Neurotrophins and neurotrophin receptors in pulmonary sarcoidosis - granulomas as a source of expression

<p>Abstract</p> <p>Background</p> <p>Pulmonary sarcoidosis is an inflammatory disease, characterized by an accumulation of CD4⁺lymphocytes and the formation of non-caseating epithelioid cell granulomas in the lungs. The disease either resolves spontaneously or develops into a chronic disease with fibrosis. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been suggested to be important mediators of inflammation and mediate tissue remodelling. In support of this, we have recently reported enhanced NGF levels in the airways of patients with pulmonary sarcoidosis. However, less is known about levels of BDNF and NT-3, and moreover, knowledge in the cellular sources of neurotrophins and the distribution of the corresponding neurotrophin receptors in airway tissue in sarcoidosis is lacking.</p> <p>Methods</p> <p>The concentrations of NGF, BDNF and NT-3 in bronchoalveolar lavage fluid (BALF) of 41 patients with newly diagnosed pulmonary sarcoidosis and 27 healthy controls were determined with ELISA. The localization of neurotrophins and neurotrophin receptors were examined by immunohistochemistry on transbronchial lung biopsies from sarcoidosis patients.</p> <p>Results</p> <p>The sarcoidosis patients showed significantly enhanced NT-3 and NGF levels in BALF, whereas BDNF was undetectable in both patients and controls. NT-3 levels in BALF were found higher in patients with non-Löfgren sarcoidosis as compared to patients with Löfgren's syndrome, and in more advanced disease stage. Epithelioid cells and multinucleated giant cells within the sarcoid granulomas showed marked immunoreactivity for NGF, BDNF and NT-3. Also, immunoreactivity for the neurotrophin receptor TrkA, TrkB and TrkC, was found within the granulomas. In addition, alveolar macrophages showed positive immunoreactivity for NGF, BDNF and NT-3 as well as for TrkA, TrkB and TrkC.</p> <p>Conclusions</p> <p>This study provides evidence of enhanced neurotrophin levels locally within the airways of patients with sarcoidosis. Findings suggest that sarcoid granuloma cells and alveolar macrophages are possible cellular sources of, as well as targets for, neurotrophins in the airways of these patients.</p

Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type.</p> <p>Results</p> <p>Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals.</p> <p>Conclusions</p> <p>We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.</p

Differential regulation of neurotrophin expression in human bronchial smooth muscle cells

BACKGROUND: Human bronchial smooth muscle cells (HBSMC) may regulate airway inflammation by secreting cytokines, chemokines and growth factors. The neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to be elevated during airway inflammation and evoke airway hyperresponsiveness. We studied if HBSMC may be a source of NGF, BDNF and NT-3, and if so, how inflammatory cytokines may influence their production. METHODS: Basal and cytokine (IL-1β, IFN-γ, IL-4)-stimulated neurotrophin expression in HBSMC cultured in vitro was quantified. The mRNA expression was quantified by real-time RT-PCR and the protein secretion into the cell culture medium by ELISA. RESULTS: We observed a constitutive NGF, BDNF and NT-3 expression. IL-1β stimulated a transient increase of NGF, while the increase of BDNF had a later onset and was more sustained. COX-inhibitors (indomethacin and NS-398) markedly decreased IL-1β-stimulated secretion of BDNF, but not IL-1β-stimulated NGF secretion. IFN-γ increased NGF expression, down-regulated BDNF expression and synergistically enhanced IL-1β-stimulated NGF expression. In contrast, IL-4 had no effect on basal NGF and BDNF expression, but decreased IL-1β-stimulated NGF expression. NT-3 was not altered by the tested cytokines. CONCLUSION: Taken together, our data indicate that, in addition to the contractile capacity, HBSMC can express NGF, BDNF and NT-3. The expression of these neurotrophins may be differently regulated by inflammatory cytokines, suggesting a dynamic interplay that might have a potential role in airway inflammation

Combined FDG-PET/CT for the detection of unknown primary tumors: systematic review and meta-analysis

The aim of this study was to systematically review and meta-analyze published data on the diagnostic performance of combined 18F-fluoro-2-deoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in the detection of primary tumors in patients with cancer of unknown primary (CUP). A systematic search for relevant studies was performed of the PubMed/MEDLINE and Embase databases. Methodological quality of the included studies was assessed. Reported detection rates, sensitivities and specificities were meta-analyzed. Subgroup analyses were performed if results of individual studies were heterogeneous. The 11 included studies, comprising a total sample size of 433 patients with CUP, had moderate methodological quality. Overall primary tumor detection rate, pooled sensitivity and specificity of FDG-PET/CT were 37%, 84% (95% CI 78–88%) and 84% (95% CI 78–89%), respectively. Sensitivity was heterogeneous across studies (P = 0.0001), whereas specificity was homogeneous across studies (P = 0.2114). Completeness of diagnostic workup before FDG-PET/CT, location of metastases of unknown primary, administration of CT contrast agents, type of FDG-PET/CT images evaluated and way of FDG-PET/CT review did not significantly influence diagnostic performance. In conclusion, FDG-PET/CT can be a useful method for unknown primary tumor detection. Future studies are required to prove the assumed advantage of FDG-PET/CT over FDG-PET alone and to further explore causes of heterogeneity

FDG PET/CT in carcinoma of unknown primary

Carcinoma of unknown primary (CUP) is a heterogeneous group of metastatic malignancies in which a primary tumor could not be detected despite thorough diagnostic evaluation. Because of its high sensitivity for the detection of lesions, combined 18F-fluoro-2-deoxyglucose positron emission tomography (FDG PET)/computed tomography (CT) may be an excellent alternative to CT alone and conventional magnetic resonance imaging in detecting the unknown primary tumor. This article will review the use, diagnostic performance, and utility of FDG PET/CT in CUP and will discuss challenges and future considerations in the diagnostic management of CUP

ESC Joint Working Groups on Cardiovascular Surgery and the Cellular Biology of the Heart Position Paper: Perioperative myocardial injury and infarction in patients undergoing coronary artery bypass graft surgery

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Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick

Background The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. Results Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. Results One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.</p