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Immunological Basis of Disease - Essay Example

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The "Immunological Basis of Disease" paper analizes such researches as "Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-β1" by Woodman, L., Siddiqui, S., Cruse, G., Sutcliffe, A., Saunders, R., Kaur, D., et al…
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Immunological Basis of Disease
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Immunological Basis of Disease Interpretation of a Research Paper Paper chosen: (Please see attached) Woodman, L., Siddiqui, S., Cruse, G., Sutcliffe, A., Saunders, R., Kaur, D., et al. (2008). Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-β1. The Journal of Immunology, 181, 5001-5007, http://www.jimmunol.org/cgi/content/full/181/7/5001 accessed on 4 November 2009. 2. Aim(s) of the paper and how these aims were addressed in general terms (20%): The major aim of the paper was to show that mast cells promote or induce the differentiation of airway smooth muscle (ASM) to a more contractile phenotype, which results in airway dysfunction and inflammation observed in asthma. This aim was achieved by following these procedures: a. Comparison of bronchial tissues from asthmatic and non-asthmatic patients by bronchial examination and biopsy. The immunohistochemistry of the proximal airway of bronchial biopsies were conducted by staining them with monoclonal antibodies against smooth muscle actin, and tryptase for mast cells, followed by image analysis. b. Measuring the effect of coculturing of mast cells and ASM on production of smooth muscle actin and TGF-β. Pure cultures of human lung mast cells and airway smooth muscle were produced. ASM cells were labelled with mABs for smooth muscle actin (SMA). ASM cells were cultured with human mast cells, and mast cells that have been sensitized with protease inhibitor and lactoferrin (to inhibit the action of tryptase, a protease produced by mast cells). Under these coculture conditions, the production of smooth muscle actin protein and gene expression were determined periodically for one week using immunofluorescence, flow cytometry and quantitative real-time PCR. Cell free supernatants of the cocultures were analysed for production of TGF-β1 and TGF-β2 using ELISA. TGF-β ( transforming growth factor beta) is a protein that controls cell proliferation and differentiation c. The effect of the addition of recombinant TGF-β on ASM proliferation and contractile activity of ASM pure cultures was also measured by examining SMA protein expression by flow cytometry, and ASM contractile activity by collagen gel analysis. 3. Choose ONE immune-based method used in the paper and, with reference to the paper and the literature, describe this method including its underlying principles. (Use diagrams where appropriate). (No more than 500 words excluding diagram(s)) (30%). An immune-based method used in the paper was ELISA, or enzyme-linked immunosorbent assay. In the paper being reviewed, ELISA was used to quantify the expression of TGF-β1 and TGF-β2 in supernates of ASM and mast cells cultures. ELISA is a very useful tool in identifying the presence or absence of proteins or other antigens. ELISA utilizes the specificity of binding of antibody to the antigen against which it was designed. The basic principle behind the method is the use of an enzyme that is covalently linked to an antibody that specifically recognizes the target antigen. The same enzyme has its own specific substrate that upon reaction, results in the production of a coloured product. If the specific antigen is present in solution, the antibody-enzyme complex will bind to the antigen; at the same time, the enzyme will catalyze its own reaction with its substrate, therefore generating a coloured product. The coloured product indicates the presence of the antigen in the sample. ELISA can be performed with monoclonal and polyclonal antibodies, although specificity of results is obtained with the use of monoclonal antibodies. ELISA is rapid, and can detect proteins of less than a nanogram. There are many types of ELISA, but they can be categorized into two general types, the indirect, and the sandwich (Figure 1) (Goldsby et al., 2000). Figure 1. Indirect and sandwich ELISA The indirect ELISA detects the presence of antibody. Antigen is coated to the wells of ELISA plates. The antigen-specific antibody is allowed to bind to the antigen followed by allowing bound antibody to bind an enzyme-linked antibody. After washing off unbound antibodies, enzyme substrate is added to produce the color. Sandwich ELISA is used to detect and quantitate the amounts of antigen. First, antibody is bound to the wells, followed by addition of the specific antigen. Then a second and different antibody is bound to the antigen. The second antibody is enzyme-linked, and reacts with its specific substrate to give the colored product. The extent of reaction allows for the quantitation of small amounts of antigen (Berg et al., 2002). The absorbance of the coloured product can be measured using an ELISA reader or spectrophotomer. 4. Discuss the immunological basis of the problem being investigated using diagrams where appropriate. (NB you may need to look up aspects of this in textbooks or papers). (No more than 500 words excluding diagram(s)). (30%) Asthma is a disease which is characterized by difficulty in breathing due to the narrowing of airways into the lungs and the production of mucus. A broad number of events including allergies, viruses, chemicals, dust, physical activity, and even emotional stress can trigger asthma. Common worldwide, asthma significantly causes morbidity and mortality. In asthma, variable airflow obstruction, airway hyperresponsiveness and inflammation, cytokine expression, eosinophilic inflammation result in disordered airway physiology (Figure 2). Figure 2. The involvement of immune cells and inflammatory agents in asthma (figure from Barnes, 2008). Figure 2 presents the immunological basis of asthma (Barnes, 2008). Allergens activate mast cells by interacting and crosslinking with mast cells surface IgE molecules. The interaction signals the release of bronchoconstrictors. The role of mast cells, residing in all body tissues, has been found to be important in causing disordered airway physiology. The keys role of mast cells in asthma are seen by their secretion of cytokines, bronchoconstrictors like histamines, and the increased production of lipid mediators leukotrienes and prostaglandins. Leukotrienes specially are potent mediators that are involved in the production of pro-inflammatory compounds and the activation of smooth muscle in animals in vitro and in vivo (Drazen & Israel, 1999). Stem-cell factor (SCF) is released by epithelial cells and is important for retaining the mast cells at the airways’ surfaces. Chemokines are also released after the allergens are engaged by dendritic cells, which are major components of the immune system. T helper 2 (TH2) cells are recruited by the chemokine receptors. The helper cells release interleukins resulting in inflammatory response. Interleukin (IL) - 4 and IL-13 stimulate B cells to produce IgE; interleukin-5 has an essential role in eosinophilic inflammation. Eosinophils are recruited by chemokine receptor CCR3. Mast cell proliferation is stimulated by IL-9. Further proliferation of TH2 in asthmatic could be promoted by defects in regulatory T cells. The constriction in bronchial airways, made up of airway smooth muscle (ASM) is the common event during an asthma attack. Airway muscle mass has been found to be increased in patients with asthma. Explanations for the increased mass have been attributed to the inflammatory environment surrounding the muscles in asthmatic patients; and the presence of smooth muscle growth factors. It is possible that the increase in mass in asthmatic patients is due to inflammatory elements surrounding the muscle (Shore, 2004). However, it has also been proposed that the smooth muscle mass of asthmatic patients are intrinsically different from non-asthmatic individuals, and that differences account for excessive airway narrowing during an asthma attack (Figure 3, adapted from Shore 2004). The origin and cause of increased muscle mass and contractility is one of the questions that the current paper being reviewed wants to answer. Figure 3. Intrinsic differences in normal and asthmatic airway smooth muscles and consequent airway narrowing during asthma attacks 5. What is your overall opinion of the paper (e.g. is it clearly written?). In terms of the methodology, are appropriate controls done? Can you suggest any other experiments? Are the conclusions drawn valid? Give reasons for your answer. (No more than 500 words) (20%) The paper is relatively well written. The abstract gives an over-all picture of the problem, the significance of the study, the necessity for the research, the hypothesis that the authors wanted to prove, and a short description of the methodology and results. The abstract also states the conclusion of the study while at the same time hinting at the work that still needs to be done in future experiments. The introduction was concise and presented the rationale and objective of the research in a straightforward manner. The state of the art on the subject matter was briefly presented. The introduction also gave a summary of the major results. The methodology was written in a very detailed and careful manner. This is ideal for other researchers who want to duplicate the experiments, but a nonprofessional reading the article will get lost in all the details. Since the journal article is available as open access online, and because the problem (asthma) being discussed is universal in nature, presentation of the methodology in a less technical voice is recommended. If this is not possible, then a short summary using popular terminology is suggested. The technical aspects required in a research study were present in the design of the experiments. The researchers used appropriate isotype controls when needed. In determining the bronchial physiology, samples were also taken from patients without asthma to compare the bronchial tissues and biopsies. The different treatments imposed on the cell cocultures also made the appropriate isotype controls, aside from varying treatments. Moreover, the study results were also subjected to statistical analysis to test for statistical validity. The paper concludes that the localization of mast cells near the airway smooth muscles bundle allows for interaction, which resulted in the alteration of the ASM phenotype to a more contractile one as measured by smooth muscle actin expression and increased muscle contractibility. This conclusion is valid based on an examination of the results, which were presented in graphs, tables, and images. All told, the results point to the effect of mast cells β-tryptase and recombinant TGF-β on the proliferation and contractility of airway smooth muscle. Image and immunochemistry of bronchial biopsies showed that there was increased mast cell within the vicinity of the airway smooth muscles of asthmatic patients. Improvements in the experiments are proposed as follows: 1. To conduct sampling of tissues from asthmatic patients regularly to understand the changes that occur in airway muscles and mast cell physiology. 2. Since the experiments were performed under cell culture conditions, it is recommended that experiments should also be carried out on asthmatic patients or in animal models. However, it is also recognized that currently, there is no reliable animal model that can be used in studying mast cell-airway smooth muscle interaction. References Barnes, P. (2008). Immunology of asthma and chronic obstructive pulmonary disease. Nature Reviews Immunology, 8, pp.183-192. Berg, J. et al (2002). Biochemistry. 5th ed. W.H. Freeman and Co. Drazen, J. and Israel, E. O. (1999) Treatment of asthma with drugs modifying the leukotriene pathway. The New England Journal of Medicine, 340 (3), pp. 197-208. Goldsby, R. et al. (2000). Kuby Immunology. 4th ed. W.H. Freeman and Company. Shore, S. (2004) Airway smooth muscle in asthma - not just more of the same. The New England Journal of Medicine, 351 (6), pp. 531-532. Woodman, L. et al. (2008) Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-β1. The Journal of Immunology, 181, pp. 5001-5007 http://www.jimmunol.org/cgi/content/full/181/7/5001 [accessed 4 November 2009]. Read More
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