Ectopic FOXP3 Expression Preserves Primitive Features Of Human Hematopoietic Stem Cells While Impairing Functional T Cell Differentiation.

FOXP3 is the transcription factor ruling regulatory T cell function and maintenance of peripheral immune tolerance, and mutations in its coding gene causes IPEX autoimmune syndrome. FOXP3 is also a cell-cycle inhibitor and onco-suppressor in different cell types. In this work, we investigate the effect of ectopic FOXP3 expression on HSC differentiation and we challenged this approach as a possible HSC-based gene therapy for IPEX. FOXP3-expressing HSC showed reduced proliferation ability and increased maintenance of primitive markers in vitro in both liquid and OP9-ΔL1 co-cultures. When transplanted into immunodeficient mice, FOXP3-expressing HSC showed significantly enhanced engraftment ability. This was due to a pronounced increase in the frequency of repopulating cells, as assessed by extreme limiting dilution assay. Likely underlying the increased repopulating ability, FOXP3 expressing HSC showed significantly enhanced expression of genes controlling stemness features. However, peripheral T cells developed in the FOXP3-humanized mice were quantitatively reduced and hyporesponsive to cytokine and polyclonal stimulation. Our findings reveal unpredicted effects of FOXP3 in the biology of HSC and may provide new tools to manipulate primitive features in HSC for clinical applications. Moreover, they formally prove the need of preserving endogenous FOXP3 regulation for an HSC-based gene therapy approach for IPEX syndrome.

Effect of storage time and natural corrosion inhibitor on carbohydrate and carboxylic acids content in canned tomato puree.

In this research compositional changes of tinplate-canned tomato purées, with or without the addition of essential onion oil were investigated. The study was focused on the analyses of carbohydrates and carboxylic acids in two groups of canned samples (with or without nitrates) to determine whether their chemical composition was affected with storage time. The measurements were performed by high performance liquid chromatography, during six months of storage. The contents of glucose, fructose and two major organic acids, citric and malic, were found in the concentration range 1.77-1.97%, 1.86-2.09%, 0.60-0.75% and 0.23-0.30%, respectively, in all canned samples. Compared to carbohydrates and organic acids, amino acids were found in minor quantities, among them, as most abundant ones were glutamic acid, arginine, aspartic and γ-amino butyric acids. The results show that contents of carbohydrates and carboxylic acids are significantly affected by the change of storage time in majority of analyzed samples. The results also indicated that the influence of essential onion oil on composition of canned tomato purée is within the range of changes due to storage time measured for all other types of cans. Therefore the addition of essential onion oil as natural efficient corrosion inhibitor, as it was found in our previous work, can be recommended for canned tomato purée.

Analysis of free and esterified ergosterol in tomato products.

A new extraction and chromatographic procedure to quantify free and esterified ergosterol in tomato products was devised. The extraction solution was composed of a dichloromethane/methanol mixture in a 2:1 (v/v) ratio. This extraction solvent allowed for higher ergosterol recovery from tomato products (an average of 25% more) compared to hexane, which is frequently employed for ergosterol extraction. Both free and esterified ergosterol were determined by HPLC reverse-phase chromatography employing a Nova-Pak C-18 column (300 x 3.9 mm), filled with 4 mm average particle size and a guard column of the same material. The elution was performed at a flow rate of 1 mL. min(-1) with a linear gradient of solvent A (methanol/water, 80:20, v/v) and solvent B (dichloromethane). The gradient, starting at sample injection, was from 0 to 50% B for 20 min for the free ergosterol analysis and additional 15 min at 50% B to analyze the ergosterol esters. This technique has proven to be more sensitive for ergosterol determination than other reported chromatographic procedures. Moreover, ergosterol esters, extracted from various fungal sources, separated well and were easily quantified.