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M Grandolfo

Publications and source records attributed to M Grandolfo.

At least 19 recordsLinked to original sources
PubMed2006

The effects induced by the sulphonylurea glibenclamide on the neonatal rat spinal cord indicate a novel mechanism to control neuronal excitability and inhibitory neurotransmission.

BACKGROUND AND PURPOSE: Using the neonatal rat spinal cord in vitro, we investigated the action of glibenclamide, a drug possessing dual pharmacological effects, namely block of K(ATP) channels and of the cystic fibrosis transmembrane conductance regulator (CFTR). EXPERIMENTAL APPROACH: Intra- and extracellular recordings were performed on motoneurons and interneurons. RT-PCR and western immunoblotting were used to determine gene and protein expression. KEY RESULTS: Glibenclamide (50 microM) facilitated mono- and polysynaptic reflexes, hyperpolarized motoneuron resting potential, increased action potential amplitude, decreased Renshaw cell-mediated recurrent inhibition, and increased network excitability by depressing GABA- and glycine-mediated transmission. The action of glibenclamide was mimicked by tolbutamide (500 microM) or the CFTR blocker diphenylamine-2,2-dicarboxylic acid (500 microM). The action of glibenclamide was independent from pharmacological inhibition of the Na(+)-K(+) pump with strophanthidin (4 microM) and was associated with a negative shift in the extrapolated reversal potential for CI(-) dependent synaptic inhibition. On interneurons, intracellularly-applied 8-bromo-cAMP elicited an inward current and resistance decrease; effects antagonized by the selective CFTR antagonist, CFTR(inh)-172 (5 microM). RT-PCR and western immunoblotting indicated strong expression of the CFTR in neonatal rat spinal cord. CONCLUSIONS AND IMPLICATIONS: These data suggest the CFTR expressed in motoneurons and interneurons of the neonatal spinal cord is involved in the control of Cl(-) homeostasis and neuronal excitability. CFTR appeared to contribute to the relatively depolarized equilibrium potential for synaptic inhibition, an important process to control hyperexcitability and seizure-predisposition in neonates.

Animals↗
PubMed2006

Early signs of motoneuron vulnerability in a disease model system: Characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice.

Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis. The mechanisms by which these mutations lead to cell loss within the spinal cord ventral horns are unknown. In the present report we used the G93A transgenic mouse model of amyotrophic lateral sclerosis to develop and characterize an in vitro tool for the investigation of subtle alterations of spinal tissue prior to frank neuronal degeneration. To this aim, we developed organotypic slice cultures from wild type and G93A embryonic spinal cords. We combined immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard in vitro conditions. By SMI32 and choline acetyl transferase immunostaining, the distribution and morphology of motoneurons were compared in the two culture groups. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motoneurons. Similar results were observed when glial fibrillary acidic protein and myelin basic protein-positive cells were examined. Cell types within the G93A slice underwent maturation and slices could be maintained in culture for at least 3 weeks when prepared from embryos. Electron microscopy investigation confirmed the absence of early signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. However, a significantly different ratio between inhibitory and excitatory synapses was present in G93A cultures, when compared with wild type ones, suggesting the expression of subtle synaptic dysfunction in G93A cultured tissue. When compared with controls, G93A motoneurons exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress prior to clear disease appearance. This in vitro disease model may thus represent a valuable tool to test early mechanisms contributing to motoneuron degeneration and potential therapeutic molecular interventions.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes.

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.

Animals↗
PubMed1999

Generation of rhythmic patterns of activity by ventral interneurones in rat organotypic spinal slice culture.

1. In the presence of certain excitatory substances the rat isolated spinal cord generates rhythmic oscillations believed to be an in-built locomotor programme (fictive locomotion). However, it is unknown whether a long-term culture of the same tissue can express rhythmic activity. Such a simplified model system would provide useful data on the minimal circuitry involved and the cellular mechanisms mediating this phenomenon. For this purpose we performed patch clamp recording (under whole-cell voltage or current clamp conditions) from visually identified ventral horn interneurones of an organotypic slice culture of the rat spinal cord. 2. Ventral horn interneurones expressed rhythmic bursting when the extracellular [K+] was raised from 4 to 6-7 mM. Under voltage clamp this activity consisted of composite synaptic currents grouped into bursts lasting 0.9 +/- 0.5 s (2.8 +/- 1.5 s period) and was generated at network level as it was blocked by tetrodotoxin or low-Ca2+-high-Mg2+ solution and its periodicity was unchanged at different potential levels. 3. In current clamp mode bursting was usually observed as episodes comprising early depolarizing potentials followed by hyperpolarizing events with tight temporal patterning. Bursting was fully suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced in amplitude and duration by N-methyl-D-aspartate (NMDA) receptor antagonism without change in periodicity. Extracellular field recording showed bursting activity over a wide area of the ventral horn. 4. Regular, rhythmic activity similar to that induced by K+ also appeared spontaneously in Mg2+-free solution. The much slower rhythmic pattern induced by strychnine and bicuculline was also accelerated by high-K+ solution. 5. The fast and regular rhythmic activity of interneurones in the spinal organotypic culture is a novel observation which suggests that the oversimplified circuit present in this culture is a useful model for investigating spinal rhythmic activity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Intercellular calcium signalling between chondrocytes and synovial cells in co-culture.

Intercellular communication allows the co-ordination of cell metabolism between tissues as well as sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks that favour the intercellular exchange of nutrients and second messengers. Heterologous intercellular communication was studied in co-cultures of articular chondrocytes and HIG-82 synovial cells by measuring mechanically induced cytosolic changes in Ca2+ ion levels by digital fluorescence video imaging. In confluent co-cultures, mechanical stimulation induced intercellular Ca2+ waves that propagated to both cell types with similar kinetics. Intercellular wave spreading was inhibited by 18alpha-glycyrrhetinic acid and by treatments inhibiting the activation of purinoreceptors, suggesting that intercellular signalling between these two cell types occurs both through gap junctions and ATP-mediated paracrine stimulation. In rheumatoid arthritis the formation of the synovial pannus induces structural changes at the chondrosynovial junction, where chondrocyte and synovial cells come into close apposition: these results provide the first evidence for direct intercellular communication between these two cell types.

Adenosine Triphosphate↗
PubMed1998

Mechanism of mechanically induced intercellular calcium waves in rabbit articular chondrocytes and in HIG-82 synovial cells.

Intercellular communication through gap junctions allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Intercellular Ca2+ signaling was investigated with digital fluorescence video imaging in primary cultures of articular chondrocytes and in HIG-82 synovial cells. In both cell types, mechanical stimulation of a single cell induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. In the absence of extracellular Ca2+, mechanical stimulation induced communicated Ca2+ waves similar to controls; however, the number of HIG-82 cells recruited decreased significantly. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte and HIG-82 cell, but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Treatment of cells with thapsigargin and with the phospholipase C (PLC) inhibitor U73122 blocked mechanically induced signal propagation. These results provide evidence that in chondrocytes and in HIG-82 synovial cells, mechanical stimulation activates PLC, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighboring cells. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate tissue responses in joint physiology.

Animals↗
PubMed1997

Vaccination campaign against hepatitis B for 12-year-old subjects in Italy.

The vaccination campaign against hepatitis B for 12-year-old subjects in Italy was evaluated in 136 health districts (USL); 55 in the north, 69 in the centre, and 12 in the south, respectively. Out of the 113,230 eligible subjects born in 1981, 105, 989, (93.6%) completed the three dose series during the period 1993-1994. However, wide geographical differences are observed: the acceptance rate was as high as 97.9% (range 89.7-100.0) and 96.6% (range 76.0-100.0) in the north and in the centre, respectively; but only 65.1% in the south (range 26.8-100.0). Subjects were recruited in the north and in the centre mostly from the list of residents, while in the south more likely only from the list of people attending school. The school attendance in the south, not as good as in the other Italian areas, particularly among people belonging to a subgroup of the population economically disadvantaged, appears to be a strong determinant of the different vaccine coverage observed. Active efforts from health services in southern areas are necessary to reach subgroups of population who escape hepatitis B vaccination and who are also at high risk of HBV infection.

Child↗
PubMed1997

Measles vaccine in egg allergic children: poor immunogenicity of the Edmoston-Zagreb strain.

Despite the fact that a number of recent studies have shown that measles/ mumps/rubella vaccine is safe for egg allergic children, many pediatricians are still concerned about immunization in egg allergic children. In Europe, a measles vaccine with the Edmoston-Zagreb strain (EZMV) grown in human fibroblast culture has been developed and recommended for children with egg allergy. However, some doubt arises on the efficacy of this strain due to its weak immunogenicity. The aim of this study was to investigate the immunogenicity of the EZMV in comparison to the measles vaccine with the Schwarz strain (SWMV) grown in a chick embryo fibroblast culture. Thirty-nine children affected by severe immediate manifestations due to IgE mediated egg allergy were enrolled. The children received at random the SWMV (Morupar, Sclavo) or the EZMV (Triviraten, Berna) in one 0.5 ml subcutaneous injection, and were checked for any immediate allergic reactions in the following 4 hours. Blood samples were taken for the detection of specific antibody response 5 months after the immunization. In SWMV seroconverted children (18/19) the geometric mean antibody titer was 3 times higher than that observed in EZMV seroconverted children (17/20) (p < 0.01). No allergic reactions occurred following the immunization with the two different vaccines. This data confirms the safety of SWMV in egg allergic children. In addition, the present study provides further data on the lower immunogenicity of the EZMV in comparison to the SWMV.

Antibodies, Viral↗
PubMed1997

An evaluation of the allergic contact dermatitis potential of colloidal grain suspensions.

BACKGROUND: Colloidal grain suspensions have been used for decades as adjuncts in the treatment of atopic dermatitis, especially in the US. In Italy, many young children have been exposed to colloidal grains. Recently, it was suggested that these bath therapies may induce allergic contact dermatitis in some young atopic children. OBJECTIVE: To evaluate the allergic skin reactions to topical oat and rice colloidal grain suspensions of normal and atopic children with and without previous exposure to colloidal grain suspensions. METHODS: A double-blind, randomized patch study. Two concentrations of oat and rice colloidal grains (0.007% and 0.7%) were applied occlusively to the backs of 65 children living in Italy, ages 6 months to 2 years (43 were atopic and 22 were normal). RESULTS: There were neither immediate urticarial nor allergic reactions in any of the 65 study subjects, atopic or nonatopic; 5 of 43 (12%) atopic subjects developed irritant reactions to the test materials. Radioallergosorbent tests (RAST) tests were performed on 55 subjects. The negative RAST test results found in the nonatopic group correlated well with nonatopic status, but positive RAST tests were found in only 8 of 35 (23%) atopic dermatitis subjects. None of the sera from positive RAST scores corresponded to subjects with irritant patch reactions. CONCLUSIONS: The data indicate that topical colloidal grains can be used as an adjunct in the management of mild atopic dermatitis in children under 2 years of age. There was no evidence of sensitization to topical colloidal grains in the group studied.

Avena↗
PubMed1996

Contact allergy to impurities in surfactants: amount, chemical structure and carrier effect in reactions to 3-dimethylaminopropylamine.

Since finding that all subjects with contact allergy to cocamidopropylbetaine give positive reactions to 3-dimethylaminopropylamine (DMPA), we wished to verify whether sensitization to other industrially-used tensioactives might also be due to content of DMPA as an impurity. We also investigated the possible "carrier action" that tensioactives might exert on minimal quantities of DMPA. Finally, we analyzed the relationship between the structure of DMPA and other chemically-correlated molecules and their sensitizing potential, with particular reference to the structure of alkylamidopropylbetaines. For this purpose, in 34 patients with contact allergy to DMPA, we tested: (i) DMPA in concentrations below the threshold limit in water and in different tensioactives; (ii) substances that employ DMPA as a reagent in their synthesis; (iii) substances similar to DMPA as regards chemically reactive groups. The study showed that: (i) DMPA remains as a quantitatively detectable impurity in all tensioactives employing it in their synthesis; (ii) some common anionic (SLES) and non-ionic (polysorbate 20) tensioactives enhance the risk of sensitization from very low doses of DMPA, presumably due to a "carrier effect;" (iii) the sensitizing chemical structures in DMPA and related molecules are the primary amine and the tertiary (dimethyl-substituted) amine groups, when separated by either 2 or 3 carbon atoms; (iv) no sensitizing action can be attributed to the functional groups present in alkylamidopropylbetaine molecules.

Cosmetics↗
PubMed1996

Occupational sensitization to epoxy resin and reactive diluents in marble workers.

10 out of 22 marble workers handling a bicomponent resin, based on epoxy resin and ortho-cresyl glycidyl ether (CGE), developed contact dermatitis and airborne contact dermatitis within 20 days to 2 months of exposure. All 22 subjects were patch tested with epoxy resin, bisphenol A, epichlorohydrin and a series of reactive diluents. The 10 symptomatic subjects were all positive to the reactive diluent CGE, and 4 of them also to epoxy resin. The other reactive diluents that gave positive reactions were phenyl glycidyl ether (7 cases), cyclohexanedimethanol glycidyl ether (2 cases), 1,6-hexanediol diglycidyl ether (2 cases) and allyl glycidyl ether (1 case). The findings of our study suggest that allergic potential is directly proportional to the electronic charge available, for all electron-rich molecules (solvents, high and low polymers, reagents) that interact with the glycidyl ether group. Lesser, but still noticeable, effects are detected when activation of the glycidyl group is related to the possible formation of intramolecular hydrogen bonds. In practice, the occupational problem was partly solved by changing the type of glycidyl ether.

Adult↗
PubMed1995

Calcium, sulfur, and zinc distribution in normal and arthritic articular equine cartilage: a synchrotron radiation-induced X-ray emission (SRIXE) study.

Calcium, sulfur, and zinc content in normal and arthritic equine cartilage have been studied by synchrotron radiation-induced X-ray emission (SRIXE). Ranging from the superficial to the columnar zone of the normal tissue, calcium and zinc concentrations are increasingly higher, whereas sulfur is at its highest concentration in the transitional zone. In the arthritic tissue, calcium concentration is at its maximum in the transitional zone, whereas zinc and sulfur distributions are relatively homogeneous. Sulfur concentration in arthritic cartilage is reduced to about one-third with respect to that in normal tissue. The possibility that zinc concentration reflects the distribution of the zinc-containing enzyme alkaline phosphatase is presented.

Animals↗
PubMed1995

Human natural killer cytotoxic activity is not affected by in vitro exposure to 50-Hz sinusoidal magnetic fields.

Epidemiological studies have suggested, but not demonstrated, a role of exposure to 50/60-Hz magnetic fields in increasing cancer risk in man (workers and the general population). A possible target of magnetic fields is the immune system. In particular, it is known that an important defence against cancer is represented by natural killer (NK) cells capable of killing cancer cell targets. To test this hypothesis, human NK cells, stimulated or not with phytohaemagglutinin or interleukin 2, were exposed to 50-Hz sinusoidal magnetic fields before or during the cytotoxicity test, and then mixed with a variety of target cancer cell lines (Daudi, Raji, U937, H14, IGROV, SW626, K562, HL60). The experiments were performed in two laboratories (Rome and Modena) by means of two different exposure systems. The results of both laboratories suggest that 50-Hz sinusoidal magnetic fields with flux densities up to 10 mT do not affect the cytotoxic activity of human NK cells.

Adult↗