Adenoviral vectors expressing siRNAs for discovery and validation of gene function

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.     

Scalp topography of the spontaneous K-complex and of delta-waves in human sleep

Objective: Together with spindles, K-complexes are well known hallmarks of stage 2 sleep (S2). However, little is known about their topographical distribution in comparison to delta-waves and to K-complexes superimposed by spindles. Patients and methods: In this study, the topographical distribution of spontaneous K-complexes and delta-waves in S2 and delta-waves in stage 4 sleep (S4) in 10 healthy young adults (aged 20 to 35 years, 7 female) was investigated. K-complexes with and without spindles in S2, delta-waves with and without spindles in S2, and delta-waves in S4 distributed all over the night were visually selected. EEG power maps and statistical parametric maps were calculated. Results: Absolute delta power of S2 K-complexes appeared to be significantly higher than of S2 delta-waves and delta power of S4 delta-waves was higher than of S2 delta-waves. In K-complexes and delta-waves, power was found to be highest over medio-frontal regions in the delta frequency band (0.5 - 4.0 Hz) with a second maximum occipitally in delta-waves, no matter whether superimposed by a spindle or not. Conclusion: K-complexes and delta-waves in S2 differ in topographical distribution. Even though in S2 delta-waves have less power, they have a similar topographical distribution in S2 and S4, supporting the hypothesis that delta-waves in S2 further develop towards delta-waves in slow wave sleep. The delta frequency components of K-complexes and delta-waves are unaffected by spindles.     

Microsatellite analysis of breast carcinoma and corresponding local recurrences

Local recurrence is a serious complication of breast carcinoma that reduces quality of life and influences prognosis. The aim of this study was to determine whether local recurrences of breast carcinoma are genetically related to the primary tumours. Forty cases of locally recurrent breast carcinomas (median onset: 3.6 years after primary surgery) were analysed: 22 patients had undergone breast-conserving therapy and 18 mastectomy. Eighteen microsatellites on chromosomes 2p, 3p, 5q, 10q, 11p, 11q, 13q, 17q, 17p, 18p were amplified by PCR using fluorescent-labelled primers, automatically detected after polyacrylamide gel electrophoresis and analysed for loss of heterozygosity (LOH) or microsatellite instability (MSI). Follow-up data were available for 39 cases with a median value of 89 months. All LOH and MSI found in the primary tumours were also present in the corresponding recurrences, indicating that they are genetically related to the primary tumours and not secondary malignancies in the same breast. MSI was found in three cases, of which one harboured MSI at more than two loci. The median value of LOH per case was significantly higher in the recurrent (four per case) compared to the primary tumours (two per case; p < 0.001, Mann-Whitney test), reflecting the genotype of tumour progression. Early local recurrence was associated with specific LOH for TP53.15 (p = 0.018, log-rank test) in the primary tumours. LOH on D13S1699 or D17S855 was associated with lymph node metastases (p = 0.024 and p = 0.019, respectively; chi-square test). In addition, tumour grade, lack of oestrogen or progesterone receptor expression, young patient age and early appearance of local recurrence significantly correlated with poor survival. The development of local recurrence despite clear resection margins may result from residual DCIS distant from the invasive carcinoma, homing of circulating tumour cells, or genetically altered, histologically normal breast tissue not immediately adjacent to the invasive carcinoma.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Hepatocellular Hyperplasia, Plasmacytoma Formation, and Extramedullary Hematopoiesis in Interleukin (IL)-6/Soluble IL-6 Receptor Double-Transgenic Mice

Cytokines interact not only with membrane anchored receptors, but also with specific soluble receptors which circulate in the bloodstream. In general, soluble cytokine receptors such as soluble tumor necrosis factor receptor, soluble interleukin 1 receptor, and soluble interleukin 4 receptor compete with their membrane-bound counterparts for the ligands and therefore act as antagonists. In contrast, soluble receptors for cytokines of the interleukin-6 (IL-6) family complex with their ligands act agonistically. Interestingly, the complex of IL-6 and the soluble interleukin 6 receptor (sIL-6R) activates target cells that do not express the membrane-bound IL-6R and therefore cannot respond to IL-6. To identify cellular responses that are due to IL-6/sIL-6R but not to IL-6 alone, IL-6/sIL-6R double-transgenic mice were generated and compared with IL-6 single-transgenic mice. IL-6/sIL-6R transgenic mice develop a severe phenotype showing 1) marked hepatocellular hyperplasia frequently surrounded by peliosis and necrosis, 2) significant acceleration and aggravation of plasmacytoma formation, and 3) excessive activation of extramedullary hematopoiesis in spleen and liver followed by a subsequent increase of all cellular components in the peripheral blood. These in vivo data suggest that the sIL-6R recruits primarily unresponsive cell populations such as hematopoietic progenitor cells and hepatocytes to IL-6-induced proliferation, but also enhances the known mitogenic effect of IL-6 on plasma cells and thereby contributes to plasmacytoma formation.     

Physical activity and plasma interleukin-6 in humans – effect of intensity of exercise

The present study included data from three marathon races to investigate the hypothesis that a relationship exists between running intensity and elevated concentrations of interleukin (IL)-6 in plasma. The study included a total of 53 subjects whose mean age was 30.6 [95% confidence interval (CI) 1.4] years, mean body mass 77.7 (95%CI 2.0) kg, mean maximal oxygen uptake (V˙O_2max) 59.3 (95%CI 1.4) ml · min⁻¹ · kg⁻¹, and who had participated in the Copenhagen Marathons of 1996, 1997 or 1998, achieving a mean running time of 206 (95%CI 7) min. Running intensity was calculated as running speed divided by V˙O_2max. The concentration of IL-6 in plasma peaked immediately after the run. There was a negative correlation between peak IL-6 concentration and running time (r=−0.30, P < 0.05) and a positive correlation between peak IL-6 concentration and running intensity (r=0.32, P < 0.05). The IL-1 receptor antagonist (IL-1ra) plasma concentration peaked 1.5 h after the run and there was a positive correlation between the peak plasma concentrations of IL-6 and IL-1ra (r=0.39, P < 0.01). Creatine kinase (CK) plasma concentration peaked on the 1st day after the run, but no association was found between peak concentrations of IL-6 and CK. In conclusion, the results confirmed the hypothesized association between plasma IL-6 concentration and running intensity, but did not confirm the previous finding of a connection between IL-6 plasma concentration and muscle damage. Accepted: 6 August 2000     

Expression of genes in normal human monocytes in response to Aspergillus fumigatus.

The objective of these studies was to investigate genes of importance in the pathogenesis of Aspergillus infections. To do so, we employed microarray methodology to explore gene expression in human monocytes infected with Aspergillus conidia as compared with unstimulated monocytes and those stimulated with lipopolysaccharide (LPS) signaling through TOLL-like receptor 4 (TLR4). We found 997 (P相似文献   

Co‐occurring medical conditions in adults with Down syndrome: A systematic review toward the development of health care guidelines. Part II

Adults with Down syndrome (DS) represent a unique population who are in need of clinical guidelines to address their medical care. Many of these conditions are of public health importance with the potential to develop screening recommendations to improve clinical care for this population. Our workgroup previously identified and prioritized co‐occurring medical conditions in adults with DS. In this study, we again performed detailed literature searches on an additional six medical conditions of clinical importance. A series of key questions (KQ) were formulated a priori to guide the literature search strategy. Our KQs focused on disease prevalence, severity, risk‐factors, methodologies for screening/evaluation, impact on morbidity, and potential costs/benefits. The available evidence was extracted, evaluated and graded on quality. The number of participants and the design of clinical studies varied by condition and were often inadequate for answering most of the KQ. Based upon our review, we provide a summary of the findings on hip dysplasia, menopause, acquired cardiac valve disease, type 2 diabetes mellitus, hematologic disorders, and dysphagia. Minimal evidence demonstrates significant gaps in our clinical knowledge that compromises clinical decision‐making and management of these medically complex individuals. The creation of evidence‐based clinical guidance for this population will not be possible until these gaps are addressed.     

c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.