Microarray Profiling of Lymphocytes in Internal Diseases With an Altered Immune Response: Potential and Methodology

Recently it has become possible to investigate expression of all human genes with microarray technique. The authors provide arguments to consider peripheral white blood cells and in particular lymphocytes as a model for the investigation of pathophysiology of asthma, RA, and SLE diseases in which inflammation is a major component. Lymphocytes are an alternative to tissue biopsies that are most often difficult to collect systematically. Lymphocytes express more than 75% of the human genome, and, being an important part of the immune system, they play a central role in the pathogenesis of asthma, RA, and SLE. Here we review alterations of gene expression in lymphocytes and methodological aspects of the microarray technique in these diseases. Lymphocytic genes may become activated because of a general nonspecific versus disease-specific mechanism. The authors suppose that in these diseases microarray profiles of gene expression in lymphocytes can be disease specific, rather than inflammation specific. Some potentials and pitfalls of the array technologies are discussed. Optimal clinical designs aimed to identify disease-specific genes are proposed. Lymphocytes can be explored for research, diagnostic, and possible treatment purposes in these diseases, but their precise value should be clarified in future investigation

Exposure to TARC alters beta2-adrenergic receptor signaling in human peripheral blood T lymphocytes

The beta(2)-adrenergic receptor (beta(2)-AR) negatively regulates T cell activity through the activation of the G(s)/adenylyl cyclase/cAMP pathway. beta(2)-AR desensitization, which can be induced by its phosphorylation, may have important consequences for the regulation of T cell function in asthma. In the present study we demonstrate that the C-C chemokine thymus and activation-regulated chemokine (TARC) impairs the ability of beta(2)-agonist fenoterol to activate the cAMP downstream effector cAMP-responsive element binding protein (CREB) in freshly isolated human T cells. The TARC-induced activation of Src kinases resulted in membrane translocation of both G protein-coupled receptor kinase (GRK) 2 and beta-arrestin. Moreover, TARC was able to induce Src-dependent serine phosphorylation of the beta(2)-AR as well as its association with GRK2 and beta-arrestin. Finally, in contrast to CREB, phosphorylation of Src and extracellular signal-regulated kinase was enhanced by fenoterol upon TARC pretreatment. In summary, we show for the first time that TARC exposure impairs beta(2)-AR function in T cells. Our data suggest that this is mediated by Src-dependent activation of GRK2, resulting in receptor phosphorylation, binding to beta-arrestin, and a switch from cAMP-dependent signaling to activation of the MAPK pathway. We propose that aberrant T cell control in the presence of endogenous beta-agonists promotes T cell-mediated inflammation in asthma

Airway inflammation contributes to health status in COPD:a cross-sectional study

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation and airway inflammation, accompanied by decreased health status. It is still unknown which factors are responsible for the impaired health status in COPD. We postulated that airway inflammation negatively contributes to health status in COPD. METHODS: In 114 COPD patients (99 male, age: 62 ± 8 yr, 41 [31–55] pack-years, no inhaled or oral corticosteroids, postbronchodilator FEV(1): 63 ± 9% pred, FEV(1)/IVC: 48 ± 9%) we obtained induced sputum and measured health status (St. George's respiratory questionnaire (SGRQ)), postbronchodilator FEV(1), hyperinflation (RV/TLC), and airway hyperresponsiveness to methacholine (PC(20)). Sputum was induced by hypertonic saline and differential cell counts were obtained in 102 patients. RESULTS: Univariate analysis showed that SGRQ total and symptom score were positively associated with % sputum macrophages (r = 0.20, p = 0.05; and r = 0.20, p = 0.04, respectively). Multiple regression analysis confirmed these relationships, providing significant contributions of % sputum macrophages (B = 0.25, p = 0.021) and RV/TLC (B = 0.60, p = 0.002) to SGRQ total score. Furthermore, SGRQ symptom score was associated with % sputum macrophages (B = 0.30, p = 0.03) and RV/TLC (B = 0.48, p = 0.044), whilst SGRQ activity score was associated with % sputum macrophages (B = 0.46, p = 0.002), RV/TLC (B = 0.61, p = 0.015), and PC(20 )(B = -9.3, p = 0.024). Current smoking and FEV(1 )were not significantly associated with health status in the multiple regression analysis. CONCLUSION: We conclude that worse health status in COPD patients is associated with higher inflammatory cell counts in induced sputum. Our findings suggest that airway inflammation and hyperinflation independently contribute to impaired health status in COPD. This may provide a rationale for anti-inflammatory therapy in this disease

Catholic Healthcare Organizations and the Articulation of Their Identity

Contains fulltext : 69947.pdf (publisher's version ) (Open Access

Measurement of specific IgE and IgG antibodies against Aspergillus fumigatus antigen in patient sera by use of enzyme immunoassays:Influence of different procedures of antigen immobilization

IgG and IgE antibody titers against Aspergillus fumigatus were measured in patient sera by enzyme immunoassays by use of antigens or allergens immobilized to different carriers. Specific-IgG antibodies were measured by a double antibody-layer enzyme immunoassay; specific IgE was determined by Phadezym-RAST (Pharmacia Diagnostics). In both cases antigens and allergens were immobilized in two ways: first by covalent binding to CNBr-activated paper discs and second by spontaneous binding to polystyrene surface of microtiter plates. Much higher IgE-antibody titers were found with allergens immobilized to paper discs when these discs were compared with microtiter plates, which could be explained by a higher allergen-binding capacity of activated paper discs. On the contrary, higher IgG antibody titers were found with antigens bound to microtiter plates when these plates were compared with paper discs. It is concluded that IgG antibodies are directed against antigenic components that are preferentially bound to polystyrene surfaces

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

<p>Abstract</p> <p>House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of <it>Dermatophagoides pteronissinus </it>with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca²⁺levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca²⁺mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca²⁺mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.</p

An integrated high-performance liquid chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases

Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 mu L crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations. (c) 2007 Elsevier B.V. All rights reserved