Lentiviral Based Gene Transduction and Promoter Studies in Human Hematopoietic Stem Cells (hHSCs).

Human hematopoietic stem cells (hHSCs) have enormous potential for clinical use in cell-based therapies, especially as a gene delivery system. Moreover, lentiviral transduction in stem cells is very often associated with low transduction efficiency and low levels of foreign gene expression. Therefore, it is important to analyze vector and promoter systems that can generate robust foreign gene expression in these cells. In this study, we evaluated and compared the ability of different commercially available promoters to drive the expression of exogenous reporter genes in hHSCs and evaluated the effect of different doses of stem cell growth factors on the expression of transgenes. We used lentivirus based vector system carrying the following promoters: 1) Human cytomegalovirus (CMV) promoter, 2) Simian virus 40 (SV40) promoter, 3) mammalian Ubiquitin C (UBC) promoter and 4) cellular polypeptide chain elongation factor 1 alpha (EF1) promoter. EF1 and CMV promoters robustly drove the expression of green fluorescence protein (GFP) reporter gene, while SV40 and UBC promoters induced very low level of GFP expression. Lentivectors containing EF1 and CMV promoters showed high-level stable GFP expression in human cord blood stem cells for 6 weeks period after post transduction. CD133+ hHSCs stimulated with higher concentration of growth factors exhibited enhancement of transduction rate. Cord blood derived CD133+ hHSCs could be effectively transduced with lentivectors under CMV or EF-1 promoters for the expression of foreign gene.

Colonic vasoactive intestinal polypeptide (VIP) in ulcerative colitis--a radioimmunoassay and immunohistochemical study.

BACKGROUND/AIMS: In this study, we present radioimmunoassay data describing the concentration of Vasoactive Intestinal Polypeptide (VIP) in both plasma and colonic biopsies, as well as immunostaining of VIPergic innervation in mucosal biopsies of normal subjects and patients with ulcerative colitis (UC). PATIENTS AND METHODS: Thirty three patients with UC and 17 healthy subjects were investigated. All UC patients suffered from active disease. Fasting circulating levels of VIP in plasma as well as tissue concentrations were measured by radioimmunoassay. For the immunohistochemistry, polyclonal antibody against VIP and the streptavidin-biotin peroxidase complex technique were carried out. RESULTS: Overall plasma VIP concentrations in the UC patients were similar to those in the controls. Significantly decreased concentrations of VIP were found in UC of rectum compared to the normal tissue. However, both plasma VIP values and tissue concentrations were found to be significantly lower in patients expressing minimal or mild active disease according to clinical activity index (AI) and histological activity index (HAI), but marked increase of plasma VIP was clear in UC patients with moderate or severe AI and HAI. There was a trend towards increased tissue concentrations of VIP in the group of patients with moderate or severe AI and HAI. Our immunohistochemical analysis of VIP fibers and nerve cell bodies revealed consistently weaker VIP-immunoreactivity in the rectum in UC patients with minimal or mild HAI. Simultaneously, in the rectal biopsies from UC patients with moderate and severe disease, the fibers in the lamina propria and ganglion cells in the submucous plexus were markedly increased in density and in degree of immunostaining. Very strong immunoreactivity was also found in inflammatory cells of the lamina propria as well as in the epithelial layer of the biopsies from UC patients with obvious disease. CONCLUSIONS: Our study shows clearly the heterogeneity in the response of VIP plasma level as well as rectum concentration and distribution in UC patients at different stages of the active disease. The possible role of VIP in the colon suggests that further studies of the alterations of this gut peptide may be useful in the understanding of UC pathophysiology.

Rat pancreatic B-cells after chronic alcohol feeding. A morphometric and fine structural study.

Quantitative analysis of the light microscopic and fine structure of rat islet B-cells was carried out in chronic alcoholism. Absolute pancreatic weight and volume were similar in groups C (control) and E (ethanol), but relative pancreatic weight in group E rat was decreased. The results for fasting blood glucose and insulin levels were similar in the two groups of animals. There was a significantly reduced total pancreatic islet volume in E rats. The total number of endocrine cells both per islet and per microns2 of islet was similar in the two groups of animals. The volume density and number of B-cells per islet and per microns2 of islet were not changed in ethanol-treated rats as compared with the control. On the other hand, diameter, surface area and volume of the B-cells and their nuclei were found to be statistically significantly decreased. Histological examination revealed that islet blood vessels were dilated in alcoholic rats. Over the 4-month period of ethanol intake a significant decrease in cell profile area, nuclear profile area and volume density of cytoplasmic granules and an increase in the profile area and volume density of endoplasmic reticulum occurred. The gross histological alteration seen in most B-cells of the ethanol-treated rats was irregularity of the nuclear envelope with deep invagination and with margination of heterochromatin and many empty granules or granules without clear electron dense crystals of insulin. The present results indicate some optical and structural abnormalities of B-cells in chronic alcoholism that may be related to cell dysfunction and may contribute, at least in part, to the endocrine pancreas functional disturbance.