In the past Marco Gobbi has collaborated on articles with Pier Luigi Tazzari and Patrizio Mazza. One of their most recent publications is Research reportIn situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots. Which was published in journal Journal of Immunological Methods.

More information about Marco Gobbi research including statistics on their citations can be found on their Copernicus Academic profile page.

Marco Gobbi's Articles: (16)

Research reportIn situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots

AbstractBromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.

Autoradiographic localization of [3H]paroxetine specific binding in the rat brain

AbstractSpecific binding of [3H]paroxetine, a selective serotonin uptake inhibitor, has recently been described. We used the autoradiographic technique to establish the regional distribution of these binding sites in the brain of control and 5,7-dihydroxytryptamine (5,7-DHT) lesioned rats.Binding levels were highest in the dorsal raphe, followed by superior colliculus, thalamus, mammillary nuclei, ventral tegmental area and substantia nigra. Values were intermediate in hippocampus, hypothalamus, caudate nucleus and median raphe and lowest in the cerebral cortex and cerebellum. The degeneration of serotonergic terminals by 5,7-DHT completely prevented specific binding in the majority of brain regions analyzed, with the exceptions of raphe nuclei, ventral tegmental area and caudate nucleus.These data indicate that [3H]paroxetine binding sites are mainly localized on serotonin nerve terminals even if it seems that they cannot always be directly correlated to 5-HT uptake.

Prognostic significance of lymphography in stage IIIs Hodgkin's disease (HD)☆

AbstractNinety-six patients with pathological stage IIIs Hodgkin's disease, uniformly treated with six cycles of MOPP and TNI, were retrospectively analysed in an effort to determine whether the lymphographic aspect of lymph nodes influence the prognosis. Case material was grouped according to the presence of lymph nodes less than 3 cm in diameter or larger at lymphography. Five-year survival and disease-free survival were 85 and 78% for patients with small lymph node involvement, compared to 48 and 30% for patients with larger lymph nodes. The comparative analysis between the lymphographic aspect and other prognostic factors shows that large lymphographic involvement is strongly correlated with the presence of large spleen involvement (P < 0.0000029), followed by stage III2 (P < 0.000612), followed by ⩾5 involved sites (P < 0.012), followed by age >40 yr (P < 0.047). Conversely, no significant correlation was found with symptoms, histology and mediastinal involvement. Modifications of current treatment for both large and small lymph node involvement are discussed.

Notes & TipsA modified protocol to prepare seed-free starting solutions of amyloid-β (Aβ)1–40 and Aβ1–42 from the corresponding depsipeptides

AbstractPreparing reliable, seed-free stock solutions of the highly amyloidogenic peptides amyloid-β (Aβ) is difficult. Besides the formation of aggregates during synthesis and storage, dissolution of the peptide is a critical step because vortexing can induce aggregation. To overcome this, synthesis of the more water-soluble depsi-Aβ1–42 peptide, from which the native sequence is easily obtained, has been suggested. We further refined this technique, including a cutoff filtration step and switching the depsipeptide in basic conditions, to stabilize the formed native peptide. The obtained solutions of native Aβ1–40 and Aβ1–42 peptides were homogeneous and aggregate free, as indicated by thioflavin T and circular dichroism analysis.

Effect of granulocyte colony-stimulating factor on secretion of prolactin, growth hormone, thyroid-stimulating hormone, and cortisol in humans

AbstractIn order to assess the endocrine effects of acute and short-term granulocyte colony-stimulating factor (G-CSF), we studied seven patients with myelo- and lymphoproliferative disorders who were given G-CSF to prevent chemotherapy-induced neutropenia. On day 0, the patients received a placebo; on days 1 to 4, they received recombinant human G-CSF (300 μg, subcutaneously). Plasma prolactin, growth hormone, thyroid-stimulating hormone, and cortisol levels were determined at baseline and hourly for 8 hours on days 0 and 1. Additional blood samples were collected at 12 hours on days 0 and 1, and at 9:00 am on days 2 to 5 of the study. After administration of G-CSF, no significant variations in circulating hormone levels were observed with respect to baseline and corresponding placebo levels. The data indicate that short-term G-CSF administration does not cause endocrine abnormalities in patients undergoing this cytokine treatment

Substrate inhibitors and blockers of excitatory amino acid transporters in the treatment of neurodegeneration: critical considerations

AbstractExcessive glutamate release (mediated by reversed uptake) or impaired reuptake contributes to the etiopathology of many neurodegenerative disorders. Thus great effort has been devoted to the discovery of agents that can interfere with high-affinity Na+-dependent glutamate transport, with the aim of finding new therapeutics against neurodegenerative diseases. We developed two different 3D-pharmacophore models for substrate inhibitors and blockers, which led to the rational design of novel and potent glutamate and aspartate analogues that selectively interact with excitatory amino acid transporters (EAAT). Our results indicated that all analysed EAAT ligands share the same orientation of the acidic functions and the protonatable nitrogen, even though the distance between the carboxylic carbons varies from 3.7 to 4.9 Å. This distance does not discriminate between substrate inhibitors and blockers, but between glutamate and aspartate derivatives. In contrasts differences in the volume distribution of the rest of the molecule with respect to the axis connecting the two carboxylic groups are responsible for the difference in activity between transportable and nontransportable inhibitors. Thus our 3D receptor interaction model for EAAT substrates and nontransportable inhibitors could lead to the rational design of selective EAAT ligands as possible neuroprotective agents. However, some critical points, such as which glutamate transporter is present on glutamatergic nerve terminals and which glutamate transporter mediates reversed glutamate uptake, still remain to be elucidated.

Short communicationRiluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1

AbstractRiluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigated whether this drug affects glutamate transporter-mediated uptake, using clonal cell lines stably expressing the rat glutamate transporters GLAST, GLT1 or EAAC1. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner; kinetic analysis indicated that the apparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate the buffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partly mediated by its activating effect on glutamate uptake.

Lipid-based nanoparticles with high binding affinity for amyloid-β1–42 peptide

AbstractThe neurotoxic beta-amyloid peptide (Aβ), formed in anomalous amounts in Alzheimer’s disease (AD), is released as monomer and then undergoes aggregation forming oligomers, fibrils and plaques in diseased brains. Aβ aggregates are considered as possible targets for therapy and/or diagnosis of AD. Since nanoparticles (NPs) are promising vehicles for imaging probes and therapeutic agents, we realized and characterized two types of NPs (liposomes and solid lipid nanoparticles, 145 and 76 nm average size, respectively) functionalized to target Aβ1–42 with high affinity. Preliminary immunostaining studies identified anionic phospholipids [phosphatidic acid (PA) and cardiolipin (CL)] as suitable Aβ1–42 ligands. PA/CL-functionalized, but not plain, NPs interacted with Aβ1–42 aggregates as indicated by ultracentrifugation experiments, in which binding reaction occurred in solution, and by Surface Plasmon Resonance (SPR) experiments, in which NPs flowed onto immobilized Aβ1–42. All these experiments were carried out in buffered saline. SPR studies indicated that, when exposed on NPs surface, PA/CL display very high affinity for Aβ1–42 fibrils (22–60 nm), likely because of the occurrence of multivalent interactions which markedly decrease the dissociation of PA/CL NPs from Aβ. Noteworthy, PA/CL NPs did not bind to bovine serum albumin. The PA/CL NPs described in this work are endowed with the highest affinity for Aβ so far reported. These characteristics make our NPs a very promising vector for the targeted delivery of potential new diagnostic and therapeutic molecules to be tested in appropriate animal models.

The binding affinity of anti-Aβ1-42 MAb-decorated nanoliposomes to Aβ1-42 peptides in vitro and to amyloid deposits in post-mortem tissue

AbstractAmyloid β (Aβ) aggregates are considered as possible targets for therapy and/or diagnosis of Alzheimer disease (AD), and nanoparticles functionalized with Aβ-specific ligands are considered promising vehicles for imaging probes and therapeutic agents. Herein, we characterized the binding properties of nanoliposomes decorated with an anti-Aβ monoclonal antibody (Aβ-MAb). The Aβ-MAb was obtained in mice by immunization with Aβ antigen followed by hybridoma fusion. Surface Plasmon Resonance (SPR) studies confirmed the very high affinity of purified Aβ-MAb for both Aβ monomers and fibrils (KD = 0.08 and 0.13 nm, respectively). The affinity of the biotinylated Aβ-MAb, used thereafter for liposome decoration, was lower although still in the low nanomolar range (KD = 2.1 and 1.6 nm, respectively). Biotin-streptavidin ligation method was used to decorate nanoliposomes with Aβ-MAb, at different densities. IgG-decorated liposomes were generated by the same methodology, as control. Vesicles were monodisperse with mean diameters 124–134 nm and demonstrated good colloidal stability and integrity when incubated with serum proteins. When studied by SPR, Aβ-MAb-liposomes, but not IgG-liposomes, markedly bound to Aβ monomers and fibrils, immobilized on the chip. KD values (calculated on Aβ-MAb content) were about 0.5 and 2 nm with liposomes at high and low Aβ-MAb density, respectively. Aβ-MAb-liposome binding to Aβ fibrils was additionally confirmed by ultracentrifugation technique, in which interactions occur in solution under physiological conditions. Moreover, Aβ-MAb-liposomes bound amyloid deposits in post-mortem AD brain samples, confirming the potential of these nanoparticles for the diagnosis and therapy of AD.

Monte Carlo method for predicting of cardiac toxicity: hERG blocker compounds

Highlights•Predictive models for cardiac toxicity are built up.•The models are calculated with the Monte Carlo method.•The CORAL software is utilized to build up the models.•The statistical quality of the models is quite good.•The models are built up in accordance with the OECD principles.

Short communicationUse of surface plasmon resonance to study the elongation kinetics and the binding properties of the highly amyloidogenic Aβ1–42 peptide, synthesized by depsi-peptide technique

AbstractA wide variety of human diseases are associated with the formation of highly organized protein aggregates termed amyloid fibrils, whose growth (elongation) is due to the assembly of the basic molecular units (monomers) in a sequential polymerization process.Surface plasmon resonance (SPR) technology has been proposed as a powerful approach to study in detail the fibril elongation of some amyloidogenic peptides. In particular, the injection of monomers over immobilized fibrils allows to follow in real time, and on a very short time-scale, the kinetics of fibril growth. In the present study we confirmed and extended this application of SPR to Aβ1–42, hampered till now by the very pronounced aggregation propensity of this peptide, involved in Alzheimer disease. We took advantage of a new synthetic strategy (“depsi-peptide” technique) which allows to obtain reliable seed-free solutions (monomers) as well as fibrils of Aβ1–42. SPR data were consistent with a “dock-and-lock” mechanism underlying Aβ1–42 elongation process. The setup of an assay monitoring the elongation kinetics is very useful for investigating potential anti-amyloidogenic compounds. Moreover, the possibility to reliably immobilize both Aβ1–42 monomers and fibrils allows to measure the binding affinities of putative ligands for these different species. The approach applied here to Aβ1–42 might well be also applied to the study of other fibrillogenic peptides/proteins or to the study of polymerization reactions in general.

New insights into the molecular mechanisms underlying sensitivity/resistance to the atypical retinoid ST1926 in acute myeloid leukaemia cells: The role of histone H2A.Z, cAMP-dependent protein kinase A and the proteasome

AbstractST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosinemonophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

A novel spirocyclic tropanyl-Δ2-isoxazoline derivative enhances citalopram and paroxetine binding to serotonin transporters as well as serotonin uptake

AbstractA group of spirocyclic tropanyl-Δ2-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC50 >> 10 μM), while some had an IC50 value in the range 5–10 μM (8a–c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3′-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5′(4′H)-isoxazole 7a, was able to enhance at a concentration of 10 μM both [3H]citalopram and [3H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of Bmax) and [3H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

AbstractPharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state.We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase.Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites.Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Fast track — ArticlesFludarabine and mitoxantrone followed by yttrium-90 ibritumomab tiuxetan in previously untreated patients with follicular non-Hodgkin lymphoma trial: a phase II non-randomised trial (FLUMIZ)

SummaryBackgroundFollicular lymphoma is the most common form of lymphoma in Europe and the USA. In this prospective, single-arm, open-labelled, multicentre non-randomised phase II trial (FLUMIZ [FLUdarabine, MItoxantrone, Zevalin] trial) we aimed to assess the efficacy and safety of fludarabine and mitoxantrone plus radioimmunotherapy in untreated patients with follicular non-Hodgkin lymphoma (NHL).MethodsPatients with stage III or IV untreated indolent follicular NHL were enrolled between June 1, 2004, and April 15, 2006, at 13 Italian institutions, and were treated with oral fludarabine (40 mg/m2 on days 1 to 3) and intravenous mitoxantrone (10 mg/m2 on day 1) every 28 days for six cycles. Patients who had at least a partial response (PR) with normal platelet counts (>100×109/L) and granulocyte counts (1·5×109/L), and bone-marrow infiltration less than 25% 4–6 weeks after completion of the sixth cycle of chemotherapy were deemed eligible for consolidation treatment 6–10 weeks after the sixth cycle with one course of yttrium-90 (90Y)-labelled ibritumomab tiuxetan (Zevalin), which consisted of an initial infusion of intravenous rituximab (250 mg/m2) on day 1 followed by a second 250 mg/m2 infusion on day 7, 8, or 9. The second infusion was followed by a weight-based dose of 90Y-ibritumomab tiuxetan, administered as a slow intravenous push over 10 min. Primary endpoints were complete response (CR) and haematological toxic effects and secondary endpoints were overall survival and progression-free survival. Responses were classified according to the International Workshop for Response Criteria for non-Hodgkin's lymphomas. Analysis was per protocol. This trial is registered as a European Standard Controlled Trial on the EudraCT website, number 2004-002211-92.Findings61 patients were enrolled in the trial and received six cycles of fludarabine and mitoxantrone, after which an overall response was noted in 98% (60 of 61) of patients (43 of 61 patients had CR and 17 of 61 patients had PR). 57 patients (43 with CR and 14 with PR) were deemed eligible for subsequent 90Y-ibritumomab tiuxetan. Of the 14 patients who had PR after the initial treatment, 12 obtained CR after 90Y-ibritumomab tiuxetan. By the end of the entire treatment regimen 55 of 57 patients achieved CR. With a median follow-up of 30 months (range 21–48), 3-year progression-free survival was estimated to be 76% (95% CI 72·3–82·4) and 3-year overall survival 100%. 36 of 57 patients had grade 3 or 4 haematological toxic effects, and blood transfusions were given to 21 of 57 patients.InterpretationThis trial has provided evidence for the feasibility, tolerability, and efficacy of fludarabine and mitoxantrone plus 90Y-ibritumomab tiuxetan in untreated patients with follicular NHL.FundingItalian Association for Leukaemias, Lymphomas, and Myeloma, Bologna, Italy.

Ranolazine ameliorates postresuscitation electrical instability and myocardial dysfunction and improves survival with good neurologic recovery in a rat model of cardiac arrest

BackgroundDuring ischemia, enhancement of the “late Na+ current” (INaL) contributes to intracellular Ca2+ overload. Dysregulation of intracellular Ca2+ homeostasis plays a critical role in the pathophysiology of cardiac arrest and cardiopulmonary resuscitation (CPR), leading to ventricular arrhythmias and left ventricle (LV) dysfunction.ObjectiveThe purpose of this study was to investigate the effects of the INaL blocker ranolazine on outcome of CPR in a rat model. We hypothesized that ranolazine might reduce postresuscitation arrhythmias and improve survival and recovery.MethodsEighteen rats were assigned to receive intravenous ranolazine 10 mg/kg or vehicle. Ventricular fibrillation was induced and untreated for 8 minutes. CPR then was performed for 8 minutes. ECG and arterial and right atrial pressures were monitored up to 3 hours after CPR. After resuscitation, LV function was monitored by echocardiography, and 72-hour survival with neurologic recovery was evaluated. Plasma was obtained for biomarkers of heart and brain injury.ResultsAll animals in the ranolazine group were resuscitated and survived up to 72 hours, whereas 72% in the vehicle group were resuscitated but 54% survived. The period of postresuscitation arrhythmia with hemodynamic instability was shorter in the ranolazine group compared to vehicle group (P < .02). Seventy-two hours after resuscitation, LV systolic and diastolic functions were better in the ranolazine group compared to vehicle (P < .05). Full neurologic recovery was observed in all ranolazine animals, whereas neurologic impairment persisted in the vehicle group (P < .02).ConclusionIn this model, ranolazine pretreatment reduced postresuscitation electrical and hemodynamic instability and improved 72-hour postresuscitation LV function and survival with good neurologic recovery.

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