Combination Therapys Effect On Human Bronchial Epithelial Cells Biology Essay

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To investigate the anti - inflammatory effect of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide on human bronchial epithelial cell line i.e. IB3 and C38 cell lines individually and in combination upon challenging the cells with Lipopolysaccharides. The existence of an anti - inflammatory effects would be of potential benefit in the treatment of group of people suffering from moderate to severe COPD.

Research Outcomes:

Establishing the increase in anti - inflammatory activity by the combination of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide. If optimisation is observed then this study can advise possible benefits of treating cystic fibrosis patients with COPD with the combination of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide.

Background:

Greene &Harris (2008) portrayed chronic obstructive pulmonary disease (COPD) or chronic obstructive lung disease (COLD) or chronic obstructive airways disease (COAD) as an array of illness that comprises of chronic bronchitis, emphysema and COPD that are chronic and gradually progressive conditions which is caused mainly by tobacco smoking or are exacerbated by it. COPD is more common in UK and Eastern Europe than in most developed countries(Greene &Harris, 2008). Green and Harris (2008) noted that in Western Europe, COPD is called as 'English Disease' that has an overall prevalence in the UK among men than women with the statistics as 4% in men aged about 50 years, 9% aged about 60 years, 12% aged about 80 years, but only 4% in women. COPD is the 5thmost source of death in the UK (National Statistics, 2006) and rank 4thin worldwide (ICC International COPD Coalition, No date). COPD is responsible for extensive morbidity that caused about 30,000 deaths in 1999 in the UK with a male: female ratio of about 5:1(Greene &Harris, 2008).

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There is no cure for COPD, but the deterioration can be slowed down by treatment. The treatments aspire in improving symptoms like breathlessness, reducing the exacerbations and improving the social quality of life. The risk of further deterioration can be reduced by quitting smoking, physiotherapy (it aids in clearing mucus from lungs and medicines that makes the airway wider and reduces inflammation). The medicines that are used for treatment are bronchodilators, corticosteroid and antibiotics. Antibiotics are used for the treatment of bacterial pathogens like Pneumococci, Haaemophilus influenzae or Moraxella catarrhalis.

In this research project Fluticasone propionate is a corticosteroid, Salmeterol xinafoate is the long - acting - β2 - agonistand Tiotropium bromide serves as a source of Muscuranic agonist. The present report describes the effect of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide drugs on human bronchial epithelial cells (HBE) cell line via an in vitrohuman model.

Fluticasone propionate is a man - made steroid of the glucocorticoid family which is related to naturally - occurring steroid hormone, cortisol or hydrocortisone, produced by adrenal glands. Glucorticoid steroids have potent anti - inflammatory actions. When used as inhaler, Fluticasone propionate goes directly to the airways of the lung. In asthmatic patients, the suppression of inflammation within the airways reduces the spasms that narrows the airways and makes getting air into and out of the lungs difficult. When used in lower doses, very little Fluticasone propionate is absorbed into the body. When higher doses are used Fluticasone propionate is absorbed and cause side effects elsewhere in the in the body.

Salmeterol xinafoate is a long acting beta 2 adrenergic agonist which acts as broncho dilator. It stimulates beta 2 receptors and increases cyclic AMP. However, Salmeterol also interacts with an exosite in the receptor. By this mechanism, Salmeterol remains associated with the receptor, thus providing a prolonged duration of action. In patients with asthma and COPD, Salmeterol is effective upto 12 h.

Tiotropium bromide is a muscarinic receptor antagonist, often referred to as an antimuscuranic or anticholinergic agent. Although it doesnot display selectivity for muscuranic recpetors, on topical application it acts mainly on M# muscuranic receptors that is located on smooth muscle cells and submucosal glands not to produce smooth muscle contraction and mucus secretion, thus producing a bronchodilatory effect. Tiotorpium bromide is indicated as daily, 24 hour, maintenance treatment of chronic obstructive pulmonary disease.

Parameters choosen for study in the present were selected because of their relationship to cellular growth and control of differentiation and because of alterations reported previously in other investigations (reference required).

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Inflammation in the airways is characterized by the presence of dense neutrophil infiltrations. Neutrophils cause progressive airway damage by the release of oxygen metabolites and proteolytic enzymes which interferes with the host defense. There are numerous inflammatory mediators which are elevated in vivo in the airway secretions of cystic fibrosis, asthma and COPD. Interleukin 8 is the chemokine and a proinflammatory mediator that is present at high levels in patients suffering from cystic fibrosis and asthma. Interleukin 8 also known as CXCL 8 is the chemokine which is produced by macrophages and other cell types such as epithelial cells and is encoded by the IL 8 gene. There are numerous receptors present on the cell surface which is capable to bind IL - 8; the most frequently studied types is the G protein coupled serpentine receptors CXCR1 and CXCR2. Expression and affinity to IL - 8 is different in two receptors (CXCR1 >CXCR2). The receptors recognize antigen patterns like LPS in gram negative bacteria. Through a chain of biochemical reactions IL - 8 is secreted. Primary function of IL - 8 is the induction of chemotaxis in its target cells.

The human bronchial epithelial cell line that is used for this experiment is IB3 -1 and C38. IB3 - 1 are an immortalised cell line that was created in 1992 from a primary culture of bronchial epithelial cells that is isolated from a patient with cystic fibrosis. The culture was transformed with a hybrid virus, adeno - 12 - SV40. The IB3 - 1 cell lines are deficient in cyclic AMP - medicated protein kinase A activation of chloride conductance, which is diagnostic of Cystic Fibrosis. Genotypically, the IB3 -1 cell line is a compound hetrozygote containing the delta F508 mutation and a nonsense mutation, W128X, with a premature termination signal. The C38 cell line was derived from the IB3 - 1 cell line. The CF phenotype present in the IB3 -1 cell lines was corrected in C38 cell line by transfection with wild type adeno - associated viral cystic fibrosis transmemberane conductance regulator.

In this project an in vitro human model system is constructed that consist of human bronchial epithelial (HBE) cells to evaluate the potential of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide through by single, dual or triple therapy to study their anti - inflammatory effect.

Method:

Cell culture: Respiratory tract - derived cell lines (IB3 and C38) obtained from the American Type Culture Collection were grown in a complete medium and will be maintained at 37'C in a humidified 5% CO2 atmosphere.

Challenging the Human bronchial epithelial cells: The human bronchial epithelial cells were challenged with LPS so that there is production of IL - 8.

Cells treatments: The human bronchial epithelial cell lines were suspended in a complete medium in the presence or absence of either Fluticasone propionate or Salmeterol xinafoate or Tiotropium bromide. Then cells were either in combination of Fluticasone propionate and Salmeterol xinafoate or Fluticasone propionate and Tiotropium bromide or Salmeterol xinafoate and Tiotropium bromide. The cell lines were then treated with combination Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide.

Exposure of cells to IL - 8:

Measurement LPS activity is by IL - 8 ELISA:

Interleukin - 8 is a member of structurally - related low molecular weight proinflammatory factors known as chemokines. IL - 8 is produced by stimulated monocytes. The IL - 8 ELISA Linked Immunosorbent Assay) kit is an in vitroenzyme linked Immunosorbent assay for the quantitative measurement of human IL - 8 cell lysate and tissue lysate. This assay an antibody specific for human IL - 8 coated on a 96 well plate. Human bronchial epithelial cells secrete chemokines IL - 8 which are soluble in the medium. Then the supernantent is transferred to ELISA well where it get bounds to immobilized antibody. The wells were washed and biotinylated anti - human IL - 8 antibody is added. After washing away unbound biotinylated antibody, HRP - conjugated streptavidin is pipetted out to the wells. The wells are washed again, a TMB substrate solution is added to the wells. The wells were washed again, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of IL - 8 bound. The stop solution changes the colour from blue to yellow and the intensity of the colour is measured at 450 nm.

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This method is highly specific as it doesnot exhibit any cross reactivity with any other cytokines such as human angionenin, BDNF, BLC, ENA - 78, FGF - 4, IL- 1 alpha, IL - 1 beta, Il- 2, IL - 3, IL - 4, IL - 5, IL - 7, IL - 9, IL - 10, IL-11, IL-12 p70, IL- 12 p40, IL-13, IL-15, IL-309, IP-10, G-CSF, GM-CSF, IFN-γ, Leptin, MCP- 1, MCP-2, MCP-3, MDC, MIP-1α, MIP-1 β, MIP-1δ, PARC, PDGF,

RANTES, SCF, TARC, TGF-β, TIMP-1, TIMP-2, TNF-α, TNF-β, TPO,

VEGF).

Cell treatment with drugs and determination of IL - 8 inhibition:

To measure the effects of Fluticasone propionate, Salmeterol xinafoate and Tiotropium bromide on IL - 8 production by IB3 -1 AND C38 cell lines. At first cells were plated and tested with different concentrations of individual drugs in 96 - well plates and incubation is period out. The next day, cells were challenged with LPS and IL - 8 activity was measured. IL - 8 diffuses into the medium.

Apoptosis assay:

The methodology for this experiment involves measurement of chemokines such as Interleukin 8 by IL8 ELISA.

Statistical analysis:

Cell survival among groups was compared

Analysis of IL - 8 exposure data for Statistical analysis will be performed with Microsoft excel Software. Results will be presented as means± SEM,

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Annotated Bibliography.

http://jimmunol.org/content/183/3/2159.full