The concentrations of IL-6, IL-17 and VEGF were significantly higher in EBC of patients with lung cancer, compared with controls, while only serum IL-6 concentration was higher in patients compared to controls. A significant correlation (r = 0.78, p = 0.001) was observed between EBC levels of IL-6 and IL-17; IL-17 was also correlated to EBC levels of the VEGF (r = 0.83, p < 0.001) and TNF-α (r = 0.62, p = 0.014). The tumor diameter was significantly correlated with EBC concentrations of VEGF (r = 0.58, p = 0.039), IL-6 (r = 0.67, p = 0.013) and IL-17 (r = 0.66, p = 0.017). Our results show a significant relationship between inflammatory and angiogenic markers, measured in EBC by a non-invasive method, and tumor mass.

The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage.

Predictors of cardiovascular disease in asthma and chronic obstructive pulmonary disease

BackgroundCardiovascular disease (CVD) is a common comorbidity in patients with chronic airway obstruction, and is associated with systemic inflammation and airway obstruction. The aim of this study was to evaluate the predictors of CVD in two different conditions causing chronic airway obstruction, asthma and COPD.MethodsLung function tests, clinical and echocardiographic data were assessed in 229 consecutive patients, 100 with asthma and 129 with COPD. CVD was classified into: pressure overload (PO) and volume overload (VO). Sub-analysis of patients with ischemic heart disease (IHD) and pulmonary hypertension (PH) was also performed.ResultsCVD was found in 185 patients (81%: 51% COPD and 30% asthmatics) and consisted of PO in 42% and of VO in 38% patients. COPD patients, as compared to asthmatics, had older age, more severe airway obstruction, higher prevalence of males, of smokers, and of CVD (91% vs 68%), either PO (46% vs 38%) or VO (45% vs 30%). CVD was associated with older age and more severe airway obstruction both in asthma and COPD. In the overall patients the predictive factors of CVD were age, COPD, and male sex; those of PO were COPD, BMI, VC, FEV1 and MEF50 and those of VO were age, VC and MEF50. In asthma, the predictors of CVD were VC, FEV1, FEV1 /VC%, and PaO2, those of PO were VC, FEV1 and FEV1 /VC%, while for VO there was no predictor. In COPD the predictors of CVD were age, GOLD class and sex, those of VO age, VC and MEF50, and that of PO was BMI. Sub-analysis showed that IHD was predicted by COPD, age, BMI and FEV1, while PH (found only in 25 COPD patients), was predicted by VO (present in 80% of the patients) and FEV1. In subjects aged 65 years or more the prevalence of CVD, PO and VO was similar in asthmatic and COPD patients, but COPD patients had higher prevalence of males, smokers, IHD, PH, lower FEV1 and higher CRP.ConclusionsThe results of this study indicate that cardiovascular diseases are frequent in patients with chronic obstructive disorders, particularly in COPD patients. The strongest predictors of CVD are age and airway obstruction. COPD patients have higher prevalence of ischemic heart disease and pulmonary hypertension. In the elderly the prevalence of PO and VO in asthma and COPD patients is similar.

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

Introduction Bio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs). Methods One-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted. Results RNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively). Conclusion RNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

Eosinophilic inflammation of chronic rhinosinusitis with nasal polyps is related to OX40 ligand expression

The aims of this study were to investigate OX40 ligand expression in sinus tissue from patients with nasal polyposis compared with patients with chronic rhinosinusitis without nasal polyps (NPs), and to determine if OX40 ligand expression is related to eosinophilic sinus infiltration. Twenty patients with chronic rhinosinusitis (11 with and nine without NPs) and seven controls were enrolled in the study. The mRNA expression of OX40 ligand and thymic stromal lymphopoietin and its receptor were analyzed. The immunoreactivity score for OX40 ligand and the eosinophil count were obtained. The mRNA expression and immunoreactivity score of OX40 ligand were higher in patients with nasal polyposis than in patients without NPs, as well as healthy controls. The mRNA expression of thymic stromal lymphopoietin and its receptor was significantly higher in nasal polyposis than in the control, but not significantly higher than in chronic rhinosinusitis without NPs. A correlation between the number of OX40 ligand-positive cells and the number of eosinophils in sinus biopsies was found only in patients with nasal polyposis. In conclusion, the thymic stromal lymphopoietin/OX40 ligand axis is up-regulated in nasal polyposis and is related to the intensity of eosinophilic inflammation.

Release of Type 2 Cytokines by Epithelial Cells of Nasal Polyps

Background. T2 inflammation of chronic rhinosinusitis with nasal polyps (CRSwNP) may be influenced by epithelial cytokines release (TSLP, IL-25, and IL-33). We investigated the release of TSLP, IL-25, and IL-33 by epithelial CRSwNP cells compared to epithelial sinus mucosa cells of patients with chronic rhinosinusitis without nasal polyps (CRSsNP). Methods. IL-25, IL-33, and TSLP were measured by ELISA in the supernatant of cell cultures derived by CRSwNP (9 patients, 6 atopic) and CRSsNP (7 patients, 2 atopic) in baseline condition and following stimulation with Dermatophagoides pteronyssinus (DP), Aspergillus fumigatus (AF), and poly(I:C). Results. CRSwNP epithelial cells released increased levels of IL-25 (from 0.12 ± 0.06 pg/ml to 0.27 ± 0.1 pg/ml, p < 0.01) and TSLP (from 0.77 ± 0.5 pg/ml to 2.53 ± 1.17 pg/ml, p < 0.001) following poly(I:C) stimulation, while CRSsNP epithelial cells released increased levels of IL-25 and IL-33 following AF and DP stimulation, respectively (IL-25: from 0.18 ± 0.07 pg/ml to 0.51 ± 0.1 pg/ml, p < 0.001; IL-33: from 2.57 ± 1.3 pg/ml to 5.7 ± 3.1 pg/ml, p < 0.001). Conclusions. CRSwNP epithelial cells release TSLP and IL-25 when stimulated by poly(I:C) but not by DP or AF, suggesting that viral infection may contribute to maintain and amplify the T2 immune response seen in CRSwNP.

Mechanism of phosphatidylserine inhibition of IgE/FcεRI-dependent anaphylactic human basophil degranulation via CD300a.

To the Editor: The process that leads to histamine release from human basophils constitutes a continuum that starts with piecemeal degranulation and eventually converts to anaphylactic degranulation (AND). These degranulation events are accompanied by mobilization of distinct cellular compartments and expression of surface molecules that are not or barely expressed on resting basophils. Two different histamine-containing compartments, which show distinct early signaling requirements and kinetics, have been described, namely, the CD203c compartment and the CD63 compartment. The appearance of CD63 is invariably associated with AND.Mechanisms that regulate basophil activation likely relate to activation conditions (IgE/FcεRI-dependent and -independent), mode of degranulation (piecemeal degranulation vs AND), and changes in functional properties of inhibitory receptors. The inhibitory receptor CD300a (IRp60) is constitutively expressed on human basophils and rapidly upregulated in response to IgE/FcεRI cross-linking; its engagement selectively suppresses AND without a significant effect on CD203c. Moreover, recently CD300a has been shown to be a receptor for phosphatidylserine (PS) and phosphatidylethanolamine expressed on the membrane of apoptotic cells. This study aimed at investigating the effect of PS recognition by CD300a on IgE/FcεRI-dependent basophilic activation. Three experimental models addressed (1) the effect of apoptotic K562 cells on IgE/FcεRI-mediated activation of basophils in whole blood, (2) the putative role of PS, and finally (3) the effect of the PS-binding molecule Annexin Vand CD300a-blocking antibody on inhibitory signals delivered by apoptotic PBMCs on separated basophils, excluding interference of other cells or humoral factors. For detailed information on experimental design, see the Methods section in the Online Repository at www.jacionline.org. Aliquots of whole blood from 5 patients and 5 controls were preincubated with serial dilutions of viable or apoptotic K562 cells (from 20 3 10/mL to 20 3 10/mL; 378C, 30 minutes) before stimulation with buffer (negative control) or with antiIgE (positive control) and, in patients, rBet v 1, the recombinant major allergen from birch (Betula verrucosa) pollen (for detailed information, see the Methods section). This resulted in a significant dose-dependent inhibition of CD63 upregulation (Fig 1, A) that was more pronounced in healthy subjects than in allergic patients (Fig 1, B; P < .05). Similar results were obtained with patients’ basophils stimulated with rBet v 1 (Fig 1, C). There was no significant effect of viable or apoptotic K562 on the spontaneous expression of CD63 and CD203c on basophils (data not shown). Fluorescein-stained liposomes, noncoated or coated with PS or phosphorylcholine, were added to magnetic bead–isolated basophils of 2 healthy donors (48C, 20minutes) and immediately assessed by flow cytometry (for detailed information about liposomes preparation, see the Methods section). Next, the functional effect of noncoated and PSand phosphorylcholine-coated liposomes was assessed by preincubating different concentrations of nonstained liposomes with isolated basophils at 378C for 30 minutes before anti-IgE stimulation. These experiments revealed that only PS-coated liposomes bind basophilic membranes (Fig 1, D) and inhibit IgE/FcεRI-mediated activation of the cells in a dose-dependent manner, starting with a ratio liposomes/basophil 20/1. As with apoptotic K562 cells, this effect was more pronounced on the CD63 compartment (Fig 1, E; see Fig E1 in this article’s Online Repository at www.jacionline.org). To study the effect of the PS-binding molecule Annexin Vand CD300a-blocking antibody (clone TX49), apoptotic autologous PBMCs (ratio PBMCs/basophil 20/1) were used (n5 3). To bind PS on the membrane of apoptotic PBMCs (aPBMCs), cells were incubated with different concentrations of recombinant human Annexin V at 378C for 30 minutes; thereafter, basophils were added for 30minutes at 378C. Subsequently, cells were stimulated with buffer or anti-IgE. Inversely, to block CD300a receptor on isolated basophils, cells were exposed to different concentrations of the monoclonal anti-human CD300a-blocking antibody TX49 at 378C for 30 minutes before adding aPBMC followed by anti-IgE activation. These experiments showed that blocking CD300a resulted in dose-dependent reversal of the magnitude of inhibition up to 77% (Fig 2, A and C; P 5 .05). Preincubation with anti-CD300a alone did not result in any significant effect on basophil activation markers (data not shown). This is not in contrast with our previous study, in which a CD300a receptor-activating antibody (clone MEM-260) was used, resulting in an inhibition of CD63 upregulation. Similarly, preincubating the cells with recombinant human Annexin V reversed the magnitude of aPBMC-induced inhibition up to 82% (Fig 2, B and D; P 5 .04). Taken together, these results led us to conclude that the interaction between PS and CD300a inhibits IgE/FcεRIdependent anaphylactic basophil degranulation. Nevertheless, the biological or clinical relevance of basophilic response/ inhibition in IgE-mediated hypersensitivity needs to be elucidated. The externalization of PS in the absence of apoptosis as a consequence of granular membrane fusion with the cell membrane involving inversion of membrane leaflet polarity and upregulation of the inhibitory receptor CD300a during basophilic degranulation on a single cell level may contribute to self-terminating mechanisms through cis recognition. Whether the finding of increased apoptosis of monocytes on day 3 of venom immunotherapy suggests the involvement of PS-CD300a interaction on the early suppression of basophils and mast cells is a hypothesis that deserves investigation. Finally, the modulation of basophilic phagocytic activity through CD300a-PS interaction seems difficult to postulate because these cells express low phagocytic activity. In conclusion, we have for the first time demonstrated that the apoptosis-related signal PS suppresses human basophil anaphylactic degranulation via the inhibitory receptor CD300a (IRp60). These data provide interesting input to study the role of phospholipids recognition in modulation of basophilic responses and endorse the role of CD300a as a pharmacologic target for the treatment of allergic disease.

Innate and lymphocytic response of birch-allergic patients before and after sublingual immunotherapy.

Functional imbalance in Th1/Th2 cell response toward allergens is a recognized hallmark of allergic patients and a major role of dendritic cells (DCs) in redirecting T-cell phenotypes after specific immunotherapy has been suggested. This study investigates the proliferative and cytokine responses of T cells cocultured with monocyte-derived DCs (MoDCs) after allergen stimulation in birch-allergic patients compared with controls and investigates whether sublingual immunotherapy (SLIT) could change the DC-driven immune response. T cells were stimulated with the major birch pollen allergen (nBet v1) and MoDCs from eight birch-allergic patients with seasonal allergic rhinitis and eight nonallergic controls. Proliferation and cytokine production were measured before and after one course of SLIT with birch allergoid. Significantly lower levels of proinflammatory (IL-1beta, p = 0.027; IL-6, p = 0.030; TNF-alpha, p = 0.019) and Th1 (interferon gamma, p = 0.032; IL-12, p = 0.05) cytokines were measured in supernatants of T cells and MoDCs cultures from allergic patients compared with nonallergic controls. After SLIT, significant increase in IL-12 (p = 0.039), IL-1beta (p = 0.040), IL-6 (p = 0.041), TNF-α (p = 0.048), and IL-10 (p = 0.048) and significant decrease in IL-13 (p = 0.001) were observed. MoDCs/T-cell cocultures, pulsed with the specific allergen, produced lower quantities of proinflammatory and Th1 cytokines in allergic patients compared with healthy subjects, suggesting an allergen-specific impairment of natural immunity and Th1 immune response. A single course of SLIT was able to enhance allergen-specific innate immunity and to modify lymphocyte response, promoting Th1 and T-cell regulatory activity.