Biomedical subjects

Stefano Manzini

Publications and source records attributed to Stefano Manzini.

8 recordsLinked to original sources

PubMed2006

Genetically modified pigs produced with a nonviral episomal vector.

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Animals↗

PubMed2006

Rubinstein-Taybi Syndrome: spectrum of CREBBP mutations in Italian patients.

BACKGROUND: Rubinstein-Taybi Syndrome (RSTS, MIM 180849) is a rare congenital disorder characterized by mental and growth retardation, broad and duplicated distal phalanges of thumbs and halluces, facial dysmorphisms and increased risk of tumors. RSTS is caused by chromosomal rearrangements and point mutations in one copy of the CREB-binding protein gene (CREBBP or CBP) in 16p13.3. To date mutations in CREBBP have been reported in 56.6% of RSTS patients and an average figure of 10% has ascribed to deletions. METHODS: Our study is based on the mutation analysis of CREBBP in 31 Italian RSTS patients using segregation analysis of intragenic microsatellites, BAC FISH and direct sequencing of PCR and RT-PCR fragments. RESULTS: We identified a total of five deletions, two of the entire gene and three, all in a mosaic condition, involving either the 5' or the 3' region. By direct sequencing a total of 14 de novo mutations were identified: 10 truncating (5 frameshift and 5 nonsense), one splice site, and three novel missense mutations. Two of the latter affect the HAT domain, while one maps within the conserved nuclear receptor binding of (aa 1-170) and will probably destroy a Nuclear Localization Signal. Identification of the p.Asn1978Ser in the healthy mother of a patient also carrying a de novo frameshift mutation, questions the pathogenetic significance of the missense change reported as recurrent mutation. Thirteen additional polymorphisms, three as of yet unreported, were also detected. CONCLUSION: A high detection rate (61.3%) of mutations is confirmed by this Italian study which also attests one of the highest microdeletion rate (16%) documented so far.

Adolescent↗

PubMed2006

Sperm-mediated gene transfer.

Since 1989, a new method for the production of transgenic animals has been available, namely sperm-mediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

Animals↗

PubMed2006

Human and murine macrophages mediate activation of MEN 4901/T-0128: a new promising camptothecin analogue-polysaccharide conjugate.

MEN 4901/T-0128 is a new cytotoxic prodrug constituted by the camptothecin analogue T-2513 bound to carboxymethyl dextran through a triglycine linker. MEN 4901/T-0128 was designed to target the active camptothecin at the tumour site. MEN 4901/T-0128 is weakly cytotoxic in vitro and thus T-2513 must be released from the conjugate to become active. Here, we demonstrated that human purified cathepsin B releases T-2513 from MEN 4901/T-0128 at pH values ranging from 3 to 5. pH dependency of this reaction suggests that cleavage of the linker should mainly occur in the lysosomes. As elevated cathepsin B activity has been described in macrophages, human tumour monocytic THP-1 cells differentiated into macrophage-like cells were used to study the cellular mechanisms responsible for MEN 4901/T-0128 antitumour activity. Here, we show that differentiated THP-1 internalizes MEN 4901/T-0128 efficiently in a time-dependent and concentration-dependent manner. After phagocytosis, THP-1 cells can cleave the prodrug and release T-2513 in the media. On the contrary, undifferentiated THP-1 cells or pancreatic ASPC-1 tumour cells, although expressing high levels of cathepsin B, are much less efficient in the release of cytotoxic moieties in the culture media. Moreover, normal murine macrophages, recovered from the peritoneal cavity or from the spleen, when activated (in vitro by 100 ng/ml phorbol 12-myristate-13-acetate and in vivo by 300 microl of 3% w/v thioglycollate solution), were able to release (after incubation with 10 microg/ml MEN 4901/T-0128) cytotoxic moieties in the culture supernatant, in an amount sufficient to kill human carcinoma A2780 cells. Thus, we suggest that tumour-associated macrophages may play a key role in the uptake of MEN 4901/T-0128, cleavage and local release of active moiety T-2513. This mechanism should support a tumour targeting of the cytotoxic moieties, allowing an improved antitumour efficacy/safety ratio for MEN 4901/T-0128.

Animals↗

PubMed2005

Involvement of nitric oxide in both central and peripheral haemodynamic effect of D/L-nebivolol and its enantiomers in rats.

The cardiovascular profile of the racemate D/L-nebivolol and its enantiomers administered by intravenous (i.v.) or by intracerebroventricular (i.c.v.) route was investigated in anaesthetized normotensive rats. D/L-Nebivolol (0.1-0.5 mg/kg) induced a dose-related reduction in blood pressure when administered by i.c.v. route. These hypotensive effects were more marked as compared to those achieved by peripheral administration of D/L-nebivolol (0.1-1 mg/kg i.v.). Both enantiomers contributed to the hypotensive effect of D/L-nebivolol by i.c.v. route, while the effects of the drug on blood pressure by i.v. route were due to the d-enantiomer. The bradycardic effect of the racemic form given i.v. was dose-related and, at the highest dose (1 mg/kg), was more pronounced as compared to i.c.v. route. D-Nebivolol was responsible for chronotropic effects by both the i.v. and i.c.v. route, although by i.c.v. route L-nebivolol also induced a reduction in heart rate. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) administered at 5 mg/kg i.v. bolus + 0.1 mg/kg/min infusion or at 2.5 mg/kg i.c.v. counteracted the effects of D/L-nebivolol (either 1 mg/kg i.v. or 0.5 mg/kg i.c.v.) on blood pressure, while it did not inhibit the cardiovascular changes induced by isoprenaline (300 ng/kg i.v.) or calcitonin gene-related peptide (CGRP; 400 ng/kg i.v.). In addition, i.c.v. effects of D/L-nebivolol on blood pressure and heart rate were not affected by pre-treatment with atropine (2 mg/kg i.v.). The present findings demonstrate that D/L-nebivolol produced haemodynamic changes following both peripheral and central administration; these latter findings are mainly due to its L-enantiomer and these effects involve the L-arginine/nitric oxide pathway.

Animals↗

PubMed2005

NF-kappaB activation contributes to anthracycline resistance pathway in human ovarian carcinoma cell line A2780.

The development of chemoresistance is a major obstacle for successful anticancer therapy. Understanding the molecular mechanisms leading to chemoresistance is a rational step to improve the therapeutic efficacy of cytotoxic drugs. Since anthracyclines play an important role in cancer chemotherapy, we have generated a human ovarian tumor cell line resistant to sabarubicin (MEN 10755), the newest anthracycline molecule in clinical development. Expression of the transporter protein MRP that affected sabarubicin uptake, and a reduced DNA topoisomerase II content in A2780/saba cells was observed. Since the poisoning of DNA topoisomerase II results in DNA damage, which is a critical signal for NF-kappaB activation, we explored if this transcription factor has a role in the chemoresistance to anthracyclines. We showed a reduced NF-kappaB activation in the resistant cell line. Moreover, qualitative changes in NF-kappaB dimer formation between the two cell lines were observed. In agreement with the hypothesis of a role of NF-kappaB in mediating drug resistance, we showed that the pharmacological inhibition of NF-kappaB activation attenuated drug resistance in A2780/saba cells whereas it had no effect in A2780 cells. Altogether, these findings show that anthracycline resistance in A2780 cell lines is due to the coexpression of several molecular mechanisms.

Anthracyclines↗

PubMed2003

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity.

(1) The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues--like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755--which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. (2) DOX induced electrocardiographic and haemodynamic alterations, like elongation of QalphaT or SalphaT intervals and suppression of isoprenaline-induced dP/dt increases, which developed in a time-dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. (3) DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed approximately 60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol approximately 40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). (4) DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe-S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe-S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. (5) These results show that (i) secondary alcohol metabolites are important determinants of anthracycline-induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

Animals↗

PubMed2002

Pharmacological profile of MEN91507, a new CysLT(1) receptor antagonist.

MEN91507 (8-[2-(E)-[4-[4-(4-fluorophenyl)butyloxy]phenyl]vinyl]-4-oxo-2-(5-1H-tetrazolyl)-4H-1-benzopyran sodium salt)) potently displaced [3H]leukotriene D(4) binding from guinea-pig lung and dimethylsulphoxide-differentiated U937 (dU937) cell membranes (K(i) 0.50 +/- 0.16 and 0.65 +/- 0.29 nM, respectively). On the other hand, MEN91507 did not display significant binding affinity for a series of receptors or channels. In functional studies on dU937 cells, MEN91507 behaved as insurmountable antagonist of leukotriene D(4)-induced calcium transients, with an apparent pK(B) of 10.25 +/- 0.15. In anaesthetized guinea-pigs, MEN91507 antagonized in a dose-dependent manner leukotriene D(4)-induced bronchoconstriction following i.v. or oral administration: the ED(50s) were 3.0 +/- 0.3 and 140 +/- 90 nmol/kg, respectively. The inhibition of leukotriene D(4)-induced bronchoconstriction by MEN91507 was long-lasting, since a dose of 0.6 micromol/kg produced 74% reduction of the response after 8 h from administration. Likewise, leukotriene D(4)-induced microvascular leakage was antagonized by MEN91507 either following i.v. or oral administration: a significant inhibitory effect was still evident at 16 h from oral administration of a dose of 6 micromol/kg. It is concluded that MEN91507 is a potent and selective antagonist of both guinea-pig and human CysLT(1) receptors; in addition, in vivo studies on guinea-pigs indicate that MEN91507 is an orally available and long-lasting antagonist of the bronchomotor and pro-inflammatory effects induced by leukotriene D(4) through the stimulation of CysLT(1) receptors.

Administration, Oral↗