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A decreased bronchial expression of secretory component (SC) was demonstrated in severe COPD, and correlated with neutrophils. Mechanisms of epithelial cell/neutrophils interactions remain however poorly understood. Calu-3 (human bronchial epithelial) cells were incubated after confluence (in triplicate conditions) with various ratios of activated neutrophils (0.5:1 to 15:1, neutrophils: Calu-3 cells). After 48hrs of co-culture supernatants were assayed for SC by ELISA. SC production by Calu-3 cells increased at intermediate neutrophil numbers (316±32 versus 193±19ng·ml–1, ratio of 5:1 versus control, mean±SEM of 3 experiments, p = 0.05). In contrast, a trend for decrease in SC was observed with high neutrophil numbers (111±19 versus 193±19ng·ml–1, ratio of 15:1 versus control, p = 0.06). The addition of secretory leukocyte protease inhibitor further increased SC upregulation at intermediate ratios, and inhibited the SC decrease at high neutrophil numbers. The mechanism of SC up-regulation by neutrophils did not implicate TNF-alpha or IL-1beta. This study provides direct evidence of a dual effect of neutrophils on epithelial SC. Our data suggest that neutrophils could differently affect epithelial immune secretory function according to the extent of neutrophil influx and/or to the reactivity of airway epithelial cells.

Background: The airway epithelium (AE) fulfils multiple functions to maintain pulmonary homeostasis, among which ensuring adequate barrier function, cell differentiation and polarization, and actively transporting immunoglobulin A (IgA), the predominant mucosal immunoglobulin in the airway lumen, via the polymeric immunoglobulin receptor (pIgR). Morphological changes of the airways have been reported in ARDS, while their detailed features, impact for mucosal immunity, and causative mechanisms remain unclear. Therefore, this study aimed to assess epithelial alterations in the distal airways of patients with ARDS., Methods: We retrospectively analyzed lung tissue samples from ARDS patients and controls to investigate and quantify structural and functional changes in the small airways, using multiplex fluorescence immunostaining and computer-assisted quantification on whole tissue sections. Additionally, we measured markers of mucosal immunity, IgA and pIgR, alongside with other epithelial markers, in the serum and the broncho-alveolar lavage fluid (BALF) prospectively collected from ARDS patients and controls., Results: Compared to controls, airways of ARDS were characterized by increased epithelial denudation (p = 0.0003) and diffuse epithelial infiltration by neutrophils (p = 0.0005). Quantitative evaluation of multiplex fluorescence immunostaining revealed a loss of ciliated cells (p = 0.0317) a trend towards decreased goblet cells (p = 0.056), and no change regarding cell progenitors (basal and club cells), indicating altered mucociliary differentiation. Increased epithelial permeability was also shown in ARDS with a significant decrease of tight (p < 0.0001) and adherens (p = 0.025) junctional proteins. Additionally, we observed a significant decrease of the expression of pIgR, (p < 0.0001), indicating impaired mucosal IgA immunity. Serum concentrations of secretory component (SC) and S-IgA were increased in ARDS (both p < 0.0001), along other lung-derived proteins (CC16, SP-D, sRAGE). However, their BALF concentrations remained unchanged, suggesting a spillover of airway and alveolar proteins through a damaged AE., Conclusion: The airway epithelium from patients with ARDS exhibits multifaceted alterations leading to altered mucociliary differentiation, compromised defense functions and increased permeability with pneumoproteinemia., (© 2024. The Author(s).)

Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods., (© 2023 Carlier et al.)

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., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: A major challenge for any glaucoma implant is their ability to provide long-term intraocular pressure lowering efficacy. The formation of a low-permeability fibrous capsule around the device often leads to obstructed drainage channels, which may impair the drainage function of devices. These foreign body-related limitations point to the need to develop biologically inert biomaterials to improve performance in reaching long-term intraocular pressure reduction. The aim of this study was to evaluate in vivo (in rabbits) the ocular biocompatibility and tissue integration of a novel suprachoroidal microinvasive glaucoma implant, MINIject™ (iSTAR Medical, Wavre, Belgium)., Results: In two rabbit studies, no biocompatibility issue was induced by the suprachoroidal, ab-externo implantation of the MINIject™ device. Clinical evaluation throughout the 6 post-operative months between the sham and test groups were similar, suggesting most reactions were related to the ab-externo surgical technique used for rabbits, rather than the implant material itself. Histological analysis of ocular tissues at post-operative months 1, 3 and 6 revealed that the implant was well-tolerated and induced only minimal fibroplasia and thus minimal encapsulation around the implant. The microporous structure of the device became rapidly colonized by cells, mostly by macrophages through cell migration, which do not, by their nature, impede the flow of aqueous humor through the device. Time-course analysis showed that once established, pore colonization was stable over time. No fibrosis nor dense connective tissue development were observed within any implant at any time point. The presence of pore colonization may be the process by which encapsulation around the implant is minimized, thus preserving the permeability of the surrounding tissues. No degradation nor structural changes of the implant occurred during the course of both studies., Conclusions: The novel MINIject™ microinvasive glaucoma implant was well-tolerated in ocular tissues of rabbits, with observance of biointegration, and no biocompatibility issues. Minimal fibrous encapsulation and stable cellular pore colonization provided evidence of preserved drainage properties over time, suggesting that the implant may produce a long-term ability to enhance aqueous outflow., Competing Interests: Competing interestsI. Grierson, D. Minckler, M.K. Rippy, N. Collignon and J. Bianco declare that they have no significant competing interests. A.J. Marshall is an employee of Healionics Corporation and is a member of the Scientific Advisory Board of iSTAR Medical SA. B. Detry is an employee of iSTAR Medical SA. M.A. Johnstone is a member of the Scientific Advisory Board of iSTAR Medical SA., (© The Author(s) 2020.)

Background: In cystic fibrosis (CF), recurrent infections suggest impaired mucosal immunity but whether production of secretory immunoglobulin A (S-IgA) is impaired remains elusive. S-IgA is generated following polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of dimeric (d-)IgA and represents a major defence through neutralisation of inhaled pathogens like Pseudomonas aeruginosa (Pa)., Methods: Human lung tissue (n = 74), human sputum (n = 118), primary human bronchial epithelial cells (HBEC) (cultured in air-liquid interface) (n = 19) and mouse lung tissue and bronchoalveolar lavage were studied for pIgR expression, IgA secretion and regulation., Findings: Increased epithelial pIgR immunostaining was observed in CF lung explants, associated with more IgA-producing plasma cells, sputum and serum IgA, especially Pa-specific IgA. In contrast, pIgR and IgA transport were downregulated in F508del mice, CFTR-inhibited HBEC, and CF HBEC. Moreover, the unfolded protein response (UPR) due to F508del mutation, inhibited IgA transport in Calu-3 cells. Conversely, pIgR expression and IgA secretion were strongly upregulated following Pa lung infection in control and F508del mice, through an inflammatory host response involving interleukin-17., Interpretation: A complex regulation of IgA secretion occurs in the CF lung, UPR induced by CFTR mutation/dysfunction inhibiting d-IgA transcytosis, and Pa infection unexpectedly unleashing this secretory defence mechanism., Funding: This work was supported by the Forton's grant of the King Baudouin's Foundation, Belgium, the Fondazione Ricerca Fibrosi Cistica, Italy, and the Fonds National de la Recherche Scientifique, Belgium., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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 < 0.0001). Both features were recapitulated in the ALI-cultured epithelium from COPD patients. Exogenous TGF-β1 reduced mucociliary differentiation while neutralizing TGF-β1 during ALI increased both specialized cell types. The COPD airway epithelium displays altered differentiation for ciliated cells, which recapitulates in vitro, at least in part through TGF-β1.

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.

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 G 2 -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., (©2018 American Association for Cancer Research.)

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 human asthma and its potential regulation by T-helper cell type 2 inflammation., Methods: Bronchial biopsies from asthma and control subjects were assayed for bronchial epithelial polymeric immunoglobulin receptor (pIgR) expression and correlated to T-helper cell type 2 biomarkers. Bronchial epithelium reconstituted in vitro from these subjects, on culture in air-liquid interface, was assayed for pIgR expression and regulation by IL-4/IL-13., Measurements and Main Results: Downregulation of pIgR protein was observed in the bronchial epithelium from patients with asthma (P = 0.0002 vs. control subjects). This epithelial defect was not observed ex vivo in the cultured epithelium from patients with asthma. Exogenous IL-13 and IL-4 could inhibit pIgR expression and IgA transcytosis. Mechanistic experiments showed that autocrine transforming growth factor-β mediates the IL-4/IL-13 effect on the pIgR, with a partial contribution of upregulated transforming growth factor-α/epidermal growth factor receptor., Conclusions: This study shows impaired bronchial epithelial pIgR expression in asthma, presumably affecting secretory IgA-mediated frontline defense as a result of type 2 immune activation of the transforming growth factor pathway.

Mechanisms explaining the propensity of a primary tumor to metastasize to a specific site still need to be unveiled, and clinical studies support a link between chronic inflammation and cancer dissemination to specific tissues. Using different mouse models, we demonstrate the role of inflammation-generated extracellular matrix fragments ac-PGP ( N -acetyl-proline-glycine-proline) on tumor cells dissemination to lung parenchyma. In mice exposed to cigarette smoke or lipopolysaccharide, lung neutrophilic inflammation produces increased levels of MMP-9 (matrix metalloproteinase 9) that contributes to collagen breakdown and allows the release of ac-PGP tripeptides. By silencing CXCR2 gene expression in tumor cells, we show that these generated ac-PGP tripeptides exert a chemotactic activity on tumor cells in vivo by binding CXCR2., Competing Interests: Declaration of conflicting interests:The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: D.C. is the founder of Aquilon Pharmaceuticals, received speaker fees from AstraZeneca, Boehringer-Ingelheim, Novartis, Mundipharma, Chiesi, and GSK and received consultancy fees from AstraZeneca, Boehringer-Ingelheim, and Novartis for the participation to advisory boards. None of these activities have any connection with oncology or development of drugs in the field of oncology.

Solid tumors comprise cancer cells and different supportive stromal cells, including mesenchymal stem cells (MSCs), which have recently been shown to enhance tumor growth and metastasis. We provide new mechanistic insights into how bone marrow (BM)-derived MSCs co-injected with Lewis lung carcinoma cells promote tumor growth and metastasis in mice. The proinvasive effect of BM-MSCs exerted on tumor cells relies on an unprecedented juxtacrine action of BM-MSC, leading to the trans-shedding of amphiregulin (AREG) from the tumor cell membrane by tumor necrosis factor-α-converting enzyme carried by the BM-MSC plasma membrane. The released soluble AREG activates cancer cells and promotes their invasiveness. This novel concept is supported by the exploitation of different 2D and 3D culture systems and by pharmacological approaches using a tumor necrosis factor-α-converting enzyme inhibitor and AREG-blocking antibodies. Altogether, we here assign a new function to BM-MSC in tumor progression and establish an uncovered link between AREG and BM-MSC., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Post-transplant food allergy (LTFA) is increasingly observed after paediatric 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 paediatric 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 analysed 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 homoeostasis in this patient group., (© 2015 John Wiley & Sons Ltd.)

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 with in 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., (Copyright ©ERS 2015.)

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.

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., (© 2014 John Wiley & Sons Ltd.)

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.

Purpose: To evaluate the antilymphangiogenic potential of multi-target tyrosine kinase inhibitor sunitinib in corneal neovascularization (NV)., Methods: Inflammatory corneal NV was induced by thermal cauterization applied in the central cornea of mice, to which sunitinib malate was daily administered by gavage or not. At days 6, 11, or 17 post cauterization, lymphatic and blood vessels, as well as inflammatory cells were immunostained and quantified in whole-mounted corneas. RT-PCRs were performed to evidence VEGF-A, VEGF-C, VEGF-D, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor (VEGFR)-1 and -2 (sVEGFR-1, sVEGFR-2) expressions. Macrophages were isolated from mice peritoneal cavity following thioglycollate injection to produce conditioned medium. The effects of sunitinib were evaluated in vitro in the aortic and lymphatic ring assays in the presence or not of macrophage conditioned medium., Results: Sunitinib treatment drastically reduced pathologic corneal lymphangiogenesis and angiogenesis. Reduced F4/80+ cell infiltration was evidenced in sunitinib-treated mice and was associated to decreased VEGF-A (by 50%, P < 0.01) and VEGF-C (by 35%, P < 0.01) expressions, while VEGF-D and sVEGFR-2 expressions were not affected. In vitro, sunitinib dose-dependently inhibited aortic ring outgrowth, but failed to affect lymphangiogenesis in the lymphatic ring assay. However, macrophage conditioned medium-enhanced angiogenesis and lymphangiogenesis were both strongly counteracted by sunitinib treatment. Mechanistically, sunitinib blocked VEGFR-2 phosphorylation induced by VEGF-A released by macrophages., Conclusions: Sunitinib exerts antihemangiogenic and antilymphangiogenic effects in vivo by reducing F4/80+ cell recruitment and interacting with their released factors.

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 FcγRI (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 FcαRI/CD89-mediated suppression of STAT1 phosphorylation. These data indicate that FcαRI ligation on human monocytes and dendritic cells inhibits IL-12 expression and type 1 activation by interfering with STAT1 activation.

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., (© 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.)

Membrane-type 4 matrix metalloprotease (MT4-MMP) expression in breast adenocarcinoma stimulates tumor growth and metastatic spreading to the lung. However, whether these pro-tumorigenic and pro-metastatic effects of MT4-MMP are related to a proteolytic action is not yet known. Through site directed mutagenesis MT4-MMP has been inactivated in cancer cells through Glutamic acid 249 substitution by Alanine in the active site. Active MT4-MMP triggered an angiogenic switch at day 7 after tumor implantation and drastically accelerated subcutaneous tumor growth as well as lung colonization in recombination activating gene-1-deficient mice. All these effects were abrogated upon MT4-MMP inactivation. In sharp contrast to most MMPs being primarily of stromal origin, we provide evidence that tumor-derived MT4-MMP, but not host-derived MT4-MMP contributes to angiogenesis. A genetic approach using MT4-MMP-deficient mice revealed that the status of MT4-MMP produced by host cells did not affect the angiogenic response. Despite of this tumor intrinsic feature, to exert its tumor promoting effect, MT4-MMP requires a permissive microenvironment. Indeed, tumor-derived MT4-MMP failed to circumvent the lack of an host angio-promoting factor such as plasminogen activator inhibitor-1. Overall, our study demonstrates the key contribution of MT4-MMP catalytic activity in the tumor compartment, at the interface with host cells. It identifies MT4-MMP as a key intrinsic tumor cell determinant that contributes to the elaboration of a permissive microenvironment for metastatic dissemination., (Copyright © 2012 UICC.)

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

Rationale: 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., Objectives: 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., Methods: 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)., Measurements and Main Results: 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., Conclusions: 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.

Background: Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures., Results: In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation., Conclusions: In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.

In this study, we evaluate the potential involvement of collagenase-3 (MMP13), a matrix metalloproteinase (MMP) family member, in the exudative form of age-related macular degeneration characterized by a neovascularisation into the choroid. RT-PCR analysis revealed that human neovascular membranes issued from patients with AMD expressed high levels of Mmp13. The contribution of MMP13 in choroidal neovascularization (CNV) formation was explored by using a murine model of laser-induced CNV and applying it to wild-type mice (WT) and Mmp13-deficient mice (Mmp13 ( -/- ) mice). Angiogenic and inflammatory reactions were explored by immunohistochemistry. The implication of bone marrow (BM)-derived cells was determined by BM engraftment into irradiated mice and by injecting mesenchymal stem cells (MSC) isolated from WT BM. The deficiency of Mmp13 impaired CNV formation which was fully restored by WT BM engraftment and partially rescued by several injections of WT MSC. The present study sheds light on a novel function of MMP13 during BM-dependent choroidal vascularization and provides evidence for a role for MSC in the pathogenesis of CNV.

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.

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system's role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.

Neutrophils have a dual affect on epithelial pIgR/SC, the critical receptor for transcellular routing of mucosal IgA, but mechanisms of pIgR/SC upregulation remain elusive. Requirements of cytokine, redox, and signalling pathways for pIgR/SC production were assessed in human bronchial epithelial (Calu-3) cells cocultured with increasing numbers of blood neutrophils. Increased SC production was observed after incubation for 48 hrs with intermediate neutrophil numbers (1.25 to 2.5 x 10(6)), was favoured by the elastase inhibitor SLPI, and correlated with increased TGF-beta production. Exogenous TGF-beta stimulated SC production with a maximal effect at 48 hrs and both TGF-beta- and neutrophil-driven SC upregulation were dependent on redox balance and p38 MAP-kinase activation. This paper shows that activated neutrophils could upregulate epithelial pIgR/SC production through TGF-beta-mediated activation of a redox-sensitive and p38 MAPK-dependent pathway. An imbalance between the two neutrophil-driven opposite mechanisms (SC upregulation and SC degradation) could lead to downregulation of pIgR/SC, as observed in severe COPD.

We report the case of a patient working in a factory producing inulin from chicory who developed rhinoconjunctivitis and asthma to the dust of dry chicory roots and oral allergy syndrome to raw fruits and vegetables. Nonspecific bronchial hyperresponsiveness was diagnosed. A provocation test with dry chicory induced acute rhinoconjunctivitis and an immediate asthmatic response with no further clinical symptoms. Skin prick test results were positive to birch pollen and fresh/dry chicory, and negative for inulin. Specific immunoglobulin (Ig) E was > 100 kU(A)/L for rBet v 1. Specific IgE were detected by immunoblotting chicory extract with the patient's serum, but not with a control serum. The main immunoreactive band corresponded to a protein with a molecular weight of approximately 17 kDa, like Bet v 1, and this immunoreactivity was effectively inhibited by preincubating serum with purified Bet v 1. This case documents occupational rhinoconjunctivitis and asthma due to IgE sensitization to inhaled chicory allergens, including one identified for the first time as a 17-kD Bet v 1 homologous protein, with secondary oral allergy syndrome to related foods.

A decrease in hemoglobin affinity for oxygen is considered an adaptive mechanism against tissue hypoxia. Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by recurrent episodes of apnea and hypopnea resulting in arterial oxygen desaturations during sleep. Maillard et al. (10) observed a right shift of the oxyhemoglobin dissociation curve (ODC) and an increase in 2,3-diphosphoglycerate (2,3-DPG) concentration ([2,3-DPG]) in 15 patients with severe OSAHS, but some had slight daytime arterial hypoxemia while breathing room air. The aim of our study was to measure the ODC and 2,3-DPG concentrations in a group of subjects normoxemic during daytime referred to our sleep laboratory for suspicion of snoring or OSAHS. The patients were recruited during a period of 6 mo. All arterial and venous blood samples were taken early in the morning within 1 h of awakening following a full-night polysomnography. ODC and 2,3-DPG were analyzed in 88 patients: 56 OSAHS (oxygen desaturation index: 27.5 +/- 24.5) and 32 non-OSAHS. We found a significant correlation between the P50 and 2,3-DPG levels in the 88 patients: r = 0.502, P < 0.001. We observed no difference between OSAHS and non-OSAHS for the P50 and for [2,3-DPG]. There was no correlation between the severity of OSAHS and either P50 or [2,3-DPG]. Finally, there was no change in these parameters measured at baseline, after 3 days and after 1 mo of treatment by nasal continuous positive airway pressure in 7 patients with OSAHS. We conclude that patients with OSAHS who are normoxemic during daytime have comparable oxyhemoglobin affinity than nonapneic subjects.

Blood oxygen transport and oxygen extraction were assessed in horses with colic. A gravity score (GS) ranging from 1 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 (PCO(2)), and oxygen (PO(2)) 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.

Allergen immunotherapy (IT) has long-term efficacy in IgE-mediated allergic rhinitis and asthma. IT has been shown to modify lymphocyte responses to allergen, inducing IL-10 production and IgG Abs. In contrast, a putative role for IgA and local TGF-beta-producing cells remains to be determined. In 44 patients with seasonal rhinitis/asthma, serum IgA1, IgA2, and polymeric (J chain-containing) Abs to the major allergen Phl p 5 were determined by ELISA before and after a 2-year double-blind trial of grass pollen (Phleum pratense) injection IT. Nasal TGF-beta expression was assessed by in situ hybridization. Sera from five IT patients were fractionated for functional analysis of the effects of IgA and IgG Abs on IL-10 production by blood monocytes and allergen-IgE binding to B cells. Serum Phl p 5-specific IgA2 Abs increased after a 2-year treatment (approximately 8-fold increase, p = 0.002) in contrast to IgA1. Increases in polymeric Abs to Phl p 5 (approximately 2-fold increase, p = 0.02) and in nasal TGF-beta mRNA (p = 0.05) were also observed, and TGF-beta mRNA correlated with serum Phl p 5 IgA2 (r = 0.61, p = 0.009). Post-IT IgA fractions triggered IL-10 secretion by monocytes while not inhibiting allergen-IgE binding to B cells as observed with IgG fractions. This study shows for the first time that the IgA response to IT is selective for IgA2, correlates with increased local TGF-beta expression, and induces monocyte IL-10 expression, suggesting that IgA Abs could thereby contribute to the tolerance developed in IT-treated allergic patients.

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.

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.

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.

The purpose of this study was to determine the effect of regulating factors on the oxygen equilibrium curve (OEC) under standard conditions and then to calculate the oxygen extraction between arterial and jugular venous blood in healthy Standardbred horses. The results were compared to those previously obtained in humans and cattle, using the same experimental method. The partial oxygen pressure at 50% saturation of haemoglobin, measured under standard conditions (standard P50), was 24.8+/-2.0 (SD of mean) mmHg. This value was similar to the cattle standard P50 (25.0+/-1.4 mmHg, SD of mean) but lower than the human standard P50 (26.6+/-1.2 mmHg, SD of mean) previously reported using the same experimental method. The effects of regulating factors on the standard OEC were also determined, and a major effect of pH and temperature was noted. In contrast, partial carbon dioxide pressure played only a minor role in horses, compared to cattle and humans. No significant correlation was found between phosphate and chloride concentrations and standard P50.

Complete dynamic oxygen equilibrium curves (OEC) on dogs and cats whole blood were measured at 33, 37 and 41 degrees C. 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, pCO2 40 mmHg and temperature 37 degrees C), 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, pCO2 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 pCO2 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 pCO2 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 pCO2 being similar in both.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii) BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.

The control of infectious bovine rhinotracheitis induced by bovine herpesvirus 1 (BoHV-1) requires sensitive and specific diagnostic assays. As BoHV-1 is antigenically and genetically related to four other alphaherpesviruses of ruminants-namely, BoHV-5, caprine herpesvirus 1 (CpHV-1), cervine herpesvirus 1 (CvHV-1) and CvHV-2-diagnostic tests able to discriminate BoHV-1 from these related viruses are needed to avoid misdiagnosis, especially because some of these viruses are able to cross the species barrier. In this study, murine monoclonal antibodies (MAbs) specific for BoHV-1, BoHV-5, CpHV-1, CvHV-1, and CvHV-2 were produced with the aim of setting up an immunofluorescence assay able to discriminate between these related herpesviruses. Produced MAbs were selected for their viral specificity by enzyme-linked immunosorbent assay and indirect immunofluorescence staining of virus-infected cells. Radioimmunoprecipitation characterization of the selected MAbs revealed that four of them are directed against glycoprotein C (gC) and one of them is directed against gD of these related viruses. The obtained results demonstrate that the antibodies produced allow an unambiguous discrimination of each of the four alphaherpesviruses related to BoHV-1.

Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC(-)/gE(+)) or E (gC(+)/gE(-)) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC(+)/gE(+) and gC(-)/gE(-)) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC(+)/gE(+) and gC(-)/gE(+)) predominated without fully outcompeting the presence of the two other detected populations (gC(+)/gE(-) and gC(-)/gE(-)), and (iii) after reactivation from latency, no gC(+)/gE(-) and gC(-)/gE(-) progeny viruses were detected, although gC(+)/gE(-) mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.

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/O(2)) 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/O(2) and He/O(2) (FIO(2) 0.3), or inversely, in random order. Measurements were taken at baseline, during bronchospasm, and after 30 min of He/O(2) inhalation., Results: Bronchospasm increased lung peak inspiratory pressure (49+/-6.9 vs 18+/-1 cm H(2)O, P<0.001), lung resistance (22.7+/-1.5 vs 6.8+/-1.5 cm H(2)O x l(-1).s, P<0.001), dynamic elastance (76+/-11.2 vs 22.8+/-4.1 cm H(2)O x l(-1), P<0.001), and work of breathing (1.51+/-0.26 vs 0.47+/-0.08, P<0.001). Arterial pH decreased (7.47+/-0.06 vs 7.32+/-0.06, P<0.001), PaCO(2) increased, and PaO(2) decreased. Multiple inert gas elimination showed an absence of shunt, substantial increases in perfusion to low VA/Q regions, and dispersion of VA/Q distribution. He/O(2) reduced lung resistance and work of breathing, and worsened hypercapnia and respiratory acidosis., Conclusions: In this model, while He/O(2) improved respiratory mechanics and reduced work of breathing, hypercapnia and respiratory acidosis increased. Close attention should be paid to monitoring arterial blood gases when He/O(2) is used in mechanically ventilated acute severe asthma.

Objective: 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., Design and Setting: Prospective, interventional study in the intensive care unit of a university hospital., Interventions: 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)., Results: 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., Conclusions: 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.

In human and veterinary medicine, arterial and venous haemoglobin oxygen saturations are often used to estimate the severity of a disease and to guide therapeutic decisions. In veterinary medicine, haemoglobin oxygen saturation (SO(2)) is usually calculated using a blood gas analyser and algorithms developed for humans. It is possible, therefore, that the values obtained in animals may be distorted, particularly in animals with a high haemoglobin oxygen affinity, like young calves. In order to verify this hypothesis, we compared the arterial (SaO(2)) and venous (SvO(2)) haemoglobin oxygen saturations calculated using three different algorithms, and the oxygen exchange fraction (OEF) at the tissue level, which is the degree of haemoglobin desaturation between arterial and venous blood (SaO(2)-SvO(2)), with the values obtained from the whole bovine oxygen equilibrium curve (OEC) determined by a reference method. The blood gas analysers underestimated SvO(2) values; consequently, the OEF was overestimated (by about 10%). Two methods of reducing these errors were assessed. As the haemoglobin oxygen affinity decreases during the first month of life in calves a relationship between PO(2) at 50% haemoglobin saturation (P50) and age was established in order to correct the calculated values of venous and arterial SO(2), taking into account the estimated position of the OEC. This method markedly reduced the error for SvO(2) and OEF. Secondly, the SO(2) was calculated using a mathematical model taking into account the age of the animal and the specific effects of pH, PCO(2), and temperature on the bovine OEC. Using this method, the mean difference between the OEF values calculated using the mathematical model and those calculated by the reference method was close to zero. The errors produced by blood gas analysers can thus be minimised in two ways: firstly, by simply introducing a P50 estimated from the age of the calf into the analyser before the measurement; and secondly, by calculating the SO(2) using a mathematical model applied to the bovine OEC.

Objective: To validate an animal model replicating the pathophysiological characteristics of severe induced bronchospasm observed in humans, with a high level of stability permitting measurements such as the assessment of ventilation-perfusion relationships with the multiple inert gas elimination technique., Design and Setting: Experimental study in an animal research laboratory., Subjects: 13 piglets (age 3-4 months) were studied and 7 underwent the complete protocol, Interventions: The animals were anesthetized and paralyzed. Mechanical ventilation was initiated in a volume-controlled mode. Ventilatory parameters were adjusted to obtain normocapnia and were maintained constant during the bronchospasm. Methacholine was administered via a synchronized nebulizer and progressively adjusted to obtain a stable twofold increase in peak inspiratory pressure., Measurements and Results: Cardiopulmonary physiological data including assessment of lung mechanics and measurement of ventilation-perfusion relationships were obtained before and during the bronchospasm. Peak inspiratory pressure increased from 19.7+/-2.9 to 44.4+/-7.1 cmH(2)O during the bronchospasm. The latter remained stable over 2 h. Respiratory mechanics, gas exchange, and ventilation-perfusion distribution changes typical of those observed in severe bronchospasm in humans were observed in all animals., Conclusions: The present experimental model replicates some of the physiopathological characteristics of severe human bronchospasm, and its stability should facilitate studies of the effects of different ventilatory modes in the setting of acute severe asthma.

The study was carried out on healthy Friesian calves (n = 10) aged between 10 and 30 days. Hypochloremia and alkalosis were induced by intravenous administration of furosemide and isotonic sodium bicarbonate. The venous and arterial blood samples were collected repeatedly. 2,3-diphosphoglycerate (2,3-DPG), hemoglobin and plasmatic chloride concentrations were determined. The red blood cell chloride concentration was also calculated. pH, PCO2 and PO2 were measured in arterial and mixed venous blood. The oxygen equilibrium curve (OEC) was measured in standard conditions. The correspondence of the OEC to the arterial and mixed venous compartments was calculated, taking blood temperature, pH and PCO2 values into account. The oxygen exchange fraction (OEF%), corresponding to the degree of blood desaturation between the arterial and mixed venous compartments and the amount of oxygen released at the tissue level by 100 mL of blood (OEF Vol%) were calculated from the arterial and mixed venous OEC, combined with PO2 and hemoglobin concentration. Oxygen delivery (DO2) was calculated using the arterial oxygen content, the cardiac output measured by thermodilution, and the body weight of the animal. The oxygen consumption (VO2) was derived from the cardiac output, OEF Vol% and body weight values. Despite the plasma hypochloremia, the erythrocyte chloride concentration was not influenced by furosemide and sodium bicarbonate infusion. Due to the alkalosis-induced increase in the 2,3-DPG, the standard OEC was shifted to the right, allowing oxygen to dissociate from hemoglobin more rapidly. These changes opposed the increased affinity of hemoglobin for oxygen induced by alkalosis. Moreover, respiratory acidosis, hemoconcentration, and the slight decrease in the partial oxygen pressure in mixed venous blood (Pvo2) tended to improve the OEF Vol% and maintain the oxygen consumption in a physiological range while the cardiac output, and the oxygen delivery were significantly decreased. It may be concluded that, despite reduced oxygen delivery, oxygen consumption is maintained during experimentally induced hypochloremic alkalosis in healthy 10-30 day old calves.