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C Arcelloni

Publications and source records attributed to C Arcelloni.

At least 19 recordsLinked to original sources
PubMed2000

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. The procedure here reported requires only three days to obtain 0.016-0.270 mg of the pure and characterised proteins, starting from 370-900 ml of culture media, and is suitable for the analysis of a large number of RANTES analogues.

Baculoviridae↗
PubMed2000

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). In the 6-24 h range, the ceftazidime concentrations between 16 and 1 microg/ml were +/-1 serial dilution of the values reported on the strip, confirming the accuracy of the E test in agar.

Agar↗
PubMed2000

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. All the cells transduced with the wild-type gene released intact proinsulin. HepG2 released a considerable amount of 65-66 split and des64,65, while primary myoblasts released all the intermediate forms and a limited amount of mature insulin. All the cells transduced with a furin-sensitive proinsulin gene released a higher amount of mature insulin (23-59% conversion yield) than the cells expressing wild-type proinsulin, whereas the total insulin was nearly constant. Only primary cells released all the cleavage products. Screening a wide variety of non-endocrine cells has revealed a large difference in the processing and release of immature and mature insulin forms, pointing to human hepatic cells as the most efficacious. Capillary electrophoresis provided on-line and in a single run a complete overview of the proinsulin metabolic pathway in different cells.

3T3 Cells↗
PubMed1999

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. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Animals↗
PubMed1999

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). Of interest, CD34(+)/CD38(+) committed progenitor cells, unlike primitive CD34(+)/CD38(-) cells, expressed SDF-1 mRNA. Supernatants from in vitro-cultured CD34(+) cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34(+) cells. Because CD34(+) cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34(+) cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34(+) cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4(+) T cells or CD34(+) cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34(+) cells. However, CXCR4 on CD34(+) cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34(+) progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34(+) cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4.

Anti-HIV Agents↗
PubMed1999

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. CE technique was useful to evaluate the purity of human SDF-1alpha from chemical synthesis and to resolve murine and human SDF-1alpha, differing by only one amino acid. Chemotactic activity of the murine SDF-1alpha was tested on the basis of CE quantification.

Animals↗
PubMed1999

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. Offspring of diabetic parents, notwithstanding normal fat content and distribution, were characterized by insulin resistance and increased intramyocellular triglyceride content in the soleus (P < 0.01) but not in the tibialis anterior (P = 0.19), but showed a normal content of saturated/unsaturated carbons in the fatty acid chain of subcutaneous adipocytes. Stepwise regression analysis selected intramyocellular triglyceride soleus content and plasma free fatty acid levels as the main predictors of whole body insulin sensitivity. In conclusion, 1H and 13C NMR spectroscopy revealed intramyocellular abnormalities of lipid metabolism associated with whole body insulin resistance in subjects at high risk of developing diabetes, and might be useful tools for noninvasively monitoring these alterations in diabetes and prediabetic states.

Adult↗
PubMed1998

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 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).

Chromatography, High Pressure Liquid↗
PubMed1998

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. aeruginosa inoculated plates (5.1 +/- 1.7 micrograms/ml, n = 6) vs. OXOID and DID (1.6 +/- 0.3 micrograms/ml, n = 12). BD plates gave also a different inhibition zone diameter (26 +/- 0.5 mm, n = 3) with respect to DID and OXOID (29 +/- 0.5 mm, n = 3).

Agar↗
PubMed1998

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. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85+/-14% (n = 5, mean+/-standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.

Electrophoresis, Capillary↗
PubMed1997

Quantitative cytometry of MHC class I digestion from living cells.

Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta2-microglobulin (beta2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). beta2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, "immature" MHC molecules.

Animals↗
PubMed1997

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. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer.

Antibiotics, Antineoplastic↗
PubMed1996

Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.

For an efficient antitumor cytotoxic response, tumor antigenic peptides need to be presented by professional antigen-presenting cells in association with MHC class I molecules. We established in vitro short-term human CTL lines from healthy and melanoma-bearing subjects, using as antigen-presenting cells autologous adherent cells after phagocytosis of latex beads coated with melanoma peptides. Melanoma peptides were obtained by acid extraction of melanoma cells that matched with donor peripheral blood mononuclear cells, at least for one HLA-A allele. The cytotoxic activity of the lines was specific for the melanoma from which peptides were obtained and for melanoma sharing HLA alleles. These results demonstrate that a complex mixture of naturally processed melanoma peptides conjugated to a phagocytic substrate that targets them into the MHC class I pathway of adherent cells can prime a CTL response in healthy subjects in vitro, and that peptides from allogeneic tumors may be used to propagate CTL in melanoma patients. Our data support the feasibility of active and passive vaccination procedures with nonliving vaccines in cancer patients.

Animals↗
PubMed1996

Ceftazidime kinetics of diffusion in inoculated agar plates.

The ceftazidime concentration in agar plates inoculated with Pseudomonas aeruginosa was determined by high-performance liquid chromatography at fixed points (3, 6, 9, 12, and 15 mm) from the disk center and at fixed times (2, 4, 6, 16, and 24 h) to study the antibiotic kinetics of diffusion. A statistical difference between the concentrations determined in the presence of microorganisms and in uninoculated plates after 16 and 24 h was evidenced and was probably ascribable to the drug hydrolysis carried out by the induced beta-lactamase.

Agar↗
PubMed1995

Behavior of frozen serum pools and lyophilized sera in an external quality-assessment scheme.

To further improve analytical accuracy in clinical chemistry, proficiency testing needs amelioration in the quality of materials tested and in target value assignment. To obtain information on the actual state-of-the-art in the Lombardy region of Italy, and to examine the behavior of different types of control materials (fresh-frozen human sera and lyophilized materials), we developed the following experimental design. Two human serum pools and two lyophilized sera were distributed to 32 laboratories for determination of glucose, creatinine, cholesterol, sodium, potassium, and gamma-glutamyltransferase (gamma-GT). Each analyte was measured in triplicate on each of 3 days. Target values for the controls were obtained with Reference Methods. The results show a good intralaboratory precision for every component but some accuracy problems for glucose, creatinine, cholesterol, and gamma-GT. Lyophilized materials showed some commutability problems for glucose, electrolytes, and gamma-GT, mainly with dry chemistry technology.

Blood↗
PubMed1995

HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue.

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

Animals↗