Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples.

Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules.

Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry.

Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used arylsulfatase extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.

Model Affitin and PEG modifications onto siRNA lipid nanocapsules: cell uptake and in vivo biodistribution improvements.

Malignant melanoma is an aggressive tumor, associated with the presence of local and/or distant metastases. The development of gene therapy by the use of small interfering RNA (siRNA) represents a promising new treatment. However, the protection of this biomolecule is necessary in order for it to be intravenously administrated, for example via its incorporation into nanomedicines. In parallel to the passive targeting usually obtained by pegylation, various studies have aimed at developing "smart" nanomedicines to efficiently deliver the drug to tumor sites. In this work, siRNA loaded lipid nanocapsules (LNCs) were modified with DSPE-polyethylene glycol (DSPE-PEG), tetraether-PEG (TE-PEG) and/or with an Affitin model, to assay multiple targeting strategies. The uptake of fluorescently labelled LNCs, nanocarrier integrity and siRNA release into human SK-Mel28 melanoma cells were studied by flow cytometry, conventional confocal microscopy and by confocal spectral imaging in a Förster Resonance Energy Transfer (FRET) mode. Surface modified siRNA LNCs were followed after human plasma incubation and after intravenous injection, in order to compare the stealth properties. Finally, the biodistribution of the different siRNA LNCs in healthy and melanoma tumor bearing mice models was assessed by in vivo biofluorescence imaging (BFI), to evaluate the potential tumor targeting ability. The post-insertion of DSPE-PEG induced a strong decrease of the internalization into melanoma cells compared to TE-PEG modification. Both PEG polymer decorations induced a great plasma protection of siRNA but only DSPE-PEG led to stealth properties, even at low concentration (5 mM). The Affitin grafting by thiolation of DSPE-PEG was validated on siRNA LNCs. DSPE-PEG-Affitin LNCs were not detected in this melanoma tumor model but did not show unspecific accumulation in organs. DSPE-PEG and TE-PEG LNCs induced a significant intratumoral accumulation of modified LNCs.

Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism.

While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites.

Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

Molecular Interactions of ß-(1→3)-Glucans with Their Receptors.

ß-(1→3)-Glucans can be found as structural polysaccharides in cereals, in algae or as exo-polysaccharides secreted on the surfaces of mushrooms or fungi. Research has now established that ß-(1→3)-glucans can trigger different immune responses and act as efficient immunostimulating agents. They constitute prevalent sources of carbons for microorganisms after subsequent recognition by digesting enzymes. Nevertheless, mechanisms associated with both roles are not yet clearly understood. This review focuses on the variety of elucidated molecular interactions that involve these natural or synthetic polysaccharides and their receptors, i.e., Dectin-1, CR3, glycolipids, langerin and carbohydrate-binding modules.

The blood of healthy individuals exhibits CD8 T cells with a highly altered TCR Vb repertoire but with an unmodified phenotype.

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vß repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vß repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25-52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vß families with altered CDR3 length distribution and increased TCR Vß/HPRT transcript ratios in all individuals tested. The increase in TCR Vß/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vß(+) T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8(+) T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vß repertoire led to the identification of different types of CD8(+) T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

Autoimmune responses against renal tissue proteins in long-term surviving allograft recipients.

Major histocompatibility complex antigens (MHC) are classical targets of recipient responses to allotransplants. However, the role of an immune response directed against autologous graft tissue determinants is poorly defined. In this study, we investigated (i) whether autologous kidney tissue extract can induce an immune response to autologous kidney proteins in normal rats, and (ii) if a similar autologous response develops in the long-term surviving LEW.1A recipients of an MHC-mismatched LEW.1W kidney (RT1(u) to RT1(a)). LEW.1A rats immunized with allo- or syngeneic soluble kidney extracts developed a T-cell response to self antigens as shown by the frequency of specific IFN-gamma-producing T cells from LEW.1A rats in the presence of extracts (ELISPOT). In contrast, they responded only marginally to dominant RT1(u) determinants. The ELISPOT against fractions of soluble autologous kidney extracts separated by an FPLC gel-filtration system indicated a preferential response to megalin, a high molecular weight protein that has been shown to be involved in experimental Heymann nephritis. In a model of long-term kidney allograft survival by anti-CD28 administration, recipients also developed humoral but not cellular responses to megalin. Our data suggest that autoimmune processes develop in long-term surviving kidney allograft recipients.

Blood CD8+ T cell responses against myelin determinants in multiple sclerosis and healthy individuals.

Patients with multiple sclerosis (MS) display significant peripheral blood CD8(+) T cell receptor biases, suggesting clonal selection. Our objective was to identify relevant myelin-derived peptides capable of eliciting responses of fresh blood CD8+ T cells in MS patients. We focused our analysis on the HLA supertypes (HLA-A3, -A2, -B7, -B27, -B44) predominant in a patient cohort. Three myelin protein (MBP, PLP and MOG) sequences were screened for HLA binding motifs and peptides were tested for their binding to HLA molecules. The cellular responses of 27 MS patients and 19 age- and sex-matched healthy controls (HC) were tested in IFN-gamma ELISPOT assays only detecting pre-committed CD8+ T cells. Sixty-nine new epitopes elicited positive responses, with MOG-derived peptides being the most immunogenic and peptides binding to HLA-A3 being the most frequent. However, MS patients and HC displayed the same frequency of autoreactive cells. The epitopes inducing the strongest responses were not those with the highest HLA binding, suggesting an effective thymic selection in MS patients. Our data extend the concept that the frequency of myelin-reactive T cells in MS patient blood is not increased compared to HC. The description of this set of myelin-derived peptides (MHC class I restricted, recognized by CD8+ T cells) offers new tools to explore the CD8+ cell role in MS.

Long-term cell monitoring of kidney recipients after an antilymphocyte globulin induction with and without steroids.

BACKGROUND: Because of several side effects, the corticosteroid usage has been minimized in kidney transplantation. The increased acute rejection episodes associated with their withdrawal may counterbalance with induction treatment using polyclonal antilymphocyte globulin (ALG). The effects of ALG on blood cell phenotype have already been the subject of several reports. However, to date, no data are available concerning the comparison of blood phenotype when ALG is given with or without steroids and no gene profiling study has been performed. METHODS: We report here on a longitudinal blood cell analysis of a selected cohort of kidney recipients enrolled in a randomized study of steroid avoidance or withdrawal (during 6 months) during ALG induction. RESULTS: In the two groups, ALG quickly and massively depleted all the T cells and natural killer cells, but not B cells. Interestingly, the lymphopenia-driven homeostatic proliferation of CD4 and CD8T cells strongly differed with persistent low CD4 (including CD25CD4) T-cell counts. Effector memory CD8T cells reappeared rapidly. ALG induced apoptosis-associated molecules and increased myeloid cell genes. However, few genes were found differentially expressed with a low fold ratio between the two groups during and at distance of corticotherapy. CONCLUSION: Thus initial steroid avoidance or withdrawal associated with ALG induction has a weak influence on phenotype and transcriptional pattern of blood leukocytes. In contrast, ALG therapy induces an early and strong depletion of all T-cell subsets with contrasted long-lasting homeostatic regulation.

Phenotypically and functionally distinct CD8+ lymphocyte populations in long-term drug-free tolerance and chronic rejection in human kidney graft recipients.

A substantial proportion of long-term kidney graft recipients, including those with a stable renal function in the absence of immunosuppressive therapy, present a skewed T cell receptor (TCR) Vbeta chain usage, essentially in the CD8+ subset. This study analyzed in more detail phenotypical and functional alterations of CD8+ lymphocytes in drug-free tolerant patients (DF-Tol) compared with recipients with chronic rejection (CR). Phenotyping revealed a significant increase in central memory and a decrease in effector CD8+ lymphocytes in DF-Tol versus CR. The expression of CD28+ and CD27+ on these effector cells was significantly decreased in CR. These profiles were stable over time and independent of treatment. Functionally, the CD8+CD28- lymphocytes were less sensitive to apoptosis than their CD8+CD28+ counterparts, without differences in polyclonal proliferation. The CD8+CD28- cells did not express GITR and FoxP3 but were characterized by high levels of preformed perforin and granzyme A, pointing toward a cytotoxic rather than a suppressor function. CD8+CD28- lymphocytes did not show antigen-specific degranulation when co-cultured with targets that bear donor HLA class I antigens, suggesting that the cytotoxicity is directed either to other determinants of the graft or to nongraft epitopes. Of interest, CD8+ cells from DF-Tol displayed the same profile as healthy individuals, indicating an increase in CD8+CD28- effector lymphocytes in CR rather than a decrease in DF-Tol. CD8+ lymphocytes from stable kidney recipients under conventional maintenance immunosuppression displayed a mixed profile, independent of treatment and time of sampling. Taken collectively, these data show a strong cytotoxicity-associated CD8+CD28- signature in CR and suggest a suppression of pathologic cytotoxicity in DF-Tol. Further prospective studies should assess whether serial CD8+ phenotyping may help to identify patients who are at risk for CR when immunosuppression is tapered.

Chronic rejection of human kidney allografts.

Human chronic rejection is an uncommon feature of chronic allograft nephropathy, the major cause of late graft loss in kidney transplantation. Chronic rejection is a complex process, mediated, at least in part, by immunological mechanisms. T cells with donor, tissue or pathogen specificities that react with donor cells damage the graft tissue in synergy with antibodies. The cellular and humoral components of the immune system may be preformed or appear at both early and late time points following kidney transplantation. Both slowly contribute to the development of vasculopathy, glomerulopathy, tubular atrophy and tissue fibrosis, as observed in chronic rejection biopsies. Characterization of the blood transcriptome of patients prone to develop chronic rejection is an objective that may soon be achieved. However, despite increasing knowledge of the pathogenesis of late allograft failure and advances in the identification of patients at high risk, chronic rejection remains poorly responsive to immunosuppressive treatment, reinforcing the implication of both immune and nonimmune causes. Efforts aimed at diminishing the incidence of risk factors, such as acute rejection episodes, transplantation injury and human leukocyte antigen mismatching, and at controlling inflammation and scar processes, are thus important in chronic rejection prevention and control.

Exhaustive depletion of graft resident dendritic cells: marginally delayed rejection but strong alteration of graft infiltration.

BACKGROUND: Donor dendritic cells (DDC) are believed to sustain direct recognition leading to acute allograft rejection. However, DDC are also required for tolerance induction in various models. METHODS: We studied the effect of DDC depletion on major histocompatibility complex (MHC) mismatched rat heart allografts in a strain combination characterized by a DDC-dependant tolerance induction. Grafts were depleted of DDC either by pretreating donors with cyclophosphamide (CyP) or by being parked in an intermediate recipient treated with cyclosporine A (CsA). RESULTS: CyP depleted 95% of resident DC and no specific donor MHC class II staining was observed in parked grafts. Parked grafts survived significantly but only moderately longer than untreated grafts (10.8+/-1.9 days vs. 6.5+/-0.5 days; P<0.05). Compared to unmodified grafts, on day 5 after transplantation, the magnitude of the graft infiltrate was dramatically decreased in DDC-depleted grafts, with IgG deposition within the grafts at the time of rejection. In parallel, the cytokine transcript levels were also lower in these grafts on day 5, but reached levels similar to those of unmodified grafts by day 7, indicating a delayed pattern of rejection. CONCLUSIONS: Taken collectively, these data suggest that DDC depletion has a greater effect on the capacity of tolerance induction than the rejection process.