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Biomedical subjects

David Ramos-Barbón

Publications and source records attributed to David Ramos-Barbón.

9 recordsLinked to original sources
PubMed2006

Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration.

Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.

Animals↗
PubMed2006

Heaves, an asthma-like equine disease, involves airway smooth muscle remodeling.

BACKGROUND: Increased airway smooth muscle mass is a prominent feature of asthmatic airway remodeling. Airway smooth muscle hyperplasia occurs in rodent models of experimental asthma, but the relevance of such finding to spontaneously occurring disease in large mammals is unknown. OBJECTIVE: We examined horses with heaves, a naturally occurring equine asthma related to sensitization and exposure to moldy hay. We hypothesized that airway remodeling occurs in heaves and shares disease mechanisms with asthma. METHODS: We quantified the airway smooth muscle mass and the numbers of proliferating and apoptotic airway smooth muscle cells in 5 horses with heaves and 5 control horses using morphometric techniques. Cell proliferation was detected in tissue sections by immunostaining for proliferating cell nuclear antigen, and apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of fragmented DNA. Both signals were colocalized with smooth muscle specific alpha-actin. RESULTS: Horses with heaves had a significant increase in the amount of smooth muscle in the airways (nearly triple that of the controls) associated with increased myocyte proliferation (7-fold proliferating cell nuclear antigen-positive airway myocytes) and apoptosis (6-fold). CONCLUSION: Heaves involves airway smooth muscle growth associated with myocyte hyperplasia, which may contribute to the growth, and increased myocyte apoptosis that may reflect a compensatory mechanism serving to limit the abnormal smooth muscle growth. CLINICAL IMPLICATIONS: Airway smooth muscle remodeling in heaves may be involved in the mechanism of airway hyperresponsiveness and chronic lung function impairment in a way comparable to human asthma.

Animals↗
PubMed2005

Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma.

Airway smooth muscle (ASM) growth contributes to the mechanism of airway hyperresponsiveness in asthma. Here we demonstrate that CD4+ T cells, central to chronic airway inflammation, drive ASM remodeling in experimental asthma. Adoptive transfer of CD4+ T cells from sensitized rats induced an increase in proliferation and inhibition of apoptosis of airway myocytes in naive recipients upon repeated antigen challenge, which resulted in an increase in ASM mass. Genetically modified CD4+ T cells expressing enhanced GFP (EGFP) were localized by confocal microscopy in juxtaposition to ASM cells, which suggests that CD4+ T cells may modulate ASM cell function through direct cell-cell interaction in vivo. Coculture of antigen-stimulated CD4+ T cells with cell cycle-arrested ASM cells induced myocyte proliferation, dependent on T cell activation and direct T cell-myocyte contact. Reciprocally, direct cell contact prevented postactivation T cell apoptosis, which suggests receptor-mediated T cell-myocyte crosstalk. Overall, our data demonstrate that activated CD4+ T cells drive ASM remodeling in experimental asthma and suggest that a direct cell-cell interaction participates in CD4+ T cell regulation of myocyte turnover and induction of remodeling.

Animals↗
PubMed2004

Proliferative aspects of airway smooth muscle.

Increased airway smooth muscle (ASM) mass is perhaps the most important component of the airway wall remodeling process in asthma. Known mediators of ASM proliferation in cell culture models fall into 2 categories: those that activate receptors with intrinsic receptor tyrosine kinase activity and those that have their effects through receptors linked to heterotrimeric guanosine triphosphate-binding proteins. The major candidate signaling pathways activated by ASM mitogens are those dependent on extracellular signal-regulated kinase and phosphoinositide 3'-kinase. Increases in ASM mass may also involve ASM migration, and in culture, the key signaling mechanisms have been identified as the p38 mitogen-activated protein kinase and the p21-activated kinase 1 pathways. New evidence from an in vivo rat model indicates that primed CD4(+) T cells are sufficient to trigger ASM and epithelial remodeling after allergen challenge. Hyperplasia has been observed in an equine model of asthma and may account for the increase in ASM mass. Reduction in the rate of apoptosis may also play a role. beta(2)-Adrenergic receptor agonists and glucocorticoids have antiproliferative activity against a broad spectrum of mitogens, although it has become apparent that mitogens are differentially sensitive. Culture of ASM on collagen type I has been shown to enhance proliferative activity and prevent the inhibitory effect of glucocorticoids, whereas beta(2)-agonists are minimally affected. There is no evidence that long-acting beta(2)-agonists are more effective than short-acting agonists, but persistent stimulation of the beta(2)-adrenergic receptor probably helps suppress growth responses. The maximum response of fluticasone propionate against thrombin-induced proliferation is increased when it is combined with salmeterol.

Adrenergic beta-Agonists↗
PubMed2004

T cell cytokines: animal models.

Allergic asthma involves a complex series of reactions within the airways that lead to inflammation and bronchoconstriction. These phenomena are closely linked to the immune response to the allergenic protein mediated by T cells with specificity for the allergen. Antigen presentation by dendritic cells initiates the allergic response, triggering the activation of CD4+ T cells predominantly. These cells secrete Th2 type cytokines, of which IL-4 and IL-5 are best understood, that act on various cells including B cells, eosinophils, macrophages and epithelial cells. These cells mediate, in turn, immunoglobulin E synthesis, and promote an eosinophil rich inflammation through the concerted action of various chemoattractant substances, both lipid-derived and proteins. CD8+ T cells are also activated and may have either anti-inflammatory or pro-inflammatory effects, depending upon the particular experimental model studied. The T cell receptor (TCR) that these cells possess has an important influence on the role they play. TCRalphabeta cells appear more likely to be pro-inflammatory and have antigen specificity whereas TCRgammadelta cells can be either pro- or anti-inflammatory, depending on species and experimental conditions and are not antigen specific. In conclusion, the magnitudes of inflammatory responses and bronchoconstriction following allergen challenge of sensitised animals are T cell driven and are determined, at least in part, by the balance of the T cell subsets that are activated.

Animals↗
PubMed2004

Airway remodeling: lessons from animal models.

Airway remodeling, an array of persistent tissue structural changes that occurs through a process of injury and dysregulated repair linked to airway chronic inflammation, is presently believed to largely account for the disease mechanisms of asthma. Increases in airway smooth muscle mass are probably the main mechanism causing airway hyperresponsiveness, and changes in the extracellular matrix may stimulate smooth muscle growth and contribute to the mechanics of airway obstruction. The various components of airway remodeling described inhuman asthma have been successfully reproduced in animal models of several species. Most of the data have been contributed by rat models of allergic sensitization and repeated challenge,transgenic mouse models of cytokine overexpression localized to the lung and, more recently, allergen-driven mouse models using wild-type inbred strains. Overall, animal model shave provided significant insights into the mechanisms of airway remodeling and recent technological developments allow us to exploit these models in new directions. However, the challenge of finding new therapeutic strategies that prevent or control airway remodeling,thus providing etiopathogenically oriented treatments for asthma, still stands. Experimental airway remodeling in animals should be an essential tool for treatment discovery in the near future.

Animals↗
PubMed2003

Airway smooth muscle growth from the perspective of animal models.

Airway smooth muscle maintains airway tone and may assist in adjusting ventilation distribution within the normal lung. Alterations in the properties or the quantity of ASM are likely responsible for some instances of airways hyperresponsiveness to bronchoconstrictive stimuli that is a characteristic of diseases such as asthma. Morphometric studies have shown an increase in the mass of ASM in human asthmatic airways. Animal models have been developed that confirm that ASM can be induced to grow by allergic sensitization and challenge. Growth is in large part by hyperplasia as measured by incorporation of bromodeoxyuridine as a marker of the S-phase of the cell cycle. T cells, in particular CD4+ cells, may participate in the stimulation of growth of ASM by allergen challenge. The growth factors responsible for the increase in ASM are as yet unidentified but two mediators associated with allergic airway responses, cysteinyl leukotrienes and endothelin, have been implicated using specific receptor antagonists. The links between T cells and the biochemical mediators of growth have not been established.

Adaptation, Physiological↗
PubMed2002

The immunomodulatory actions of prostaglandin E2 on allergic airway responses in the rat.

PGE(2) has been reported to inhibit allergen-induced airway responses in sensitized human subjects. The aim of this study was to investigate the mechanism of anti-inflammatory actions of PGE(2) in an animal model of allergic asthma. BN rats were sensitized to OVA using Bordetella pertussis as an adjuvant. One week later, an aerosol of OVA was administered. After a further week, animals were anesthetized with urethan, intubated, and subjected to measurements of pulmonary resistance (R(L)) for a period of 8 h after OVA challenge. PGE(2) (1 and 3 micro g in 100 micro l of saline) was administered by insufflation intratracheally 30 min before OVA challenge. The early response was inhibited by PGE(2) (3 micro g). The late response was inhibited by both PGE(2) (1 and 3 micro g). Bronchoalveolar lavage fluid from OVA-challenged rats showed eosinophilia and an increase in the number of cells expressing IL-4 and IL-5 mRNA. These responses were inhibited by PGE(2). Bronchoalveolar lavage fluid levels of cysteinyl-leukotrienes were elevated after OVA challenge and were reduced after PGE(2) to levels comparable with those of sham challenged animals. We conclude that PGE(2) is a potent anti-inflammatory agent that may act by reducing allergen-induced Th2 cell activation and cysteinyl-leukotriene synthesis in the rat.

Adjuvants, Immunologic↗