Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts

BACKGROUND: Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR). We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. METHODS AND RESULTS: At the mRNA level, human bronchial epithelial (HBE) cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (α1, δ, and ε) significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by α-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. CONCLUSIONS: These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine

Immunohistochemical detection and regulation of α5 nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis

<p>Abstract</p> <p>Background</p> <p>α₅nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of α₅nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of α₅nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays.</p> <p>Results</p> <p>Steady expression of α₅nAChR subunits was observed in distal lung epithelial cells during development while proximal lung expression significantly alternates between abundant prenatal expression, absence at PN4 and PN10, and a return to intense expression at PN20. α₅expression was most abundant on luminal edges of alveolar type (AT) I and ATII cells, non-ciliated Clara cells, and ciliated cells in the proximal lung at various periods of lung formation. Expression of α₅nAChR subunits correlated with cell differentiation and reporter gene assays suggest expression of α₅is regulated in part by FoxA2, with possible cooperation by GATA-6.</p> <p>Conclusions</p> <p>Our data reveal a highly regulated temporal-spatial pattern of α₅nAChR subunit expression during important periods of lung morphogenesis. Due to specific regulation by FoxA2 and distinct identification of α₅in alveolar epithelium and Clara cells, future studies may identify possible mechanisms of cell differentiation and lung homeostasis mediated at least in part by α₅-containing nAChRs.</p

CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II

The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2) and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II). AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response

Aspergillus antigen induces robust Th2 cytokine production, inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2

BACKGROUND: Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma. METHODS: To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response. RESULTS: We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines. CONCLUSION: We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

<p>Abstract</p> <p>Background</p> <p>Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application.</p> <p>Methods</p> <p>To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens.</p> <p>Results</p> <p>TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures.</p> <p>Conclusion</p> <p>Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.</p

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BACKGROUND: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. METHODS: To examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice. RESULTS: In bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues. CONCLUSION: The data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival

Toward osteogenic differentiation of marrow stromal cells and in vitro production of mineralized extracellular matrix onto natural scaffolds

Uncorrected proofTissue engineering has emerged as a new interdisciplinary field for the repair of various tissues, restoring their functions by using scaffolds, cells, and/or bioactive factors. A temporary scaffold acts as an extracellular matrix analog to culture cells and guide the development of new tissue. In this chapter, we discuss the preparation of naturally derived scaffolds of polysaccharide origin, the osteogenic differentiation of mesenchymal stem cells cultured on biomimetic calcium phosphate coatings, and the delivery of biomolecules associated with extracellular matrix mineralization

Gammaherpesvirus-Induced Lung Pathology Is Altered in the Absence of Macrophages

The purpose of this study was to examine the lung pathogenesis of murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemokine receptor CCR2, an important receptor for macrophage recruitment to sites of inflammation. BALB/c and CCR2 −/− mice were inoculated intranasally (i.n.) with MHV-68 and samples were collected during acute infection (6 dpi) and following viral clearance (12 dpi). Immunohistochemistry was used to determine which cells types responded to MHV-68 infection in the lungs. Lung pathology in infected BALB/c mice was characterized by a mixed inflammatory cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi. Immunohistochemistry showed intense positive staining for macrophages. CCR2 −/− mice showed greater inflammation in the lungs at 12 dpi than did BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the alveoli. Immunohistochemistry demonstrated much less macrophage infiltration in the CCR2 −/− mice than in the BALB/c mice. These studies show that CCR2 is involved in macrophage recruitment in response to MHV-68 infection and illustrates how impairments in macrophage function affect the normal inflammatory response to this viral infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41345/1/408_2004_Article_2535.pd

The influence of learning methodologies on the understanding of Architectural Technologies

The present research looks at teaching/learning tools for Architectural Technologies, based on the studio outcome after using the same learning methodologies with two groups of Year 3 students from two different schools of Architecture. Since these groups of students have joined architectural courses in two different schools (one in Ireland and the other in Spain), we shall not presume that being at the same stage they must have achieved the same expertise using various means of expression. Therefore, why should we consider that using the same teaching/learning tools will be equally appropriate or successful with both groups of students? Are the results of a teaching/learning methodology related to the student’s means of conveying his understanding of Architectural Technologies? This paper investigates the influence of using different learning tools on the understanding of Architectural Technologies and aims at a better understanding of the learning methodologies applied to architectural technologies