白血病骨髓移植病人全血微量元素铜的变化及其意义

为了研究白血病骨髓移植病人全血微量元素铜的变化及其意义 ,用原子吸收光谱法检测了正常对照组与白血病骨髓移植患者预处理前及移植后骨髓空虚期全血微量元素铜的含量。结果表明 ,正常对照组全血铜浓度与白血病骨髓移植患者预处理前全血铜浓度差异无显著性 (P >0 0 5 ) ,而与骨髓空虚期全血铜浓度有显著性差异 (P <0 0 5 ) ,说明白血病缓解后血铜接近正常对照组水平 ,而白血病骨髓移植患者预处理后骨髓空虚期全血铜含量高于正常对照组 ,说明预处理影响微量元素铜的代谢 ,本文就其影响机理进行了研究     

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion‐pairing HPLC‐MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post‐transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high‐performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB‐C₁₈ column (3.0 × 150 mm, 5 μm) with addition of an ion‐pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r² > 0.98) were obtained for most analytes. Intra‐ and inter‐day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.     

Transplantation of betacellulin-transduced islets improves glucose intolerance in diabetic mice

Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic β cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes β-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes.     

Bruch’s-Mimetic Nanofibrous Membranes Functionalized with the Integrin-Binding Peptides as a Promising Approach for Human Retinal Pigment Epithelium Cell Transplantation

Background: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch’s membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes. Methods: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch’s-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome. Results: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation. Conclusions: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.     

The potential use of mesenchymal stem cells in hematopoietic stem cell transplantation

In the last 10 years, mesenchymal stem cells (MSCs) have emerged as a therapeutic approach to regenerative medicine, cancer, autoimmune diseases, and many more due to their potential to differentiate into various tissues, to repair damaged tissues and organs, and also for their immunomodulatory properties. Findings in vitro and in vivo have demonstrated immune regulatory function of MSCs and have facilitated their application in clinical trials, such as those of autoimmune diseases and chronic inflammatory diseases. There has been an increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT), including hematopoietic stem cell engraftment and the prevention and treatment of graft-versus-host disease (GVHD), and their therapeutic potential has been reported in numerous clinical trials. Although the safety of clinical application of MSCs is established, further modifications to improve their efficacy are required. In this review, we summarize advances in the potential use of MSCs in HSCT. In addition, we discuss their use in clinical trials of the treatment of GVHD following HSCT, the immunomodulatory capacity of MSCs, and their regenerative and therapeutic potential in the field of HSCT.     

Discovery of structurally diverse natural product antagonists of chemokine receptor CXCR3

The chemokines (CXCL9, CXCL10 and CXCL11) and associated CXCR3 receptor are expressed during the inflammatory process from multiple sclerosis, atherosclerosis or organ transplantation resulting in the recruitment of lymphocytes leading to tissue damage. It is hypothesized that blocking of the ligand/CXCR3 receptor interaction has potential to provide opportunity for development of agents that would block tissue rejection. In this paper, four classes of natural product inhibitors (IC₅₀ ranging 0.1–41 M) have been described that block the CXCR3 receptor interaction of IP-10 ligand. These include a cyclic thiopeptide (duramycin), polyketide glycosides (roselipins), steroidal glycosides (hypoglausin A and dioscin) and a novel alkyl pyridinium alkaloid that were isolated by bioassay-guided fractionation of the organic extracts derived from actinomycete, fungal, plant and marine sources and discovered using ¹²⁵ I IP-10/CXCR3 binding assay. Duramycin was the most potent with an IC₅₀ of 0.1 M. Roselipins 2A, 2B and 1A showed IC₅₀ values of 14.6, 23.5, and 41 M, respectively. Diosgenin glycosides dioscin, hypoglaucin A and kallstroemin D exhibited IC₅₀ values of 2.1, 0.47 and 3 M, respectively. A novel cyclic 3-alkyl pyridinium salt isolated from a sponge displayed a binding IC₅₀ of 0.67 M.     

Determination of Immunosuppressive Pharmaceuticals in Whole Blood Following Kidney Transplantation by High-performance Liquid Chromatography–Tandem Mass Spectrometry

Cyclosporine, tacrolimus, sirolimus, and everolimus are commonly used immunosuppressants following organ transplantation. Their monitoring is used to determine the optimal dose for therapeutic effectiveness and minimize toxicity. High-performance liquid chromatographic–tandem mass spectrometry with positive electrospray ionization and multiple reaction monitoring mode was validated for the determination of cyclosporine A, tacrolimus, sirolimus, and everolimus in whole blood. A C18 analytical column was employed with a gradient elution of pH 4.0 aqueous 10?mmol/L ammonium acetate and acetonitrile. For the pretreatment of whole blood, simple protein precipitation was used with methanol:zinc sulfate. The calibration curves were linear from 20.0 to 1000?ng/mL for cyclosporine A, 1.0 to 50?ng/mL for tacrolimus and sirolimus, and 1.0 to 30?ng/mL for everolimus. The intra-assay precision and inter-assay precision were less than 15%. The method provides reliable and reproducible results according to the linearity, precision, accuracy, recovery, and matrix effects. The method has been introduced to routine clinical practice in Slovakia for the determination of immunosuppressants in patients after kidney transplantation.     

Bilirubin protects grafts against nonspecific inflammation-induced injury in syngeneic intraportal islet transplantation

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1β, TNF-α and IFN-γ) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1β, TNF-α, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.     

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.     

CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode

Cytidine 5′‐triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography‐mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V_max and K_m of CTPS and verified that an inhibition of the enzyme activity was induced after 3‐deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis‐Menten kinetics for lysates of resting cells were K_m=280±310 μmol/L for UTP, V_max=83±20 pmol/min and, for lysates of activated PBMCs, K_m=230±280 μmol/L for UTP, V_max=379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.