Search

Supplementary Video S1: PF-228's effect on NCI-446 SCLC cell line migration. NCI-H446 cell lines were grown to confluence in 12-well plates. Cell monolayers were wounded using a micropipette tip and floating cells were washed off with PBS. After overnight incubation, PF-228 at a concentration of 3µM or DMSO was added to culture medium. Cell movements within wounded area were monitored for 12h starting from the time drug was added using a Zeiss Axiovert 200M microscope (Zeiss, Thornwood, NY) at x200 magnification. Images were captured every 15-minutes from five different fields randomly selected in each well. They show that PF-228 decreased migration velocity in NCI-H446 cell lines.

Supplementary Figure S1: Western blot densitometric quantifications. Supplementary Figure S2: FAK Inhibitor 14 (Inh14) and PF-562,271 (PF-271)'s effects on FAK expression/activity, cell proliferation, and apoptosis in NCI-H446 SCLC cell lines. Supplementary Figure S3: PF-573,228 (PF-228)'s effects on FAK expression/activity, cell proliferation, and apoptosis in NCI-H82 SCLC cell lines transduced with FAK shRNA. Supplementary Figure S4: phospho-Paxillin expression in NCI-H446 SCLC cell line where FAK has been inhibited with pharmacological and genetic methods.

Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non–receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1–5 μmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2–M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.

BackgroundChronic obstructive pulmonary disease (COPD) is a devastating lung disease, representing the third cause of mortality worldwide. In COPD, the bronchial epithelium displays several structural and functional abnormalities affecting barrier integrity, cell polarity, and differentiation, as well as epithelial-to-mesenchymal transition (EMT) and inflammation. Although COPD is currently considered an irreversible disease, the (ir)reversible nature of epithelium changes ex vivo remains poorly known.MethodsThe persistence of COPD epithelial abnormalities was addressed in very long-term (10 weeks) primary cultures of air/liquid interface (ALI)-reconstituted airway epithelium from non-smoker controls, smoker controls, and COPD patients. The role of inflammation was also explored by stimulating ALI cultures with a cytokine mix consisting of TNF-α, IL-6 and IL-1β. Finally, the cellular niche holding epithelial memory was studied by exploiting a single cell RNA-sequencing database.ResultsAlmost all epithelial defects (barrier dysfunction, impaired polarity, lineage abnormalities) observed in smokers and COPD patients persisted in vitro up to week 10, except IL-8/CXCL-8 release and EMT which declined over time. Cytokine treatment induced COPD-like changes and reactivated EMT in COPD cells. Progenitor cells of large and small airways, namely basal and club cells, exhibited EMT-related signatures reminiscent of features observed in situ.ConclusionsThe airway epithelium from smokers and COPD patients displays a memory of its native state and previous injuries by cigarette smoking, which is multidimensional and sustained for years. This memory probably resides in progenitor cells of the airway epithelium.

Background In cystic fibrosis, the respiratory epithelium is the target tissue of both the genetic abnormality of the disease and of external aggressions, notably by pathogens (Pseudomonas aeruginosa). A detailed characterisation of the cystic fibrosis bronchial epithelium is however lacking, as most previous studies focused on the nasal epithelium or on cell lines. This study aimed to characterise the abnormal phenotype and epithelial-to-mesenchymal transition in cystic fibrosis bronchial epithelium and to evaluate in cell cultures whether abnormalities persist ex vivo. Methods Explant lung tissues (n = 44) were assessed for bronchial epithelial cell phenotyping by immunostaining. Human bronchial epithelial cells were derived from basal cells isolated from cystic fibrosis patients or control donors and cultured in air-liquid interface for 2, 4 or 6 weeks. Results Enhanced mucin 5AC and decreased β-tubulin expression were observed in cystic fibrosis airways reflecting a decreased ciliated/goblet cell ratio, associated with increased number of vimentin-positive cells, indicating epithelial-to-mesenchymal transition process. These features were recapitulated in vitro, in cystic fibrosis-derived reconstituted epithelium. However, they were not induced by CFTR inhibition or Pseudomonas infection, and most abnormalities tended to disappear in long-term culture (6 weeks) except for increased fibronectin release, an epithelial-to-mesenchymal transition marker. Conclusions This study provides new insights into airway epithelial changes in cystic fibrosis, which are imprinted through an acquired mechanism that we could not relate to CFTR function.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, mainly due to cigarette smoking, which represents the third cause of mortality worldwide. The mechanisms driving its epithelial salient features remain largely elusive. We aimed to evaluate the activation and the role of the canonical, β-catenin-dependant WNT pathway in the airway epithelium from COPD patients. METHODS: The WNT/β-catenin pathway was first assessed by WNT-targeted RNA sequencing of the air/liquid interface-reconstituted bronchial epithelium from COPD and control patients. Airway expression of total and active β-catenin was assessed in lung sections, as well as WNT components in laser-microdissected airway epithelium. Finally, we evaluated the role of WNT at the bronchial epithelial level by modulating the pathway in the reconstituted COPD epithelium. FINDINGS: We show that the WNT/β-catenin pathway is upregulated in the COPD airway epithelium as compared with that of non-smokers and control smokers, in targeted RNA-sequencing of in vitro reconstituted airway epithelium, and in situ in lung tissue and laser-microdissected epithelium. Extrinsic activation of this pathway in COPD-derived airway epithelium inhibited epithelial differentiation, polarity and barrier function, and induced TGF-β-related epithelial-to-mesenchymal transition (EMT). Conversely, canonical WNT inhibition increased ciliated cell numbers, epithelial polarity and barrier function, whilst inhibiting EMT, thus reversing COPD features. INTERPRETATION: In conclusion, the aberrant reactivation of the canonical WNT pathway in the adult airway epithelium recapitulates the diseased phenotype observed in COPD patients, suggesting that this pathway or its downstream effectors could represent a future therapeutic target. FUNDING: This study was supported by the Fondation Mont-Godinne, the FNRS and the WELBIO. ispartof: EBIOMEDICINE vol:61 ispartof: location:Netherlands status: published

Introduction: Secretion of immunoglobulin A (IgA) constitutes one of the frontline protection of the lung against inhaled pathogens. IgA is actively transported from the basolateral pole up to the apical pole of epithelial cells by the polymeric Immunoglobulin Receptor (pIgR), where a cleavage releases IgA bound to the extracellular part of the receptor called secretory component (SC). Little is known regarding IgA/pIgR regulation in the acute diseased lung. In particular, the regulation of the IgA system by hypoxia is unexplored. Methods: The effects of hypoxia on broncho-epithelial cells were studied in a model of human respiratory epithelium reconstituted in vitro upon culture in air/liquid interface (ALI). After full differentiation, the cultured bronchial epithelia were submitted to 1% O2 in an hypoxic chamber for 12 days, upon resting or stimulated (IL-1β, IL-6, TNFα) conditions. Results: In fully ALI-differentiated cultures from 15 control subjects, hypoxia induced a significant decrease of pIgR expression (Western Blot, p=0.0017), SC release in the apical washes (ELISA, 620 ± 797 ng/ml vs 4145 ± 4178 ng/ml) and pIgR mRNA expression (qPCR, p=0.031), irrespectively of inflammatory cytokines. In addition, hypoxia also induced a dedifferentiation as evidenced by cell morphology and epithelial-to-mesenchymal transition (EMT) signatures, including decreased E-cadherin expression (WB, p=0.016). In addition, pharmacological stabilization of HIF-1α recapitulated these features. Conclusion: In a model of reconstituted human airway epithelium, hypoxia alters pIgR expression and cell differentiation, probably through the HIF-1α pathway.

Rationale: Asthma is associated with increased lung IgE production, but whether the secretory IgA system is affected in this disease remains unknown.Objectives: We explored mucosal IgA transport in...

Contexte La carotte (Apiaceae) est responsable de syndromes allergiques oraux (SAO) chez les patients sensibilises aux pollens de bouleau et/ou armoise. Pourquoi certains patients developpent-ils une reaction severe ? Methode Quatorze patients atteints de pollinose ont ete repartis en trois groupes : (I) 7 avec une anaphylaxie a l’ingestion de carotte, (II) 3 ayant un SAO a la carotte et (III) 4 tolerent la carotte. Realisation d’une anamnese standardisee, tests cutanes (pneumallergenes, tests natifs carotte crue et cuite et autres Apiaceae), spIgE (Immunocap® : rBet v 1, rBet v 2, rBet v 6, nArt v 1, nArt v 3, CCD MUX F3, rPhl p 4, rPhl p 5b), Western Blots aux extraits carotte crue et cuite, armoise, bouleau, fleole, puis tests d’inhibitions par rBet v1, extrait bouleau et armoise. Resultats Douze patients sont co-sensibilises bouleau et graminees (avec PR-10 prot a l’avant plan). Dans le groupe (I), 1 est mono sensibilise aux graminees et 1 a l’armoise. Dans le groupe (I), 6/7 patients ont des cofacteurs (effort, alcool…). Les Western Blots a la carotte cuite montrent une immunofixation entre 16–19 kDa chez les patients 3, 7 du groupe I et 9 du groupe II, avec une inhibition par rBet v1 menant a son extinction ± complete. Une bande ∼40 kDa pour les extraits carotte est retrouvee chez les patients 1,3,4,5 du groupe I et patient 8 du groupe 2, bande non inhibee par l’extrait bouleau et armoise. Enfin, fixation de proteines de hauts poids moleculaires, de repartition homogene au sein des 3 groupes, probablement responsables de reactions croisees multiples de type CCD. Conclusion Ce travail laisse d’une part suspecter une resistance plus elevee de la PR-10 de la carotte (Dau c1) avec possible repercussion sur la clinique, bien que l’ensemble des reactions severes ne puissent lui etre imputees. D’autre part, identification d’une proteine ∼40 kDa de nature inconnue, avec une implication clinique potentielle vu sa repartition non homogene. Enfin, le role de cofacteur lors d’une reaction severe est retrouve.

In COPD, epithelial changes are prominent features in the airways, such as goblet cell hyperplasia and squamous metaplasia. In contrast, it remains unclear whether ciliated cells are reduced and which pathways dysregulate epithelial differentiation. We hypothesized that bronchial epithelial cell lineage specification is dysregulated in COPD because of an aberrant reprogramming through transforming growth factor (TGF)-β1. Surgical lung tissue from 81 COPD and 61 control (smokers and non-smokers) patients was assessed for bronchial epithelial cell phenotyping by immunohistochemistry, both in situ and in vitro in reconstituted air-liquid interface (ALI) cultures. The role of TGF-β1 was studied in vitro. COPD epithelium in large airways, when compared to controls, showed decreased β-tubulin IV + ciliated cells (4.4%, 2.5–8.8% versus 8.5%, 6.3–11.8% of surface staining, median and IQR, p = 0.0009) and increased MUC5AC + goblet cells (34.8%, 24.4–41.9% versus 10.3%, 5.1–17.6%, p in vitro, at least in part through TGF-β1.

Rationale: The airway epithelium (AE) in chronic obstructive pulmonary disease (COPD) displays phenotypic changes including inflammation and epithelial-to-mesenchymal transition (EMT). We addressed whether STAT3, previously linked to both inflammation and EMT, could be activated and mediate EMT in COPD-AE. Methods: AE reconstituted in air-liquid interface (ALI) cultures of human tissue from COPD patients versus (non-)smoker controls, recapitulating the abnormal phenotype observed in situ, were assessed up to 10 weeks for inflammation (IL-8/CXCL-8), EMT (fibronectin, vimentin) and STAT3 activation. Results: We observed increased IL-8/CXCL-8 production (at 1, 2 and 4 weeks) by smoker controls and COPD AE. Similarly, vimentin expression (at 1, 2 and 5 weeks) and fibronectin release (at 1, 2 and 4 weeks) were increased in COPD AEs compared with non-smoker controls. Upregulation of IL-8/CXCL-8, vimentin and fibronectin disappeared at 7 or 8 weeks. STAT3 phosphorylation (Tyr-705) was increased in COPD AEs at 5 weeks (Fig. 1), but no more at 8 weeks. Conclusions: These data show that COPD airway epithelial cells display increased STAT3 activation in vitro, that is concomitant with persistent inflammatory (IL-8/CXCL-8) and EMT features (vimentin, fibronectin) at 5 weeks, and which all regressed at later time-points. Whether STAT3 links inflammation and EMT will be further investigated in this setting.

RATIONALE: Accumulation of B cells and lymphoid follicles (LFs) has been described in chronic obstructive pulmonary disease (COPD) airways, but the functional status of lung B cells remains poorly known. OBJECTIVES: To characterize LFs for expression of IgA, the main mucosal antibody. METHODS: The presence of B cells and LFs, including intrafollicular IgA expression, were determined in the lung from patients with COPD (n = 37) versus control subjects (n = 34) by immunohistochemistry. We also evaluated follicular IgA responses in the lungs from mice infected with Pseudomonas aeruginosa (PAO1) (n = 10 per group) and in smoking mice. MEASUREMENTS AND MAIN RESULTS: Whereas in smokers B-cell numbers slightly increased, robust increases in B-cell and LF numbers (mainly in distal airways) were only observed in severe COPD. Most follicular B cells were IgM+ (70-80%), but IgA+ (and not IgG+) B-cell numbers were increased in LFs from severe COPD compared with control subjects (twofold, 44.7% vs. 25.2%), and this was significant in distal but not proximal airways. Follicular IgA response was also observed in PAO1-infected mouse lungs, but not after smoke exposure. Moreover, follicular IgA expression associated with expression of IL-21, which was very potent to activate immunoglobulin production in vitro. CONCLUSIONS: This study shows that IgA production occurs in peribronchiolar LFs from severe COPD, where IL-21-producing T cells are present, and presumably represents a feature of exacerbated mucosal adaptive immune responses against microbial and/or self-antigens. ispartof: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE vol:199 issue:5 pages:592-602 ispartof: location:United States status: published

To compare the effects of He/O(2) and external PEEP (PEEPe) on intrinsic PEEP (PEEPi), respiratory mechanics, gas exchange, and ventilation/perfusion (V(A)/Q) in mechanically ventilated COPD patients.Prospective, interventional study in the intensive care unit of a university hospital.Ten intubated, sedated, paralyzed, mechanically ventilated COPD patients studied in the following conditions: (a) baseline settings made by clinician in charge, air/O(2), ZEEP; (b) He/O(2), ZEEP; (c) air/O(2), ZEEP; (d) air/O(2), PEEPe 80% of PEEPi. Measurements at each condition included V(A)/Q by the multiple inert gas elimination technique (MIGET).PEEPi and trapped gas volume were comparably reduced by He/O(2) (4.2+/-4 vs. 7.7+/-4 cmH(2)O and 98+/-82 vs. 217+/-124 ml, respectively) and PEEPe (4.4+/-1.3 vs. 7.8+/-3.6 cmH(2)O and 120+/-107 vs. 216+/-115 ml, respectively). He/O(2) reduced inspiratory and expiratory respiratory system resistance (15.5+/-4.4 vs. 20.7+/-6.9 and 19+/-9 vs. 28.8+/-15 cmH(2)O l(-1)s(-1), respectively) and plateau pressure (13+/-4 vs. 17+/-6 cmH(2)O). PEEPe increased airway pressures, including total PEEP, and elastance. PaO(2)/FIO(2) was slightly reduced by He/O(2) (225+/-83 vs. 245+/-82) without significant V(A)/Q change.He/O(2) and PEEPe comparably reduced PEEPi and trapped gas volume. However, He/O(2) decreased airway resistance and intrathoracic pressures, at a small cost in arterial oxygenation. He/O(2) could offer an attractive option in COPD patients with PEEPi/dynamic hyperinflation.

Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non–receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1–5 μmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2–M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.

Airway inflammation is a constant feature in chronic obstructive pulmonary disease (COPD) but its role in driving abnormal broncho-epithelial phenotype remains unclear. We aimed to assess the contribution of epithelial inflammation to functional and structural changes of the airway epithelium (AE), including epithelial-to-mesenchymal transition (EMT). Methods: AE was reconstituted in air-liquid interface (ALI) cultures of human primary broncho-epithelial cells from COPD (n=12) versus control (n=12) subjects, recapitulating the abnormal phenotype in situ. The AE was treated by a cytokine mix (IL-1β, IL-6, TNF-α, 5ng/ml) up to 5 weeks, before assessing functional (transepithelial electric resistance (TER)), inflammatory (IL-8/CXCL-8 and IL-1α production), and differentiation features (polymeric immunoglobulin receptor (pIgR), secretory component (SC)), including EMT (fibronectin, vimentin). Results: Inflammatory conditioning (as compared to control) induced decreases in TER (2,787±280 vs 852±75 Ω/cm², mean±SEM of 24 experiments at 4wks, p

The bronchial epithelium in chronic obstructive pulmonary disease (COPD) displays hyperplastic goblet- and basal cells, decreased ciliated cells and epithelial-to-mesenchymal transition (EMT) features. Whether these features are irreversible is incompletely understood. We aimed to delineate the plasticity of the abnormal broncho-epithelial phenotype in COPD. Methods: The airway epithelium (AE) reconstituted in air-liquid interface (ALI) cultures of human primary broncho-epithelial cells from COPD (vs controls), recapitulating the abnormal phenotype observed in situ, was assessed up to 10 weeks for functional (transepithelial electric resistance (TER)), inflammatory (IL-8/CXCL-8 and IL-1α production) and differentiation features including EMT (fibronectin, vimentin). Results: COPD AE show a persistent decrease in TER versus controls (p

Despite their relevance to mucosal defense, production of IgA and the function of lung B-cells remain unknown in chronic obstructive pulmonary disease (COPD).We assessed IgA synthesis in the lungs of COPD (n=28) and control (n=21) patients, and regulation of B-cells co-cultured within vitro-reconstituted airway epithelium.In COPD lung tissue, synthesis of IgA1 was increased, which led to its accumulation in subepithelial areas.In vitro, the COPD bronchial epithelium imprinted normal human B-cells for increased production of IgA (mainly IgA1) and maturation into CD38+plasma cells. These effects were associated with upregulation of TACI (transmembrane activator and CAML interactor) and were observed under resting conditions, while being partly inhibited upon stimulation with cigarette smoke extract. Interleukin (IL)-6 and BAFF (B-cell activating factor)/APRIL (a proliferation-inducing ligand) were upregulated in the COPD epithelium and lung tissue, respectively; the IgA-promoting effect of the COPD bronchial epithelium was inhibited by targeting IL-6 and, to a lower extent, by blocking TACI.These data show that in COPD, the bronchial epithelium imprints B-cells with signals promoting maturation into IgA-producing plasma cells through the action of two epithelial/B-cell axes, namely the IL-6/IL-6 receptor and BAFF-APRIL/TACI pathways, while cigarette smoke partly counteracts this IgA-promoting effect.

Introduction: Immunoglobulin (Ig) A protects mucosal surfaces from inhaled pathogens and antigens. While serum and sputum IgA are increased, it remains unknown whether polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of IgA is affected in cystic fibrosis (CF). Aims: We wondered whether broncho-epithelial pIgR-mediated transport of IgA is altered and could contribute to lung infection by Pseudomonas aeruginosa (PA). Methods: Lung tissue from 19 CF patients, as compared to 17 controls, was assayed for pIgR immunostaining. Sputum samples from 49 CF patients and 46 controls were assessed for IgA, secretory (S)-IgA, IgM, S-IgM, secretory component (SC) and PA-specific IgA and IgM, as well as neutrophil elastase (NE) activity. IgA and IgM. Total and PA-specific IgA and IgM were also measured in serum. Results: Increased epithelial pIgR immunostaining was observed in CF compared to controls, in large (56% versus 8, mean, p Conclusion: We report that, in contrast with COPD, broncho-epithelial pIgR expression is enhanced in CF and associates with an increased production of sputum IgA and IgM, including PA-specific antibodies, indicating that humoral immunity is upregulated in the CF lung.

INTRODUCTION: Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may identify membrane-associated proteins (MAPs) specific to SCLC, advance our understanding of SCLC biology, and discover new biomarkers of SCLC. METHODS: MAP lysates were prepared from three SCLCs, three non-small-cell lung cancers, and three immortalized normal bronchial epithelial cell lines and coanalyzed by DIGE. Subsequent protein identification was performed by mass spectrometry. Proteins were submitted to Ingenuity Pathway Analysis. Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC). RESULTS: Principal component analysis on the global DIGE data set demonstrated that the four replicates derived from each of the nine cell lines clustered closely, as did samples within the same histological group. One hundred thirty-seven proteins were differentially expressed in SCLC compared with non-small-cell lung cancer and immortalized normal bronchial epithelial cells. These proteins were overrepresented in cellular/tissue morphology networks. Dihydropyrimidinase- related protein 2, guanine nucleotide-binding protein alpha-q, laminin receptor 1, pontin, and stathmin 1 were selected as candidate biomarkers among MAPs overexpressed in SCLC. Overexpression of all candidates but RSSA in SCLC was verified by WB and/or IHC on tissue microarrays. These proteins were significantly associated with SCLC histology and survival in univariables analyses. CONCLUSION: DIGE analysis of a membrane-associated subproteome discovered overexpression of dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, RUVB1, and stathmin 1 in SCLC. Results were verified by WB and/or IHC in primary tumors, suggesting that investigating their functional relevance in SCLC progression is warranted. Association with survival requires further validation in larger clinical data sets. Copyright © 2014 by the International Association for the Study of Lung Cancer.

Rationale: Chronic obstructive pulmonary disease (COPD) is a lung disorder associated with aberrant immune and structural responses of the airways to inhaled toxics. While accumulation of B cells and formation of lymphoid follicles (LF) have been described in COPD airways, the functional status of lung B cells remains poorly known. Objectives: The aim of this study was to further characterize LF observed in COPD airways, in terms of IgA expression. Methods: The presence of LF and CD20 and CD21 expression were determined within the lung from COPD patients (n=21) versus controls (n=18), while intra-follicular IgA expression was assessed by immunohistochemistry (quantified by TissueIA software, Leica microsystems). Results: A significant increase was observed both in the percentage of B cells among total cells as well as the number of peri-bronchial and parenchymal LF in lung tissue of severe COPD patients as compared to controls (respectively p=0.02 and p=0.03). While these LF were clearly observed in COPD, 47 % of these were displaying evidence of CD21+ follicular DCs. In another hand, IgA+ B-cell numbers were up-regulated in LF from severe COPD patients compared with controls, particularly in distal airways (p=0.02). Conclusions: This study shows that IgA production occurs within LF from severe COPD, presumably representing an end-stage feature of exacerbated mucosal immune responses against cigarette smoke and/or chronic infection. In addition, these LF are observed in the absence of CD21+ follicular DCs.

As previously reported by others for immunoglobulin (Ig)G, we observed that IgA can induce interleukin (IL)-10 expression in human monocytes. In this study, we explored the molecular mechanisms of IL-10 induction by IgA in monocytes and monocyte-derived dendritic cells (MD-DCs). Monomeric IgA induced IL-10 production in monocytes and this production was further increased upon IgA cross-linking. Similar IL-10 responses were observed in monocytes and autologous MD-DCs, and were inhibited (by approximately 77%) by preincubation with a blocking mAb to FcalphaRI. IL-10 induction by IgA correlated with activation of MAPKinases ERK1/2, p38 and JNK, whereas only p38-inhibitor SB-203580 inhibited IL-10 induction. Upon IgA stimulation, AP-1, NFkappaB and Sp1 transcription factors were activated and inhibitors of NFkappaB and of Sp1 suppressed IgA-driven transcriptional activation of IL-10. In addition, p38 MAPK activation appeared that it was required to control nuclear translocation of NFkappaB and Sp1 upon IgA stimulation. Therefore, in human monocytes and MD-DCs the mechanisms of IL-10 induction by IgA involve p38 MAPK-dependent recruitment of both NFkappaB and Sp1.Immunology and Cell Biology advance online publication, 19 January 2010; doi:10.1038/icb.2009.120.

Blood oxygen transport and oxygen extraction were assessed in horses with colic. A gravity score (GS) ranging from I to 3 was attributed to each colic case with healthy horses used as controls. Jugular venous and carotid arterial blood samples were collected and concentrations of 2,3-diphosphoglycerate, adenosine triphosphate, inorganic phosphate and chloride were determined. pH and partial pressures of carbon dioxide (PCO2), and oxygen (PO2) were also measured. Oxygen equilibrium curves (OEC) were constructed under standard conditions and oxygen extraction ratios calculated. Haemoglobin oxygen affinity measured under standard conditions (P50(std)) was unchanged in colic horses compared with healthy controls. Horses with the highest GS, i.e. 3 had lower blood pH values than healthy animals. Arterial and venous partial pressures of oxygen at 50% haemoglobin saturation (P50(a) and P50(v)) were significantly higher in horses suffering from colic (GS = 3) than in healthy horses. The oxygen extraction ratio was also significantly increased in colic horses with a GS of 3. A rise in the oxygen extraction ratio detected in the most severely affected animals seemed to reflect the compensatory properties of the oxygen transport system where extraction of oxygen from the blood increases when systemic oxygen delivery decreases, as might be anticipated in horses with colic. (C) 2007 Elsevier Ltd. All rights reserved.

We aimed to study whether IgA or IgG could modulate dendritic cells through the Notch pathway. Blood myeloid DC were isolated from controls or patients with allergic rhinitis (sensitized to house dust mite, Dermatophagoides pteronyssinus , Der p) and assayed for Notch ligand, Delta-Like 4 (DLL4) expression and co-cultured with allogeneic CD4 + T cells. An upregulation of DLL4 was observed in IgA-treated, but not IgG-treated DCs. In co-culture of DCs and T cells, pulsing DCs with IgA downregulated IL-13, IL-5 and IFN-γ responses, but upregulated IL-10. The suppressive effect of IgA was mediated by DLL4 not by IL-10. In contrast, IL-10 was required for the inhibition by IgG-DCs. Altogether, IgA imprints myeloid DCs with suppressive effects on CD4 + T cell responses to Th2 and Th1 antigens through activation of DLL4/Notch pathway, whereas IgG does not induce DLL4 expression, its inhibitory effects mainly relying on IL-10.

Background: Chronic airway diseases like asthma and chronic obstructive pulmonary disease (COPD) are associated with abnormal mucosal responses to inhaled materials. In COPD, while immunoglobulin (Ig)A synthesis is up-regulated, expression of pIgR/SC (polymeric immunoglobulin receptor/secretory component) is markedly decreased, presumably resulting in impaired defense mechanisms against pathogens. Objectives: We explored IgA production and transport in human severe asthma and its potential regulation by the Th2/IL-13 pathway. Methods: Bronchial biopsies and in vitro-reconstituted bronchial epithelium from these biopsies, re-differentiated upon air-liquid interface (ALI-HBEC), were assayed for pIgR expression and regulation by IL-13. Results: We identified a downregulation of IgA1 and IgA2 mRNA synthesis in biopsies from severe asthma vs controls (p=0.002 for IgA1 and p=0.034 for IgA2). A downregulation of pIgR protein (but not mRNA) was also observed. IL-13 stimulation of ALI-HBEC induced, besides eotaxin-3 and periostin, a downregulation of SC release while pIgR mRNA levels were not affected, further suggesting post-transcriptional regulation of pIgR by IL-13. Conclusion: Altogether, this study shows impaired lung IgA responses in severe asthma and identifies IL-13 as potential mechanism, suggesting that targeting this cytokine could restore frontline IgA-mediated lung defence in chronic Th2-mediated airway disease.

BACKGROUND: Post transplant food allergy (LTFA) is increasingly observed after pediatric liver transplantation (LT). Although the immunopathology of LTFA remains unclear, immunoglobulin (Ig) E seems to be implicated. OBJECTIVE: To study humoral and cellular immunity in pediatric LT patients in search for factors associated with LTFA, and compare with healthy controls (HC) and non-transplant food allergic children (FA). METHODS: We studied serum Ig levels in 29 LTFA, 43 non-food allergic LT patients (LTnoFA), 21 FA patients and 36 HC. Serum specific IgA and IgE against common food allergens in LTFA, IgA1 , IgA2 and joining-chain containing polymeric IgA (pIgA) were measured. Peripheral blood mononuclear cells were analyzed by flow cytometry for B and T cell populations of interest. RESULTS: Serum IgA and specific IgA were higher in LTFA compared to LTnoFA. LTFA patients had the highest proportion of circulating T follicular helper cells (cTfh). The percentage of cTfh correlated positively with serum IgA. Unique in LTFA was also the significant increase in serum markers of mucosal IgA and the decrease in the Th17 subset of CXCR5- CD4+ cells compared to HC. Both LT patients exhibited a rise in IgA+ memory B cells and plasmablasts compared to HC and FA. CONCLUSIONS: LT has an impact on humoral immunity, remarkably in those patients developing FA. The increase in serum markers of mucosal IgA, food allergen specific IgA and cTfh cells observed in LTFA, point towards a disturbance in intestinal immune homeostasis in this patient group. This article is protected by copyright. All rights reserved.

In chronic obstructive pulmonary disease (COPD), epithelial changes and subepithelial fibrosis are salient features in conducting airways. Epithelial-to-mesenchymal transition (EMT) has been recently suggested in COPD, but the mechanisms and relationship to peribronchial fibrosis remain unclear. We hypothesised that de-differentiation of the COPD respiratory epithelium through EMT could participate in airway fibrosis and thereby, in airway obstruction.Surgical lung tissue and primary broncho-epithelial cultures (in air–liquid interface (ALI)) from 104 patients were assessed for EMT markers. Cell cultures were also assayed for mesenchymal features and for the role of transforming growth factor (TGF)-β1.The bronchial epithelium from COPD patients showed increased vimentin and decreased ZO-1 and E-cadherin expression. Increased vimentin expression correlated with basement membrane thickening and airflow limitation. ALI broncho-epithelial cells from COPD patients also displayed EMT phenotype in up to 2 weeks of culture, were more spindle shaped and released more fibronectin. Targeting TGF-β1 during ALI differentiation prevented vimentin induction and fibronectin release.In COPD, the airway epithelium displays features of de-differentiation towards mesenchymal cells, which correlate with peribronchial fibrosis and airflow limitation, and which are partly due to a TGF-β1-driven epithelial reprogramming.

Rationale: In chronic obstructive pulmonary disease (COPD), epithelial changes are prominent features in conducting airways, with goblet cell hyperplasia and squamous metaplasia. In contrast, it remains unclear whether ciliated cell numbers are reduced and which pathways dysregulate epithelial specification. Objectives: We hypothesized that specification of ciliated cells could be dysregulated in the COPD airway epithelium as a result of an aberrant reprogramming through TGF-. Methods: Surgical lung tissue from 91 COPD and control (smokers and non-smokers) patients was assessed for epithelial cell phenotyping. Primary bronchial epithelium from the same patients, reconstituted in vitro in air-liquid interface (ALI) cultures, was assayed for cell lineages and for the role of transforming growth factor (TGF)-β1. Measurements and main results: COPD bronchial epithelium, when compared to controls, showed decreased -tubulin+ ciliated cells (4.3% ± 3.1%, mean ± SD, of positive area, in severe COPD versus 8.5% ± 2.1% in controls) and increased MUC5AC+ goblet cells (34.8% ± 15.4% in severe COPD vs 11.3% ± 8.7% in controls). Cultured ALI epithelium from COPD patients displayed similar features, which correlated with airflow limitation. Exogenous TGF-β1 reduced muco-ciliary specification while targeting TGF-β1 during ALI culture restored differentiation into ciliated cells. Conclusions: the COPD airway epithelium displays altered specification for ciliated cells, whilst goblet cells increase. Altered ciliogenesis correlate with disease severity and is recapitulated in vitro through an intrinsic reprogramming, at least partly mediated by TGF-1. Goblet cell hyperplasia also occurs and persists in vitro, but involves TGF--independent mechanisms.

STUDY OBJECTIVES: To trace the entire oxyhemoglobin dissociation curve (ODC) in a cohort of cirrhotic patients in stable condition who were candidates for orthotopic liver transplantation (OLT). DESIGN: Prospective cohort study. SETTING: A large academic hospital. PATIENTS AND METHODS: We traced the entire ODC in whole blood in standard conditions (pH 7.4; PCO2, 40 mm Hg; temperature, 37 degrees C) for 50 cirrhotic candidates for OLT (27 men and 23 women) and 50 age- and height-matched healthy subjects (27 men and 23 women). All subjects were nonsmokers or ex-smokers for at least 5 years. We also measured 2,3 diphosphoglycerate (2,3 DPG) in RBCs, plasma ions, and arterial blood gases in all subjects according to standard methods. Mixed venous blood was also obtained from the 50 cirrhotic patients. RESULTS: Mean ODC was the same in the two groups. However, for the cirrhotic patients, the dispersion of the PO2 values of oxygen saturation percentage (SO2%) from 20 to 80% was significantly larger (p < 0.01 to p < 0.0001). In the cirrhotic patients, the mean PO2 for half-saturation of hemoglobin (P50) was 7.11 + 0.14 mEq/L chloride (p < 0.001) plus 0.36 mEq/L inorganic phosphate (p < 0.05) plus 0.25 micromol/gram of hemoglobin (gHb) 2,3 DPG (p < 0.00002) in absolute numerical values. Sodium, potassium, and calcium, three plasma ions disturbed in cirrhotic patients, did not contribute to determine the mean P50. DISCUSSION: In patients with cirrhosis, increased dispersion of PO2 values for a given level of SO2% may be related to four factors: (1) an observed alteration of the enzymes controlling the phosphoglycerate shunt; (2) hypothyroidism, which may affect 7 to 20% of patients with primary biliary cirrhosis; (3) the type of ongoing treatment, eg, diuretics and/or propranolol; and (4) plasma ion disturbances. CONCLUSIONS: We describe the ODC by three indexes: shape, position, and an index of dispersion of the PO2 values for a given level of SO2%. In addition, when the latter is increased, we suggest that other factors than pH, temperature, carbon dioxide, and inorganic phosphates are acting on the position of the ODC.

Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.

Twelve diarrhoeic calves were treated intravenously with an isotonic solution containing sodium bicarbonate, and their oxygen equilibrium curves (OECS) were calculated under standard conditions and compared with those of a group of healthy calves. The relationships between the OECS for arterial and venous blood and the oxygen extraction ratio were investigated. In the diarrhoeic calves, the affinity of haemoglobin for oxygen, measured under standard conditions, was increased compared with the healthy animals. During the infusion, the standard partial oxygen pressure at 50 per cent saturation of haemoglobin (P50) values stayed below the values recorded in the healthy animals. At the end of the infusion the mean standard P50 of the diarrhoeic calves was lower than before the infusion. The combined effects of all the regulating factors on blood oxygen binding resulted in the OECS of the arterial and jugular venous blood of the diarrhoeic calves remaining unchanged compared with the healthy calves. However, the administration of the infusion decreased the P50 of both the arterial and venous blood to below the value recorded in the healthy calves. Oxygen extraction by the tissues was impaired in the diarrhoeic calves throughout the infusion, and they remained dehydrated and depressed until 120 minutes after the infusion began.

Complete dynamic oxygen equilibrium curves (OEC) on dogs and cats whole blood were measured at 33, 37 and 41 degreesC. OEC were also run at three partial carbon dioxide pressures (20, 40 and 80 mmHg) as well as at five pH levels (7.2, 7.3, 7.4, 7.5 and 7.6). 2,3- diphosphoglycerate (DPG) concentrations were determined. Results were compared to those previously published in humans, using the same experimental method [Comp. Biochem. Physiol. 106 (1993) 687]. In standard conditions (pH 7.4, pCO(2) 40 mmHg and temperature 37 degreesC), the partial oxygen pressure at half-saturation of haemoglobin (p50) was 30.0 +/- 1.3 mmHg for dogs and 34.1 +/- 1.8 mmHg for cats. Cat's OEC was thus rightshifted compared to dog's OEC, itself rightshifted compared to human OEC. 2,3-DPG concentrations were higher in dogs than in men until they were very low in cats. Contrary to that observed in human medicine, no significant correlation was identified between standard p50 and canine 2,3-DPG values. Influence of pH, pCO(2) and temperature on the OEC was saturation dependent. In dogs, Delta log p50/Delta pH was equal to -0.370, Delta log p50/Delta log pCO(2) was 0.093 and Delta log p50/Delta T was 0.020. In cats, Delta log p50/Delta pH was equal to -0.405, Delta log p50/Delta log pCO(2) was 0.080 and Delta log p50/Delta T was 0.016. Practically, temperature and pH variations exert a lesser influence in domestic carnivores than in humans, effect of pCO(2) being similar in both. (C) 2004 Elsevier Ltd. All rights reserved.

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I(L)) segments have been reported for some class D herpesviruses. Inefficient formation of class D I(L) genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I(L) genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I(L) orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I(L) genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur.

Objective: To explore the consequences of helium/oxygen (He/O2) inhalation on respiratory mechanics, gas exchange, and ventilation-perfusion (VA/Q) relationships in an animal model of severe induced bronchospasm during mechanical ventilation. Design: Prospective, interventional study. Setting: Experimental animal laboratory, university hospital. Interventions: Seven piglets were anesthetized, paralyzed, and mechanically ventilated, with all ventilator settings remaining constant throughout the protocol. Acute stable bronchospasm was obtained through continuous aerosolization of methacholine. Once steady-state was achieved, the animals successively breathed air/O2 and He/O2 (FIO2 0.3), or inversely, in random order. Measurements were taken at baseline, during bronchospasm, and after 30min of He/O2 inhalation. Results: Bronchospasm increased lung peak inspiratory pressure (49±6.9 vs 18±1cmH2O, P

In Eurasian perch (Perca fluviatilis), females grow significantly faster than males. Moreover, gonadal development has a significant negative impact on somatic growth and fillet yield. In order to induce sterility, triploidy induction was attempted by subjecting fertilised eggs to heat shocks. Different combinations of temperature (28, 30, 34, 35 and 36 °C), duration (2, 5, 10 and 25 min) and time of shock initiation (TI = 3, 5 and 7 min post-fertilisation) were tested. Flow cytometry analysis was used to assess ploidy level of control and heat-shocked larvae. Low intensity (28–30 °C) and long duration (10 and 25 min) shocks lead to significantly higher survival (44 ± 26%) and triploidisation (71 ± 26%) rates than high intensity (34–36 °C) and short duration (2 and 5 min) shocks (17 ± 19% and 21 ± 26%, respectively). The most effective conditions for efficient triploidy induction were low intensity shock of 30 °C, applied 5 min post-fertilisation for 25 min. This treatment led to the production of all-triploid populations (100%) with up to 43% survival rate.

The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown.To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium.Surgical lung tissue and primary bronchial epithelium (cultured in air-liquid interface, ALI) obtained from a large series of patients (n = 116) were studied for pIgR expression and regulation.pIgR immunostaining in the bronchial epithelium is decreased in severe COPD. In contrast, pIgR transcription was up-regulated in smokers with or without COPD. In ALI (vs. submerged) cultures, pIgR expression was strongly induced, whereas pIgR expression and IgA-transcytosis capacity were decreased in cultures from subjects with severe COPD as compared with control subjects. In addition, COPD cultures released more transforming growth factor-β1 (TGF-β1), reflecting increased epithelial TGF-β1 immunostaining in COPD lung tissue. Finally, besides inducing epithelial dedifferentiation, exogenous TGF-β1 dose-dependently inhibited pIgR production, whereas pIgR increased on blockade of TGF-β1 activity during ALI differentiation.pIgR down-regulation in COPD correlates with disease severity, and the bronchial epithelium reconstituted in vitro from these patients retains its aberrant imprinting for pIgR expression. This study also links pIgR down-regulation to TGF-β-driven reprogramming of the bronchial epithelium, which results in impaired lung IgA immunity in patients with COPD.

Three different levels of hyperchloremia were induced in healthy Friesian calves to study the effects of chloride on blood oxygen transport. By infusion, the calves received either 5 ml/kg of 0.9% NaCl (low-level hyperchloremia; group A), 5 ml/kg of 7.5% NaCl (moderate hyperchloremia; group B), or 7.5 ml/kg of 7.5% NaCl (high-level hyperchloremia; group C). Blood was sampled from the jugular vein and the brachial artery. Chloride concentration, hemoglobin content, arterial and venous pH, [Formula: see text], and[Formula: see text] were determined. At each time point (0, 15, 30, 60, and 120 min), the whole blood oxygen equilibrium curve (OEC) was measured under standard conditions. In groups B and C, hyperchloremia was accompanied by a sustained rightward shift of the OEC, as indicated by the significant increase in the standard [Formula: see text] at 50% hemoglobin saturation. Infusion of hypertonic saline also induced relative acidosis. The arterial and venous OEC were calculated, with body temperature, pH, and [Formula: see text] values in arterial and venous blood taken into account. The degree of blood desaturation between the arterial and the venous compartments [O2exchange fraction (OEF%)] and the amount of oxygen released at tissue level by 100 ml of bovine blood (OEF vol%) were calculated from the arterial and venous OEC combined with the [Formula: see text] and hemoglobin concentration. The chloride-induced rightward shift of the OEC was reinforced by the relative acidosis, but the altered[Formula: see text] values combined with the lower hemoglobin concentration explained the absence of any significant difference in OEF (% and vol%). We conclude that infusion of hypertonic saline induces hyperchloremia and acidemia, which can explain the OEC rightward shift observed in arterial and peripheral venous blood.

Gustin, P., B. Detry, A. Robert, M. L. Cao, F. Lessire, C. Cambier, V. Katz, M. Ansay, A. Frans, and T. Clerbaux.Influence of age and breed on the binding of oxygen to red blood cells of bovine calves. J. Appl. Physiol. 82(3): 784–790, 1997.—The influence of somatic growth and genetic selection on the whole blood oxygen equilibrium curve (OEC) was measured under standard conditions in double-muscled and dairy calves during their first 3 mo of life. Crossbreed animals were also investigated. Hemoglobin, 2,3-diphosphoglycerate (DPG), Cl, and Piconcentrations were also measured. The percentage of fetal hemoglobin (HbF) was determined. The influence of exogenous Cl, Pi, and pH on the OEC was also assessed. The[Formula: see text] at 50% hemoglobin saturation (P50) increased during somatic growth, probably because of the increase in DPG recorded in double-muscled neonates and to the progressive disappearance of HbF in both breeds. The oxygen exchange fraction (OEF%) was used to assess the combined influence of the OEC shift and OEC shape changes on blood oxygen desaturation under standard conditions, when the[Formula: see text] decreases within a physiological range. The OEF% showed an increase during the first month, then a stabilization. The effects of Cl, Pi, and pH in Friesian calves were similar as in adult cattle. Double-muscled neonates had a lower P50, OEF% values, and DPG concentrations and higher hemoglobin and Cl concentrations than Friesian neonates. The Piconcentration and the percentage of HbF were similar in both breeds. The pH and the Cl concentration had significantly less effect on the OEC in double-muscled than in Friesian calves. Crossbreed animals exhibited intermediate parameter values, between those recorded for double-muscled and Friesian calves. All differences between breeds progressively disappeared during the first month. These data show that blood function changes markedly in calves during the first month of life and that genetic selection can alter blood function.

BACKGROUND: Myeloid dendritic cells (mDCs) and costimulatory molecules such as ICOSL/B7H2 play a pivotal role in murine experimental asthma, while little is known in human allergic disease. The aim of this study was to characterize the phenotype and ICOSL expression of mDCs from allergic rhinitis patients (AR) and their functional correlates on mDC regulation of T cell responses. METHODS: Human blood myeloid, CD1c(+) DCs were isolated from AR or healthy controls. Expression of costimulatory molecules inducible costimulatory ligand (ICOSL) and programmed death ligand 1 (PD-L1) was analysed in blood mDCs by flow cytometry and in nasal tissue biopsies by dual immunostaining. Blood mDCs were cocultured with (allogeneic) CD4(+) T cells before immunoassays for cytokine responses. RESULTS: mDCs from AR patients expressed a lower level of ICOSL, in both blood and nasal tissue. mDCs from AR were constitutively primed to induce Th2 cytokines and TNF in allogeneic CD4(+) T cells, while no difference was observed for IFN-γ or IL-10. Production of IL-10 and IL-12 did not differ between AR and control mDCs. Blockade of ICOSL in control DCs up-regulated IL-13 but not IFN-γ in cocultures with T cells, while PD-L1 blockade up-regulated both IL-13 and IFN-γ. CONCLUSIONS: Our data show that mDCs from patients with AR display impaired expression of ICOSL, and this defect licenses mDCs to promote aberrant IL-13- and IL-5-producing Th2 cell responses.

We showed that IgA induces IL-10 in monocytes and dendritic cells. Because reciprocal inhibition exists between IL-10 and IL-12, we explored whether IgA could regulate this other immunoregulatory cytokine. In human monocytes and monocyte-derived dendritic cells preincubated with IFN-γ before stimulation by LPS, suppression of p40 and IL-12p70 production was observed upon IgA treatment during IFN-γ priming. Washout experiments and inhibition of IFN-γ-induced CXCL10 (IP-10) and FcgRI (CD64) indicated that inhibition by IgA occurred at both the LPS and IFN-γ levels. Inhibition was not affected by blockade of IL-10 or MAPK but involved FcaRI/CD89-mediated suppression of STAT1 phosphorylation. These data indicate that FcaRI ligation on human monocytes and dendritic cells inhibits IL-12 expression and type 1 activation by interfering with STAT1 activation. Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved.

BACKGROUND: Myeloid (m) and plasmacytoid (p) dendritic cells (DCs) regulate immune responses to allergens, whereas it remains unclear whether abnormal DC function characterizes patients with airway allergy and whether putative dysfunction exists only in target organs. To evaluate DC function from patients with allergic rhinitis (AR), we assessed nasal, cutaneous as well as blood DCs after in vivo and in vitro allergen challenge, respectively. METHODS: DCs were immunostained in nasal and skin tissues, and cytokine expression was assessed by dual immunofluorescence. Cytokine production and regulation of cocultured peripheral CD4+ T cells were assayed by ELISA. RESULTS: In AR patients, local allergen challenge resulted in increases in pDC and mDC numbers at 8 h in the nasal mucosa and at 8-48 h in the skin. Defects in IL-10 and IFN-α were observed in both organs from AR. Blood mDCs from AR exhibited reduced IL-10 and IL-12 expression. The capacity of activated pDCs from AR to produce IFN-α and to trigger IL-10 by allogeneic CD4(+) T cells was diminished, whereas mDCs from these patients supported Th2- and Th17-cell differentiation. CONCLUSION: In allergic rhinitis, DCs are altered not only locally but also in the systemic circulation. mDCs and pDCs increased in airway and skin tissues exposed to the allergen and displayed reduced production of IL-10 and 'type 1 signals' (IL-12, IFN-α) both locally and in blood. Functional studies showed that this results in preferential Th2/Th17-cell polarization and impaired generation by blood DCs of IL-10+ T cells, linking systemic DC dysfunction and biased T-cell responses.

IgE is synthesized locally, in the bronchial mucosa, in both atopic and non-atopic asthma. The present study of bronchial secretions from asthma patients confirms that part of this IgE is specific to house dust mite (Dermatophagoïdes pteronyssinus) allergens, irrespective of atopy. In addition, it reveals that this IgE response correlates with periostin, but not to eosinophils, in sputum from these patients, probably in relation to their common IL-4/IL-13 triggering pathway.

Intrinsic asthma was described by Rackemann as asthma without allergy. Local IgE production has been documented in intrinsic asthma, but antigen specificity of this response remains elusive.We investigated (1) the presence of dust mite-specific IgE in sputum of patients with intrinsic asthma, (2) their clinical/immunological relevance, and (3) their functionality.Specific IgE to Dermatophagoides pteronyssinus (Der p) and to recombinant major allergens (rDer p1 and rDer p2) were assayed by ELISA in sputum samples from patients with intrinsic versus atopic asthma and control subjects. Whole-lung challenge was performed with Der p for clinical and inflammatory readouts. Functionality of local IgE to trigger effector cells was assessed using basophil activation test (surface expression of CD203c).Both total IgE and Der p-specific IgE levels are increased in patients with intrinsic asthma compared with healthy nonatopic patients. However, no immediate asthmatic responses were observed in patients with intrinsic asthma after Der p exposure. These sputum Der p-specific IgE do, however, recognize major allergens Der p1 and Der p2 and are able to trigger activation of blood basophils from atopic donors.We confirm that IgE production occurs in intrinsic asthma and show that part of this IgE recognizes Der p antigens. However, this IgE reactivity does not translate into clinical responses to Der p exposure, despite specificity to major allergens and functionality to activate effector cells in vitro. We postulate that a second signal that promotes IgE-mediated asthmatic responses through FcεRI is lacking in intrinsic asthma.

1. The entire oxygen dissociation curve (ODC) and the effects of temperature, pH and 2,3-diphosphoglycerate (DPG) on this curve, have been compared in four mammalians: man, dog, horse and cattle. 2. If the oxyphoric capacities are similar between these species (around 1.39 ml O-2/gHb), their P50, measured in standard conditions, i.e. at pH 7.4; pCO(2) 40 mmHg and T 37 degrees C, varies between 23.8 (+/-0.8) mmHg for the horse, 25.0 (+/-1.4) mmHg for cattle, 26.6 (+/-1.2) for man and 28.8 (+/-2.6) mmHg for the dog. 3. The higher dispersion of the dog's P50 is due to difference between breeds; in seven breeds investigated, the P50 ranges from 25.8 (spaniel) to 35.8 (hound). 4. We noted no sex difference in the four species. 5. The DPG level is confirmed to be low in cattle (