Peripheral T cell tolerance occurs early during spontaneous prostate cancer development and can be rescued by dendritic cell immunization.

In the tumor-prone transgenic adenocarcinoma mouse prostate (TRAMP) mouse model we followed the fate of the immune response against the SV40 large T antigen (Tag) selectively expressed in the prostate epithelium during the endogenous transformation from normal cells to tumors. Young (5-7-week-old) male TRAMP mice, despite a dim and patchy expression of Tag overlapping foci of mouse prostate intraepithelial neoplasia, displayed a strong Tag-specific cytotoxic T lymphocyte (CTL) response after an intradermal injection of peptide-pulsed dendritic cells (DC). This response was weaker than the one found in vaccinated wild-type littermates, and was characterized by a reduced frequency and avidity of Tag-specific CTL.

Body weight and glucose metabolism have a different effect on circulating levels of ICAM-1, E-selectin, and endothelin-1 in humans.

Background: Endothelial dysfunction and inflammation are present in both type 2 diabetes mellitus (T2DM) and obesity. In this paper we compared the role of weight loss and of glycaemic control in determining circulating levels of ICAM-1, endothelin-1 (ET-1), and E-selectin in patients with morbid (grade 3) obesity.

Methods And Results: ICAM-1, E-selectin, and ET-1 were higher in obese patients (n=96) than in lean controls (n=30); among obese patients, the three molecules were higher in T2DM patients (n=26) than in patients with normal (NGT, n=43) or impaired (IGT, n=27) glucose tolerance.

Contribution of abnormal insulin secretion and insulin resistance to the pathogenesis of type 2 diabetes in myotonic dystrophy.

Objective: Myotonic dystrophy (MyD), the most common adult form of muscular dystrophy, is often complicated by diabetes. MyD is dominantly inherited and is due to heterozygosity for a trinucleotide repeat expansion mutation in a protein kinase gene able to induce derangement of RNA metabolism responsible of an aberrant insulin receptor expression.

Research Design And Methods: To assess insulin sensitivity and secretion before the onset of diabetes, we studied 10 MyD patients, 10 offspring of type 2 diabetes (OFF), and 10 healthy subjects with no family history of diabetes (control subjects) with dual X-ray energy absorption, euglycemic-hyperinsulinemic clamp (40 mU/[m(2).

High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction.

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected.

Structural determinants of CCR5 recognition and HIV-1 blockade in RANTES.

Certain chemokines act as natural antagonists of human immunodeficiency virus (HIV) by blocking key viral coreceptors, such as CCR5 and CXCR4, on the surface of susceptible cells. Elucidating the structural determinants of the receptor-binding and HIV-inhibitory functions of these chemokines is essential for the rational design of derivative molecules of therapeutic value. Here, we identify the structural determinants of CCR5 recognition and antiviral activity of the CC chemokine RANTES, showing that critical residues form a solvent-exposed hydrophobic patch on the surface of the molecule.

Quantification of gentamicin in Mueller-Hinton agar by high-performance liquid chromatography.

The aim of this study was to optimise a method for gentamicin determination in an agar matrix and to investigate if and how agar composition can affect the gentamicin diffusion kinetics during the agar diffusion tests for antibiotics sensitivity. Gentamicin was separated by RP-HPLC and detected at 365 nm after pre-column derivatization with 1-fluoro-2,4-dinitrobenzene. Recovery (> or = 79%), linearity (r2 > or = 0.

Development and characterization of pituitary GH3 cell clones stably transfected with a human proinsulin cDNA.

Successful beta-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted beta cells. Availability of non-beta cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter.

Enhancement of the HIV-1 inhibitory activity of RANTES by modification of the N-terminal region: dissociation from CCR5 activation.

Although selected chemokines act as natural inhibitors of human immunodeficiency virus (HIV) infection, their inherent proinflammatory activity may limit a therapeutic use. To elucidate whether the antiviral and signaling functions of RANTES can be dissociated, several recombinant analogues mutated at the N terminus were generated and functionally compared with the wild-type (WT) molecule, as well as with three previously described mutants. Substitution of selected residues within the N-terminal region caused a marked loss of antiviral potency.

Comparison of capillary electrophoresis with HPLC for diagnosis of factitious hypoglycemia.

Background: The diagnosis of "factitious hypoglycemia" is essentially based on the disclosure of hypoglycemic agents in blood or urine. The aim of this study was to evaluate the performance of capillary electrophoresis (CE) as a quantitative method for determination of chlorpropamide, tolbutamide, glipizide, gliclazide, and glibenclamide in serum.

Methods: Serum samples (1 mL), with internal standard added, were purified by solid-phase extraction on OASIS(TM) HLB cartridges (Waters), dried under reduced pressure, and reconstituted with 30-60 microL of acetonitrile:H(2)O.

Evaluation of ceftazidime concentration released in agar from an E test strip.

The aim of this study was to evaluate, using high-performance liquid chromatography, the concentration of ceftazidime in agar released from an E test strip, sampling at the edge of the strip at different points (1, 2, 4, 8, 16, 32, 64, and 128 microg/ml) at 6, 15, and 24 h after its deposition on uninoculated plates. From 6 to 24 h, the ceftazidime concentration in agar increased at the graduations 1, 2, and 4 microg/ml (+140, +82, and +58%, respectively), remained fairly constant at 8 microg/ml (-1.9%), and decreased at 16, 32, 64, and 128 microg/ml (-25, -44, -36, and -58%, respectively).

Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment by capillary electrophoresis.

The aim of this study was to compare the metabolic pathway to mature insulin through the intermediate forms (32-33 split, 65-66 split, des31,32 and des64,65) in human or murine cells engineered for the release of wild-type human proinsulin and in a genetically mutated one, in the search for a new approach for an insulin-dependent diabetes mellitus cure by gene therapy. Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and NIH3T3) were transduced either with a retroviral vector coding for wild-type proinsulin or for a genetically mutated one, carrying cleavage sites sensitive to furin. The pattern of all the proinsulin cleavage products released into the cell culture supernatants was analyzed by capillary electrophoresis.

Multi-step purification strategy for RANTES wild-type and mutated analogues expressed in a baculovirus system.

RANTES (regulated on activation, normal T cell expressed and secreted), a C-C chemokine, is one of the major HIV-suppressive factors produced by CD8+ T cells. Wild-type RANTES and genetically modified analogues were expressed in a baculovirus system and purified from cell culture supernatants employing a multi-step strategy based on affinity and RP-HPLC. Quantification and purity control of the final proteins were carried out by capillary electrophoresis using the synthetic or the recombinant wild-type RANTES as a reference.

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker.

Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H-13C nuclear magnetic resonance spectroscopy assessment in offspring of type 2 diabetic parents.

Insulin resistance is the best prediction factor for the clinical onset of type 2 diabetes. It was suggested that intramuscular triglyceride store may be a primary pathogenic factor for its development. To test this hypothesis, 14 young lean offspring of type 2 diabetic parents, a model of in vivo insulin resistance with increased risk to develop diabetes, and 14 healthy subjects matched for anthropomorphic parameters and life habits were studied with 1) euglycemic-hyperinsulinemic clamp to assess whole body insulin sensitivity, 2) localized 1H nuclear magnetic resonance (NMR) spectroscopy of the soleus (higher content of fiber type I, insulin sensitive) and tibialis anterior (higher content of fiber type IIb, less insulin sensitive) muscles to assess intramyocellular triglyceride content, 3) 13C NMR of the calf subcutaneous adipose tissue to assess composition in saturated/unsaturated carbons of triglyceride fatty acid chains, and 4) dual X-ray energy absorption to assess body composition.

High-performance liquid chromatographic purification and capillary electrophoresis quantification of the chemokine stromal cell-derived factor-1.

Chemokines are members of the chemotactic cytokines family implicated in various immunoregulatory functions. The CXC-chemokine stromal cell derived factor-1 (SDF-1alpha) was purified from the culture medium of murine bone marrow stromal cell line (MS-5) by affinity and reversed-phase liquid chromatography. Yield and purity were assessed by capillary electrophoresis (CE) with reference to the human SDF-1alpha from recombinant DNA technology.

Human CD34(+) cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus.

Human CD34(+) hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34(+) cells, although it was expressed at lower density on MPB with respect to BM CD34(+) cells. Freshly isolated and in vitro-cultured CD34(+) cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR).

High-performance liquid chromatographic method for measuring total plasma homocysteine levels.

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.

Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms.

Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm X 50 microm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage.

Is the direct quantitation of antibiotics in agar by high-performance liquid chromatography useful?

The direct quantification of antibiotics in agar allows one to study the quality of the agar matrix, the kinetics of diffusion and the bacteria-antibiotic interaction. Mueller-Hinton agar (MHA) plates from three manufacturers were tested using HPLC and the disc diffusion test of ceftazidime (CAZ). Notable differences in the chromatographic profiles of MHA plate extracts from OXOID, DID and Becton Dickinson (BD) were shown, with a higher CAZ concentration after 24 h a 6 mm in BD P.

Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction.

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum.