Current Issue : April - June Volume : 2015 Issue Number : 2 Articles : 6 Articles
Background: Basement matrices such as Matrigelââ??¢ and Geltrexââ??¢ are used in a variety of cell culture assays of\nanchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix\nrecommended for these assays (approximately 150 ?l/cm2) are costly, limit working distances for microscopy, and require\ncell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer\nangiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.\nResults: Geltrexââ??¢ basement matrix at 5 ?l/cm2 in 24-well (10 ?l) or 96-well (2 ?l) plates supports endothelial cell\ndifferentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working\ndistances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily.\nUsing MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the\n3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial\ntotal RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial\ncells without the need to initially detach cells from their supporting matrix.\nConclusions: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation\nof endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with\nthe added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and\nhigh-resolution microscopy....
Background: The organic chemicals present in Asian sand dust (ASD) might contribute to the aggravation of lung\neosinophila. Therefore, the aggravating effects of the Tar fraction from ASD on ovalbumin (OVA)-induced lung\neosinophilia were investigated.\nMethods: The Tar fraction was extracted from ASD collected from the atmosphere in Fukuoka, Japan. ASD\ncollected from the Gobi desert was heated at 360�°C to inactivate toxic organic substances (H-ASD). ICR mice were\ninstilled intratracheally with 12 different test samples prepared with Tar (1 ?g and 5 ?g), H-ASD, and OVA in a\nnormal saline solution containing 0.02% Tween 80. The lung pathology, cytological profiles in the bronchoalveolar\nlavage fluid (BALF), inflammatory cytokines/chemokines in BALF and OVA-specific immunoglobulin in serum were\ninvestigated.\nResults: Several kinds of polycyclic aromatic hydrocarbons (PAHs) were detected in the Tar sample. H-ASD + Tar\n5 ?g induced slight neutrophilic lung inflammation. In the presence of OVA, Tar 5 ?g increased the level of\neosinophils slightly and induced trace levels of Th2 cytokines IL-5 and IL-13 in BALF. Also mild to moderate goblet\ncell proliferation and mild infiltration of eosinophils in the submucosa of airway were observed. These pathological\nchanges caused by H-ASD + OVA were relatively small. However, in the presence of OVA and H-ASD, Tar, at as low\na level as 1 ?g, induced severe eosinophil infiltration and proliferation of goblet cells in the airways and significantly\nincreased Th2 cytokines IL-5 and IL-13 in BALF. The mixture showed an adjuvant effect on OVA-specific IgG1\nproduction.\nConclusions: These results indicate that H-ASD with even low levels of Tar exacerbates OVA-induced lung\neosinophilia via increases of Th2-mediated cytokines. These results suggest that ASD-bound PAHs might contribute\nto the aggravation of lung eosinophila....
Background: Primary pulmonary lymphoma (PPL) is rare and easily misdiagnosed because of the lack of typical\nclinical features. It most commonly involves elderly patients aged between 60 and 70 years, and pathological\ndiagnosis depends mainly on chest surgery rather than bronchial mucosal biopsy. Via percutaneous needle\naspiration biopsy of the lung of a 33-year-old woman, which had distinct tissue eosinophilia, we diagnosed a rare\ncase of rapidly growing large B cell lymphoma.\nMethods: Bronchial mucosal biopsy and computed tomographyââ?¬â??guided percutaneous needle aspiration biopsy\nwere performed to determine the nature of the lesion, and we identified its immunophenotype using\nimmunohistochemistry. We used BIOMED-2 gene rearrangement PCR to determine lymphocyte clonality; laser\nmicrodissection was used to confirm the clonality of suspicious malignant lymphocytes.\nResults: Morphologically, the lesion was composed of a large number of eosinophilic cells and a few lymphoid\ncells. Immunohistochemical staining revealed a few CD1?-positive cells, but they were S-100ââ?¬â??negative. The small\nlymphoid cells predominantly expressed CD3; the large lymphoid cells expressed CD20 and some scattered large\nlymphoid cells expressed Pax5. However, molecular studies confirmed clonal immunoglobulin heavy chain (IGH)-D\ngene rearrangement in Pax5ââ?¬â??positive large B lymphocytes.\nConclusions: This is the first recorded case of T- cell/histiocyte-rich large B cell lymphoma with tissue eosinophilia\nof the lung. It highlights the unusual morphological features of PPL that might be mistaken for eosinophilic granuloma\nor parasitic infection. In addition, IGH and T cell receptor gene rearrangement play important roles in differentiating rare\nB cell lymphoma from lung spaceââ?¬â??occupying lesions with abundant eosinophils or T cell infiltration....
Background: Right ventricular dysfunction in COPD is common, even in the absence of pulmonary hypertension.\nThe aim of the present study was to examine the effects of high intensity interval training (HIIT) on right ventricular\n(RV) function, as well as pulmonary blood vessel remodeling in a mouse model of COPD.\nMethods: 42 female A/JOlaHsd mice were randomized to exposure to either cigarette smoke or air for 6 hours/day,\n5 days/week for 14 weeks. Mice from both groups were further randomized to sedentariness or HIIT for 4 weeks.\nCardiac function was evaluated by echo cardiography and muscularization of pulmonary vessel walls by\nimmunohistochemistry.\nResults: Smoke exposure induced RV systolic dysfunction demonstrated by reduced tricuspid annular plane systolic\nexcursion. HIIT in smoke-exposed mice reversed RV dysfunction. There were no significant effects on the left\nventricle of neither smoke exposure nor HIIT. Muscularization of the pulmonary vessels was reduced after exercise\nintervention, but no significant effects on muscularization were observed from smoke exposure.\nConclusions: RV function was reduced in mice exposed to cigarette smoke. No Increase in pulmonary vessel\nmuscularization was observed in these mice, implying that other mechanisms caused the RV dysfunction. HIIT\nattenuated the RV dysfunction in the smoke exposed mice. Reduced muscularization of the pulmonary vessels due\nto HIIT suggests that exercise training not only affects the heart muscle, but also has important effects on the\npulmonary vasculature....
Background: Up to 50% of septic patients develop acute kidney injury (AKI). The\npathomechanism of septic AKI is poorly understood. Therefore, we established an\ninnovative rodent model to characterize sepsis-induced AKI by standardized colon\nascendens stent peritonitis (sCASP). The model has a standardized focus of\ninfection, an intensive care set up with monitoring of haemodynamics and\noxygenation resulting in predictable impairment of renal function, AKI parameters\nas well as histopathology scoring.\nMethods: Anaesthetized rats underwent the sCASP procedure, whereas sham\nanimals were sham operated and control animals were just monitored invasively.\nHaemo dynamic variables and blood gases were continuously measured. After 24 h,\nanimals were reanesthetized; cardiac output (CO), inulin and PAH clearances\nwere measured and later on kidneys were harvested; and creatinine, urea,\ncystatin C and neutrophil gelatinase-associated lipocalin (NGAL) were analysed.\nAdditional sCASP-treated animals were investigated after 3 and 9 days.\nResults: All sCASP-treated animals survived, whilst ubiquitous peritonitis and\nsignificantly deteriorated clinical and macrohaemodynamic sepsis signs after 24 h\n(MAP, CO, heart rate) were obvious. Blood analyses showed increased lactate\nand IL-6 levels as well as leucopenia. Urine output, inulin and PAH clearance\nwere significantly decreased in sCASP compared to sham and control. Additionally,\nsignificant increase in cystatin C and NGAL was detected. Standard parameters like\nserum creatinine and urea were elevated and sCASP-induced sepsis increased\nsignificantly in a time-dependent manner. The renal histopathological score of\nsCASP-treated animals deteriorated after 3 and 9 days.\nConclusions: The presented sCASP method is a standardized, reliable and\nreproducible method to induce septic AKI. The intensive care set up, continuous\nmacrohaemodynamic and gas exchange monitoring, low mortality rate as well\nas the opportunity of detailed analyses of kidney function and impairments are\nadvantages of this setup. Thus, our described method may serve as a new\nstandard for experimental investigations of septic AKI....
Background: In an earlier study we demonstrated the feasibility to create tissue engineered venous scaffolds\nin vitro and in vivo. In this study we investigated the use of tissue engineered constructs for ureteral replacement in\na long term orthotopic minipig model. In many different projects well functional ureretal tissue was established\nusing tissue engineering in animals with short-time follow up (12 weeks). Therefore urothelial cells were harvested\nfrom the bladder, cultured, expanded in vitro, labelled with fluorescence and seeded onto the autologous veins,\nwhich were harvested from animals during a second surgery. Three days after cell seeding the right ureter was\nreplaced with the cell-seeded matrices in six animals, while further 6 animals received an unseeded vein for ureteral\nreplacement. The animals were sacrificed 12, 24, and 48 weeks after implantation. Gross examination, intravenous\npyelogram (IVP), H&E staining, Trichrome Masson�s Staining, and immunohistochemistry with pancytokeratin\nAE1/AE3, smooth muscle alpha actin, and von Willebrand factor were performed in retrieved specimens.\nResults: The IVP and gross examination demonstrated that no animals with tissue engineered ureters and all\nanimals of the control group presented with hydronephrosis after 12 weeks. In the 24-week group, one tissue\nengineered and one unseeded vein revealed hydronephrosis. After 48 weeks all tissue engineered animals and\nnone of the control group showed hydronephrosis on the treated side. Histochemistry and immunohistochemistry\nrevealed a multilayer of urothelial cells attached to the seeded venous grafts.\nConclusions: Venous grafts may be a potential source for ureteral reconstruction. The results of so far published\nureteral tissue engineering projects reveal data up to 12 weeks after implantation. Even if the animal numbers of\nthis study are small, there is an increasing rate of hydronephrosis revealing failure of ureteral tissue engineering\nwith autologous matrices in time points longer than 3 months after implantation. Further investigations have to\nprove adequate clinical outcome and appropriate functional long-term results....
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