Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. Results: A significant MHC Class I-restricted, anti-IE1-specificCTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation. Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens

Red blood cells protect albumin from cigarette smoke\u2013induced oxidation

Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke-induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins

Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

his protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed

A step-by-step protocol for assaying protein carbonylation in biological samples

Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience

N-acetylcysteine ethyl ester as GSH enhancer in human primary endothelial cells: A comparative study with other drugs

Several drugs are currently in use as glutathione (GSH) enhancers in clinical, pre-clinical and experimental research. Here we compare the ability of N-acetylcysteine (NAC), 2-oxothiazolidine-4-carboxylic acid (OTC), glutathione ethyl ester (GSH-EE) and N-acetylcysteine ethyl ester (NACET) to increase the intracellular concentration of GSH using primary human umbilical vein endothelial cells (HUVEC) as in vitro model. Our experiments highlighted that NACET is largely the most efficient molecule in increasing the intracellular levels of GSH, cysteine, and γ-glutamylcysteine. This is because NACET is lipophilic and can freely cross plasma membrane but, inside the cell, it is de-esterified to the more hydrophilic NAC, which, in turn, is trapped into the cell and slowly transformed into cysteine. The higher availability of cysteine is matched by an increase in GSH synthesis, cysteine availability being the rate limiting step for this reaction. Surprisingly, the increase in GSH concentration was not linear but peaked at 0.5 mM NACET and gradually decreased when cells were treated with higher concentrations of NACET. We demonstrated that this puzzling ceiling effect was due to the fact that NAC released from NACET turned out to be a competitive inhibitor of the enzyme glutamate-cysteine ligase, with a Ki value of 3.2 mM. By using a cell culture medium lacking of cysteine and methionine, we could demonstrate that the slight increase in intracellular levels of cysteine and GSH induced by NAC in HUVEC grown in standard medium was due to the reduction of the cystine present in the medium itself there rather than to the action of NAC as Cys pro-drug. This fact may explain why NAC works well as GSH enhancer at very high concentrations in pre-clinical and in vitro studies, whereas it failed in most clinical trials

Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum

We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories