What have transgenic and knockout animals taught us about respiratory disease?

Over the past decade there has been a significant shift to the use of murine models for investigations into the molecular basis of respiratory diseases, including asthma and chronic obstructive pulmonary disease. These models offer the exciting prospect of dissecting the complex interaction between cytokines, chemokines and growth related peptides in disease pathogenesis. Furthermore, the receptors and the intracellular signalling pathways that are subsequently activated are amenable for study because of the availability of monoclonal antibodies and techniques for targeted gene disruption and gene incorporation for individual mediators, receptors and proteins. However, it is clear that extrapolation from these models to the human condition is not straightforward, as reflected by some recent clinical disappointments. This is not necessarily a problem with the use of mice itself, but results from our continued ignorance of the disease process and how to improve the modelling of complex interactions between different inflammatory mediators that underlie clinical pathology. This review highlights some of the strengths and weaknesses of murine models of respiratory disease

Regional differences in the pattern of airway remodeling following chronic allergen exposure in mice

BACKGROUND: Airway remodeling present in the large airways in asthma or asthma models has been associated with airway dysfunction in humans and mice. It is not clear if airways distal to the large conducting airways have similar degrees of airway remodeling following chronic allergen exposure in mice. Our objective was to test the hypothesis that airway remodeling is heterogeneous by optimizing a morphometric technique for distal airways and applying this to mice following chronic exposure to allergen or saline. METHODS: In this study, BALB/c mice were chronically exposed to intranasal allergen or saline. Lung sections were stained for smooth muscle, collagen, and fibronectin content. Airway morphometric analysis of small (0–50000 μm(2)), medium (50000 μm(2)–175000 μm(2)) and large (>175000 μm(2)) airways was based on quantifying the area of positive stain in several defined sub-epithelial regions of interest. Optimization of this technique was based on calculating sample sizes required to detect differences between allergen and saline exposed animals. RESULTS: Following chronic allergen exposure BALB/c mice demonstrate sustained airway hyperresponsiveness. BALB/c mice demonstrate an allergen-induced increase in smooth muscle content throughout all generations of airways, whereas changes in subepithelial collagen and fibronectin content are absent from distal airways. CONCLUSION: We demonstrate for the first time, a systematic objective analysis of allergen induced airway remodeling throughout the tracheobronchial tree in mice. Following chronic allergen exposure, at the time of sustained airway dysfunction, BALB/c mice demonstrate regional differences in the pattern of remodeling. Therefore results obtained from limited regions of lung should not be considered representative of the entire airway tree

Blood neutrophil activation markers in severe asthma: lack of inhibition by prednisolone therapy

BACKGROUND: Neutrophils are increased in the airways and in induced sputum of severe asthma patients. We determined the expression of activation markers from circulating neutrophils in severe asthma, and their supressibility by corticosteroids. METHODS: We compared blood neutrophils from mild, moderate-to-severe and severe steroid-dependent asthma, and non-asthmatics (n = 10 each). We examined the effect of adding or increasing oral prednisolone (30 mg/day;1 week). RESULTS: Flow cytometric expression of CD35 and CD11b, but not of CD62L or CD18, was increased in severe asthma. F-met-leu-phe increased CD11b, CD35 and CD18 and decreased CD62L expression in all groups, with a greater CD35 increase in severe asthma. In severe steroid-dependent asthma, an increase in prednisolone dose had no effect on neutrophil markers particularly CD62L, but reduced CD11b and CD62L on eosinophils. Phorbol myristate acetate-stimulated oxidative burst and IL-8 release by IL-1β, lipopolysaccharide and GM-CSF in whole blood from mild but not severe asthmatics were inhibited after prednisolone. There were no differences in myeloperoxidase or neutrophil elastase release from purified neutrophils. CONCLUSION: Because blood neutrophils in severe asthma are activated and are not inhibited by oral corticosteroids, they may be important in the pathogenesis of severe asthma

Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

Allergen-specific immunotherapy provides immediate, long-term and preventive clinical effects in children and adults: the effects of immunotherapy can be categorised by level of benefit -the centenary of allergen specific subcutaneous immunotherapy

Allergen Specific Immunotherapy (SIT) for respiratory allergic diseases is able to significantly improve symptoms as well as reduce the need for symptomatic medication, but SIT also has the capacity for long-term clinical effects and plays a protective role against the development of further allergies and symptoms. The treatment acts on basic immunological mechanisms, and has the potential to change the pathological allergic immune response. In this paper we discuss some of the most important achievements in the documentation of the benefits of immunotherapy, over the last 2 decades, which have marked a period of extensive research on the clinical effects and immunological background of the mechanisms involved. The outcome of immunotherapy is described as different levels of benefit from early reduction in symptoms over progressive clinical effects during treatment to long-term effects after discontinuation of the treatment and prevention of asthma. The efficacy of SIT increases the longer it is continued and immunological changes lead to potential long-term benefits. SIT alone and not the symptomatic treatment nor other avoidance measures has so far been documented as the therapy with long-term or preventive potential. The allergic condition is driven by a subset of T-helper lymphocytes (Th2), which are characterised by the production of cytokines like IL-4, and IL-5. Immunological changes following SIT lead to potential curative effects. One mechanism whereby immunotherapy suppresses the allergic response is through increased production of IgG4 antibodies. Induction of specific IgG4 is able to influence the allergic response in different ways and is related to immunological effector mechanisms, also responsible for the reduced late phase hyperreactivity and ongoing allergic inflammation. SIT is the only treatment which interferes with the basic pathophysiological mechanisms of the allergic disease, thereby creating the potential for changes in the long-term prognosis of respiratory allergy. SIT should not only be recognised as first-line therapeutic treatment for allergic rhinoconjunctivitis but also as secondary preventive treatment for respiratory allergic diseases

Effects of nitrogen fertilizer and elevated CO<sub>2</sub> on dry matter production and yield of rice cultivars

A pot experiment was conducted at Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur during the year 2003 to find out the dry matter production and yield of rice cultivars under different nitrogen levels and growing conditions. Thirty-day old single seedlings were transplanted in pot and were placed in 3 growing conditions, such as i) Open top chamber (OTC) with elevated CO2 (570 ± 50 ppm), ii) OTC with ambient CO2 (360 ± 50 ppm), and iii) open field condition. The three nitrogen levels used were, i) control, ii) optimum dose, and iii) supra optimum dose. Three rice cultivars used in the experiment were, i) BRRI dhan 39, ii) Khashkani, and iii) Shakkarkhora. Rice yield and dry matter production respond significantly to different environments. Increasing atmospheric CO2 increased grain yield. Stem dry weight, leaf dry weight, leaf sheath dry weight and root dry weight were increased in elevated CO2 than ambient CO2 and field condition. BRRI dhan 39 gave highest yield (50.82 g/plant) at supra optimum N level in elevated CO2. Local variety gave similar result under elevated CO2 in optimum and supra optimum N levels. The lowest yield (15.09 g/plant) was produced by Shakkorkhora in field condition with no nitrogen application. Key Words: Nitrogen; elevated CO2; yield; dry matter.DOI: 10.3329/bjar.v34i2.5804Bangladesh J. Agril. Res. 34(2): 313-322, June 2009</jats:p

Effects Of CO&lt;sub&gt;2&lt;/sub&gt; And Nitrogen Levels On Yield And Yield Attributes Of Rice Cultivars

A pot experiment was conducted at Bangbandhu Sheikh Mujibur Rahman Agricultural University during July–December of 2003 to determine the effect of rice varieties under CO2 enrichment and different levels of nitrogen supply. Plants were grown from seedling to maturity inside open top chamber under elevated CO2 (570 ±50) ppm, ambient CO2 (~360ppm) and open field condition. Cultivars responded considerably under different nitrogen levels. Increasing atmospheric CO2 directly stimulated photoynthesis and plant growth resulting in increased grain yield. Among the cultivars, BRRIdhan 39 gave the highest yield (50.82 g/plant1) at supra optimum N level and elevated CO2. Local varieties gave similar results under elevated CO2 in optimum and supra optimum N level. The lowest yield was produced by the local variety Shakkorkhora (15.09 g) under ambient CO2 with no nitrogen application. Keywords: CO2 enrichment; nitrogen level; rice cultivars DOI: http://dx.doi.org/10.3329/bjar.v36i2.9247 BJAR 2011; 36(2): 213-221</jats:p

Localization of β 2-adrenoceptor messenger RNA in human and rat lung using in situ hybridization: Correlation with receptor autoradiography

We have used in situ hybridization to study the localization of mRNA encoding the β 2-adrenoceptor in tissue sections of the human and rat lung and compared this with the distribution of β 2-receptor binding sites using receptor autoradiography. To localize β 2-receptor mRNA, a [ 32P]labeled antisense RNA probe (riboprobe) was generated from human or rat β 2-receptor cDNA. A similar distribution of β 2-receptor mRNA was identified in both species. The highest intensity of β 2-receptor mRNA was detected in smooth muscle of small airways, airway epithelium and pulmonary blood vessels. Lower intensity of β 2-receptor mRNA was identified in smooth muscle of large airways, and alveolar epithelium (presumably type I and type II pneumocytes). No significant hybridization signal was detected in interstitial tissue. The specificity of the hybridization signal was confirmed with a sense probe (having identical sequence to the mRNA) and preincubation with RNase A, and by Northern blot analysis which revealed a single band of mRNA of 2.2 kb. There was a correspondence between mRNA localization and the distribution of β 2-receptors visualized by [ 125I]iodocyanopindolol autoradiographically in the presence of CGP 20712 (a β 1-selective antagonist). However, alveolar walls that showed a high β 2-receptor density had relatively low levels of mRNA. This cellular heterogeneity may reflect differences in RNA stability or transcription rate in different lung cells. This approach opens up new options in the investigation of the regulation of pulmonary β 2-receptor gene expression in health and disease.link_to_subscribed_fulltex