Improved lentiviral vectors for Wiskott-Aldrich syndrome gene therapy mimic endogenous expression profiles throughout haematopoiesis.

Wiskott-Aldrich syndrome (WAS) gene therapy requires highly efficient and well-controlled vectors. Here we studied the performance of a lentiviral vector (LV) harbouring a 500-bp fragment of the WAS proximal promoter (WW), which we previously characterized as haematopoietic-specific and capable of restoring WAS phenotype in patients' T cells. We used an LV (WE) expressing eGFP to evaluate whether this promoter was following the expression pattern of endogenous WASp. Transgene expression was analysed in WE-transduced hCD34+ population and its progeny after in vitro and in vivo differentiation in the Rag2-/-, gammac-/- humanized mouse. We revealed very poor expression from the WE internal promoter in macrophages and erythroid cells. Therefore, we designed a novel LV including a fragment of the alternative WAS promoter in WE vector (AWE). This new vector sustained high transgene levels along the whole lymphoid lineage in vivo. Most importantly, the performance of AWE vector was highly superior to WE vector since AWE clearly improved transgene levels in in vitro and in vivo hCD34+-derived macrophages, erythroid cells, megakaryocytes and B cells while supporting a high expression in human T cells. This emphasizes that it is a suitable LV backbone for gene therapy of haematopoietic diseases such as WAS.

Nitrogenous compounds of interest in clinical nutrition.

The term "conditionally essential" (or semi-essential), initially applied to amino acids, has been generalized to other nutrients. A conditionally essential nutrient is a compound usually produced in adequate amounts by endogenous synthesis but that is exogenously required under certain circumstances. Thus, arginine, glutamine, cysteine, glycine, carnitine, choline, and polyamines are conditionally essential compounds. In addition, dietary nucleotides are considered semi-essential since some rapidly growing tissues such as the gut, bone marrow, and lymphocytes, preferentially use preformed purine and pyrimidine bases for nucleic acid synthesis. This review discusses the study of conditionally essential nitrogenous nutrients of interest in clinical nutrition. Among them we highlight arginine, involved in endothelial, immune, gastrointestinal, and renal functions, in reproduction, neonatal development, wound healing, and tumorigenicity; glutamine, necessary for maintaining bowel integrity, and with beneficial effects on catabolic states such as sepsis, infection, trauma, and cancer; and nucleotides, implicated in cell growth and differentiation, and with various effects on lipid metabolism, intestinal microbiota, and immune system.

Exogenous nucleosides modulate the expression of rat liver extracellular matrix genes in single cultures of primary hepatocytes and a liver stellate cell line and in their co-culture.

BACKGROUND & AIMS: We have previously reported the antifibrotic effect of dietary nucleotides in cirrhotic rats. In this work, we used primary rat hepatocytes, a liver stellate cell line (CFSC-2G) and co-cultures of both cell types to investigate the effects of exogenous nucleosides on the gene expression of various extracellular matrix components and on markers of liver function, and to ascertain whether the effects found in vivo are due to CFSC-2G, hepatocytes, or are the consequence of cell-cell interactions. RESULTS: Nucleosides enhanced fibronectin, laminin, and alpha1(I) procollagen levels in CFSC-2G and hepatocytes, as well as collagen synthesis and secretion in CFSC-2G. In contrast, nucleosides lowered fibronectin, laminin and alpha1(I) procollagen levels, and decreased collagen synthesis in co-cultures. Matrix metalloproteinase-13 content and collagen secretion increased in co-cultures incubated with nucleosides. Albumin increased in hepatocytes and co-cultures incubated in the presence of nucleosides. CONCLUSIONS: Nucleosides modulate the production of extracellular matrix in single cultures of hepatocytes and of CFSC-2G, and in co-cultures. This effect seems to be regulated at the translational level. The opposite behavior of single cultures and co-cultures is probably due to the fact that the latter model reproduces many of the physical and functional relationships observed in vivo between hepatocytes and stellate cells.