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Biomedical subjects

E Boschetti

Publications and source records attributed to E Boschetti.

At least 19 recordsLinked to original sources
PubMed1996

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.

Animals↗
PubMed1996

Correlation between clinical and morphologic findings in unstable angina.

This study was undertaken to verify the hypothesis that the discrepant findings in published reports on the prevalence of thrombus in unstable angina depend on the inclusion of different clinical subsets in the various studies. We therefore correlated the clinical characteristics of patients included under the label of unstable angina with the morphologic features assessed by coronary angiography and intravascular ultrasound, and with histopathologic findings of atherectomy specimens. Fifty-eight patients with unstable angina (class B of the Braunwald classification) undergoing coronary arteriography followed by either coronary angioplasty (n = 20) or directional coronary atherectomy (n = 38) were studied. Fifteen patients were in class IB and 43 were in class II to IIIB. Among these 43 patients with angina at rest, 28 had ST-segment elevation during pain, and 15 had ST-segment depression, and 26 developed negative T waves on the baseline electrocardiogram (ECG) as a result of prolonged or repeated episodes of resting chest pain. Intravascular ultrasound examination of the culprit lesion was performed in 43 patients before the interventional procedure, and histopathologic analysis of atherectomy specimens was performed in 38 patients. Complex lesion morphology by angiography was observed in 31 patients (53%) without any significant relation to various clinical subsets. Patients in Braunwald class IB had more calcific plaques than patients in class II to IIIB (p < 0.001). Among patients with angina at rest, those with negative T waves on the baseline ECG, as well as those with transient ST elevation during pain, had a significantly higher incidence of noncalcific lesions (p = 0.001 for both). Analysis of atherectomy specimens revealed acute coronary lesions (thrombus and/or intraplaque hemorrhage) in 18 patients (47%). The incidence of acute coronary lesions was significantly higher in patients with than without negative T waves on the baseline ECG (p = 0.005), and increased further when negative T waves were combined with ST elevation during pain (p = 0.001). Multivariate analysis revealed that the presence orf negative T waves on the baseline ECG was the only explanatory variable related to the presence of acute coronary lesions by histology (p = 0.03). Patient subsets included in the broad spectrum of unstable angina have different morphologic features and incidence of acute coronary lesions by histology. These data provide an explanation for the discrepant findings in published reports on the relevance of thrombus formation in the pathogenesis of unstable angina.

Angina, Unstable↗
PubMed1995

Prolonged antithrombin activity of low-molecular-weight heparins. Clinical implications for the treatment of thromboembolic diseases.

BACKGROUND: The mechanism for the efficacy of once- or twice-daily subcutaneous injections of low-molecular-weight heparins (LMWHs) for the treatment of venous thromboembolism has been difficult to explain. The confusion exists because the observation from experimental studies that the antithrombin activity of LMWHs is necessary for their antithrombotic effect is inconsistent with the reported short half-life of the antithrombin activity of LMWHs. Previous pharmacokinetic studies were performed with lower doses of LMWHs than have been used in contemporary trials, and antithrombin activity was assessed with the barely sensitive chromogenic assay. METHODS AND RESULTS: We performed a pharmacokinetic study to compare the relative half-lives of prophylactic and therapeutic doses of LMWHs assessing antithrombin activity with both the chromogenic and a more sensitive assay (plasma thrombin neutralization assay). An eight-way cross-over randomized study in healthy volunteers was performed. Enoxaparin (20 and 40 mg and 1 and 2 mg/kg) and nadroparin (7500 and 10,000 ICU and 225 and 450 ICU/kg) were administered subcutaneously. The maximal peak activity for aPTT ratio was 1.7. A dose-dependent peak activity was found for both antifactor Xa and antithrombin activities. Disappearance time of these activities after the highest dose of both LMWHs was longer than 16 hours. Overall mean antifactor Xa activity half-life was 4.6 hours. Overall mean antithrombin activity half-life was longer than 4 hours. CONCLUSIONS: Our results provide an explanation for the effectiveness of LMWHs administered either once or twice daily. High and sustained plasma antithrombin activity is achieved when LMWHs are administered in therapeutic doses used in contemporary trials with only a moderate prolongation of the aPTT.

Adult↗
PubMed1995

In vitro toxicity assays for dye ligands used in affinity chromatography.

Some reactive textile dyes have been used for years as biomimetic ligands in protein purification. There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. Therefore, toxicological data are necessary to quantify the level of this hazard. This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies. Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10-62.5 micrograms/ml in several assays.

Chromatography, Affinity↗
PubMed1995

Specific sorbent to remove solvent-detergent mixtures from virus-inactivated biological fluids.

A method involving a sorbent designed to capture specifically virus-inactivating solvent-detergent mixtures is described. Its specificity allows the adsorption of these undesirable chemicals with a high capacity in order to treat large amounts of inactivated biological fluids in small-sized columns. Typically, the volume of sorbent that can be used repeatedly is between one quarter and one tenth of the sample volume to be treated. Cleaning and sanitization can be done by classical methods while strong oxidizing agents can also be used to sterilize the sorbent.

Body Fluids↗
PubMed1995

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports.

HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.

Chromatography, High Pressure Liquid↗
PubMed1994

Manufacture of recombinant proteins with safe and validated chromatographic sorbents.

Purification of recombinant proteins to achieve homogeneity, purity, consistency and potency as required for therapeutic proteins and in vivo diagnostics is performed under stringent and validated conditions. As liquid chromatography is one of the major technologies used for this purpose, it has to be carried out according to special regulatory guidelines. One of the reported aspects is the long-term consistency of a chromatographic process and validation of its operation; other aspects described are more sorbent oriented. In-place cleaning and sterilization are also very important aspects, the efficiency of which is dependent on the chosen working conditions and the chemical nature of the sorbents. Drastic cleaning may deteriorate the chromatographic matrices, releasing chemicals that may contaminate the biologicals of interest, which modifying the behaviour of the chromatographic columns. Moreover, leachable compounds, when present, could have adverse effects in case of high toxicity. Determination of leaching levels and toxicity tests are part of the validation steps to turn chromatographic separations into consistent, effective and safe production processes for biologicals.

Animals↗
PubMed1994

Interferon-alpha is effective in the treatment of HIV-1-related, severe, zidovudine-resistant thrombocytopenia. A prospective, placebo-controlled, double-blind trial.

OBJECTIVE: To determine the effect of interferon-alpha for severe, zidovudine-resistant, HIV-1-related thrombocytopenia. DESIGN: Prospective, randomized, double-blind, placebo-controlled, multicenter, crossover trial. SETTING: Outpatient clinics in Central Northern Italy. PATIENTS: 15 sequential patients positive for HIV-1 with platelet counts less than 25 x 10(9)/L who were refractory to 1 month of full-dose (1000 mg/d) zidovudine. INTERVENTION: Interferon-alpha (3 million units) or placebo (1 mL saline) three times a week subcutaneously for 4 weeks, followed by a 4-week washout period. Patients were then switched to the alternative treatment for the next 4 weeks, followed by another 4 weeks of washout, and they were randomly assigned to either sequence of treatment. Patients received zidovudine (200 mg three times daily) throughout the study. MEASUREMENTS: The primary end point was the platelet count (measured weekly). Secondary end points were qualitative assessment of the platelet response; bleeding time; p24 antigen in serum; CD4/CD8 counts; beta 2-microglobulin in serum; and platelet-associated IgG. RESULTS: Interferon-alpha significantly increased platelet counts in the 12 patients who completed the study (baseline level, 15.6 +/- 7.1 x 10(9)/L; after 4 weeks of interferon-alpha therapy, 82.2 +/- 52.2 x 10(9)/L). The estimated increase in the platelet count after interferon-alpha compared with placebo was 60.0 x 10(9)/L (95% CI, 23.2 to 96.8 x 10(9)/L). The increase was already statistically significant after 3 weeks (66.6 +/- 49.7 x 10(9)/L) and remained significantly increased 1 week after discontinuing interferon-alpha therapy (58.2 +/- 45.0 x 10(9)/L). Placebo did not modify the platelet count. The bleeding time was significantly shortened by interferon-alpha. Four of 12 patients who had more serious alterations of some measures reflecting disease severity did not respond to interferon-alpha. No relevant side effects were observed. CONCLUSIONS: Interferon-alpha is a safe and effective treatment for zidovudine-resistant, HIV-related thrombocytopenia.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Amino acid derivatives of 5-ASA as novel prodrugs for intestinal drug delivery.

In an attempt to obtain site-specific delivery of 5-ASA in the intestinal tract, we have determined the extent of absorption and metabolism of a number of novel 5-ASA derivatives, namely, (N-L-glutamyl)-amino-2-salicylic acid (1), (N-L-aspartyl)-amino-2-salicylic-acid (2), 5-aminosalicyl-L-proline-L-leucine (3), and 5-(N-L-glutamyl)-aminosalicyl-L-proline-L-leucine (4), which are selectively cleaved by intestinal brush border aminopeptidase A and carboxypeptidases. These novel prodrugs, 5-ASA, and sulfasalazine were administered to adult Fisher rats (N = 30) and to animals that had undergone prior colostomy (N = 30). Urine and feces were collected at timed intervals for 48 hr and the metabolites, 5-ASA, and N-acetyl-5-ASA were measured by high-performance liquid chromatography. The absorption and metabolism of all compounds were essentially identical in colostomized and normal animals. 5-ASA exhibited a rapid proximal intestinal absorption as evidenced by the high cumulative urinary excretion (> 65%) and low fecal excretion. Sulfasalazine, as expected, exhibited a lower urinary recovery (< 35%) and higher fecal excretion of 5-ASA and its metabolite. The novel glutamate and aspartate derivatives (1 and 2) behaved similarly to sulfasalazine, while administration of the proline-leucine derivative (3) resulted in urinary and fecal recovery values intermediate with respect to those observed with 5-ASA and sulfasalazine. 5-(N-L-Glutamyl)-aminosalicyl-L-proline-L-leucine yielded the highest fecal recovery of 5-ASA and its N-acetyl derivative, indicating a more efficient delivery to the distal bowel. Amino acid derivatives of 5-ASA appear to be potentially useful prodrugs for the site-specific delivery of 5-ASA to different regions of the intestinal tract.

Aminopeptidases↗
PubMed1994

Toxicity studies on native Procion Red HE-3B and released dye from affinity material exposed to degradative chemical conditions.

Leached ligands from chromatographic packing material submitted to drastic regeneration conditions can contaminate pure biological preparations. These contaminants could have adverse effects from a toxicology point of view that are very poorly documented in liquid chromatography for protein separation. Investigations on toxicity level have been made on released material from immobilized Procion Red HE-3B, after formal identification of the nature of the leached chemical material. Toxicity investigations in vitro involved a number of tests on living cells (eucaryotic and procaryotic) covering different aspects. Behaviour of cells in regular cultures, polyploïdia induction, genotoxicity as well as mechanisms of endocytosis have been studied. Results showed no toxic effects within the range of concentration of dye and dye derivatives studied. Genotoxicity studies in particular did not show any toxic effect over a range of concentration much higher than the regular level of dye leakage from the sorbent.

Cell Division↗
PubMed1994

Toxicity studies on Reactive Blue-2 leached from affinity material exposed to extreme chemical conditions.

Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2. Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions. Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration. Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures. Genotoxicity studies did not evidence any toxic effect in E. Coli cultures of dyes themselves or of the same dyes after metabolic activation.

Cell Adhesion↗
PubMed1994

Synthesis and separation of optically active compounds. Part I.

Biological processes involve a high degree of stereoselectivity. Considering that pharmacological activity is very often associated with only one enantiomer, regulation authorities have defined new guidelines in requiring chiral drugs to be marketed as pure individually safe and effective enantiomers. If large scale production of pure stereoisomers has met until recently a great difficulty to become economically acceptable, the current state of the art in asymmetric synthesis and chiral separation, provides however many industrial applications. Among the methods currently used, asymmetric synthesis, using chiral auxiliaries or chiral catalysts such as enzymes or metal complexes, allows to create new asymmetric centers, starting from prochiral molecules. Regarding racemate resolution, asymmetric synthesis offers the real economical advantage to produce exclusively the targeted enantiomer. However, resolution methods allowing access to both enantiomers, are very useful at preparative scale for new chiral drugs when preclinical studies are requested for each enantiomer. Large scale racemate resolution can be achieved by crystallization, enzymatic resolution and even more recently by liquid chromatography. Although the "unwanted" enantiomer is also produced, the feasibility of its racemization for recycling has made resolution methods widely used in industry. It is clear today that in most cases, pharmacologically active molecules having an asymmetric center will have to be marketed as optically pure drugs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1994

Synthesis and separation of optically active compounds. Part II.

This review is the second part of an article devoted to the synthesis and separation of optically active compounds. This article deals mainly with chiral chromatography which represents an important component of the state of art for the preparation of enantiomers, as illustrated by a description and a classification of the main packing materials available to date. The various phases are based on different principles of molecular recognition and show advantages and drawbacks according to the molecules to separate. The preparative aspect of this method is particularly emphasized here and in the same context, trends and future developments are mentioned.

Chromatography, High Pressure Liquid↗
PubMed1994

[Studies of in vitro and in vivo toxicity of dyes used in affinity chromatography].

Some reactive textile dyes are used for years as biomimetic ligands in protein purification. The reluctance to use these performant systems in large scale for therapeutically applicable proteins is related with the possible dye leakage and consequently with problems of contamination. Therefore, toxicology data are necessary to quantify the level of danger in association with sensitive assays. This study deals with a series of in vitro toxicity studies with eucaryotic cells (growth, polyploidia, ...) as well as with procaryotic cells (E. coli) for genotoxic studies. Both approaches demonstrated a total absence of toxicity for all ranges of concentrations investigated for Reactive Blue 2, Reactive Red 120 and their derivatives. Additional experiments done in vivo by the administration of dye solutions to a series of mice confirmed the non toxic character of these dyes in vitro.

Animals↗
PubMed1993

Features of thrombi and diagnostic accuracy of impedance plethysmography in symptomatic and asymptomatic deep vein thrombosis.

Impedance plethysmography (IPG) has high sensitivity and specificity in patients with symptomatic deep vein thrombosis (DVT) while it fails to detect asymptomatic DVT. The aim of this study was to determine whether the features of thrombi such as location, size and occlusiveness could explain the different accuracy of IPG in symptomatic and asymptomatic DVT patients. One-hundred and seventeen consecutive outpatients with a clinical suspicion of DVT and 246 consecutive patients undergoing hip surgery were admitted to the study. In symptomatic patients IPG was performed on the day of referral, followed by venography, while in asymptomatic patients IPG was performed as a surveillance programme, followed by bilateral venography. A venography proved DVT was observed in 37% of the symptomatic patients and 34% of the asymptomatic limbs. A significantly higher proportion of proximal DVTs was found in symptomatic patients than in asymptomatic patients (78% vs 46%; p = 0.001). The mean Marder score, taken as an index of thrombus size, was significantly higher in symptomatic patients than in asymptomatic patients (19.0 vs 9.6; p = 0.0001). A significantly higher proportion of occlusive DVTs was observed in symptomatic than in asymptomatic patients (69% vs 36%; p = 0.001). We conclude that the unsatisfactory diagnostic accuracy of IPG in asymptomatic DVT is due to the high prevalence of distal, small and non occlusive thrombi. Such thrombi are unlikely to cause a critical obstruction of the venous outflow and therefore to produce a positive IPG.

Dermatan Sulfate↗
PubMed1993

[Chromatographic sorbents for the preparative separation of proteins].

Chromatography separation steps are technologically very well accepted in downstream bioprocessing, but progresses are expected on productivity level as well as on cleanability. At large production scale, the driving force for purification processes is the throughput intended as the amount of material separated at a given velocity and for a defined level of purity. The possibility to play on some phases of the separation cycle and on the variation of dynamic binding capacity are clearly useful to diminish the separation time while maintaining high levels of loads. On the other hand, to sanitize properly the sorbents, it has been found that only very strong solutions such as alkaline ethanol or alternated acidic-alkaline solutions are effective. In this general situation, special sorbents are now proposed, withstanding stringent operational situations.

Chromatography, Liquid↗