Adenovirus 5 and chimeric adenovirus 5/F35 employ distinct B-lymphocyte intracellular trafficking routes that are independent of their cognate cell surface receptor

Gene transfer applications with adenovirus (Ad) type 5 are limited by its native tropism, hampering their use in several cell types. To address this limitation, several Ad vectors bearing chimeric fiber have been produced to take advantage of the different cellular receptors used by other subgroups of Ads. In this study, we have compared the transduction efficiency of Ad5 and the chimeric Ad5/F35 in primary human B lymphocytes and B-cell lines as a function of the developmental stage. We found that transduction efficiencies of the two Ads differ independently of their targeted cellular receptor but are related to the intracellular localization of the virus. In efficiently transduced cells, Ads were localized in early endosomes or cytosol, whereas in poorly transduced cells they were localized within late endosomes/lysosomes. Finally, we demonstrate that treatment of cells with phosphatase inhibitors known to redirect endocytosis towards caveolae, increased Ad5/F35 transduction efficiency.     

Comfort Theory: a unifying framework to enhance the practice environment

The application of theory to practice is multifaceted. It requires a nursing theory that is compatible with an institution's values and mission and that is easily understood and simple enough to guide practice. Comfort Theory was chosen because of its universality. The authors describe how Kolcaba's Comfort Theory was used by a not-for-profit New England hospital to provide a coherent and consistent pattern for enhancing care and promoting professional practice, as well as to serve as a unifying framework for applying for Magnet Recognition Status.     

Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome

Lessard M, Chouiali A, Drouin R, Sébire G, Corbin F. Quantitative measurement of FMRP in blood platelets as a new screening test for fragile X syndrome. The fragile X syndrome usually results from CGG repeats expansion and methylation of the FMR1 gene leading to the absence of expression of its encoded protein, fragile X mental retardation protein (FMRP). Therefore, its diagnosis is traditionally based on the detection of these molecular alterations. As an alternative, FMRP-based screening methods have been proposed over the years. Most of them are based on immunohistochemistry analyses applied to a restricted number of lymphocytes (100) or hair roots (10-20) with limited diagnosis potential. In this study, we describe a truly quantitative approach using a new model, the blood platelet, which can be recovered easily with very high purity (99.9%). FMRP levels in platelets were first measured in a control population (n = 124) and reference values were established. FMRP measurements were also performed in confirmed fragile X subjects. Receiver operating characteristic curve analysis has shown that our test can easily discriminate fragile X males and females from controls (area under curve, AUC = 0.948). Cognitive functions were also assessed in these individuals using age-specific Wechsler Intelligence Scales for Children and the Vineland Adaptive Behavior Scales. A proportional relationship between FMRP levels, intelligence quotient and adaptive behavior was observed among fragile X individuals, suggesting that our test would be able to detect fragile X cases and may predict cognitive functions.     

Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells.     

Modifications in apoprotein C composition of very low density lipoproteins in type IV hyperlipoproteinemic subjects after dietetic triglyceridemia reduction

Variations in peptide composition of apoprotein C from very low density lipoproteins were studied in 10 type IV hyperlipoproteinemics, before and after triglyceridemia normalization through appropriate diet. The subjects were sensitive to carbohydrates or alcohol : a reduction or elimination of the inducing nutrient led to a decrease in serum triacylglycerol levels, correlated with an increase in the proportion of apo CII and a decrease in the proportion of apo CIII1. Apo CIII2 was not affected. This led to an increased ratio, apo CII/apo CIII1. The percentage of variation of different lipid (triacylglycerols and cholesterol) and protein (apoproteins CII, CIII2) parameters were also studied.     

Efficient gene transfer into normal human B lymphocytes with the chimeric adenoviral vector Ad5/F35

The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than na?ve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.     

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.     

Abnormalities of cardiac repolarization in multiple sclerosis: Relationship with a model of allergic encephalomyelitis in rat

Ventricular repolarization was investigated for the first time in 48 multiple sclerosis (MS) patients using measurement of QTc interval on standard electrocardiographic recordings. The repolarization process was prolonged significantly in MS compared to control subjects (P = 0.0001). This result was confirmed with an animal model of MS, i.e., the experimental allergic encephalomyelitis in rat. The contribution of prolonged QT to syncopal attack or sudden cardiac death in MS patients need further investigation. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:940–942, 1998.