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Biomedical subjects

R Ciccarelli

Publications and source records attributed to R Ciccarelli.

At least 37 records · Page 2Linked to original sources
PubMed1998

The trophic effects of purines and purinergic signaling in pathologic reactions of astrocytes.

This article reviews the effects of extracellular purine bases, nucleosides, and nucleotides as intracellular signaling molecules with trophic effects on cells after insults to the brain and spinal cord. Astrocytes are the principal source of extracellular purines in brain after injury, ischemia, or trauma. In vitro and in vivo extracellular purines have both immediate and long-term trophic effects, including stimulation of astrocyte and neuronal differentiation, mitosis, morphogenesis, apoptosis, and stimulation of growth and trophic factor synthesis. The effects of the nucleoside adenosine and the nucleotide adenosine triphosphate (ATP) are mediated principally via specific receptors on the cell surface coupled to a series of signaling cascades. Unlike adenosine and ATP, guanosine and guanosine triphosphate (GTP) do not act at classical purine receptors. However, they exert similar effects on astrocytes, apparently by causing the astrocytes to release large amounts of adenosine and ATP over prolonged periods. The release of adenosine and ATP may be related to the effects of guanosine on the purine nucleoside transporters in the cell membrane, whereas the release of ATP may be due to the effects of GTP on the ATP-binding cassette (ABC) proteins. Physiologically, the effects of guanosine are important because this nucleoside, unlike adenosine, remains elevated for prolonged periods after brain injury.

Adenosine↗
PubMed1998

[Three-dimensional imaging with computerized tomography. Etiologic considerations and methods for studying temporo-mandibular joints].

INTRODUCTION: The temporomandibular joint (TMJ) had always been studied with conventional tomography and, more recently, with Magnetic Resonance Imaging. Computed Tomography (CT) can demonstrate periarticular soft tissues only. Recently, the introduction of three-dimensional (3D) CT reconstructions with dedicated softwares permitted to demonstrate functional changes in the TMJ with a simple and accurate measurement system. MATERIAL AND METHODS: We examined 347 patients with TMJ disorders using two different 3rd generation CT units and acquiring two series of scans, with the mouth closed and in maximum opening. 3D reconstructions on predefined planes were obtained with a dedicated software and integrated with the electronic measurements of the following morphologic and functional parameters: zygomatic tubercle height, axis of sagittal inclination of the mandibular condyle, TMJ movement range and changes in torsion/dislocation of condylar angle. RESULTS AND DISCUSSION: CT showed the TMJ easily. This examination lasts about 10 minutes, plus 10-15 more minutes for 3D reconstructions. Some artifacts were found on 3D images--thresholding artifacts, defined as pseudoforamina and pseudocalcifications. Neverthless, 3D imaging can easily demonstrate an articular district which is very complex in both morphology and function, supporting or replacing similar subconscious processes made by radiologists themselves. This technique was well accepted by all our patients and functional results were always in agreement with clinical findings. Moreover, CT is the only technique showing both TMJ sides at the same time. Our measurement system was easy to perform, repeatable and comparable also thanks to its potentials of standardization on different CT units. CONCLUSIONS: CT with 3D reconstructions appears the gold standard technique for functional TMJ studies. Even though it provides no information on the joint disk--which MRI does--3D CT permits easy and accurate measurements of both TMJ sides which can support clinical findings.

Humans↗
PubMed1997

Opposite influence of the metabotropic glutamate receptor subtypes mGlu3 and -5 on astrocyte proliferation in culture.

In non-synchronized, subconfluent secondary cultures of rat cortical astrocytes, the selective group-I metabotropic glutamate (mGlu) receptor agonist 3,5-dihydroxyphenylglycine (DHPG) increased [methyl-3H]-thymidine incorporation. This effect was mediated by the activation of the mGlu5 receptor, which was shown to be present by either RT-PCR or Western blot analysis. The mixed mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine reduced the increase in both intracellular Ca2+ and [methyl-3H]-thymidine incorporation produced by DHPG. In contrast, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), a potent and selective agonist of group-II mGlu receptors, reduced [methyl-3H]-thymidine incorporation in non-synchronized astrocyte cultures. The antiproliferative effect of DCG-IV was prevented by the selective group-II mGlu receptor antagonist (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV). The opposite effect of DHPG and DCG-IV on astrocyte proliferation was confirmed in cultures deprived of serum for 48 hours and then stimulated to proliferate with either epidermal growth factor (EGF) or the metabolically stable ATP analogue adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP). We conclude that activation of mGlu5 receptors enhances proliferation in cultured astrocytes, whereas activation of a receptor with pharmacological characteristics similar to those of mGlu2/3 receptors reduces proliferation.

Animals↗
PubMed1997

Nucleic acid immunization of chimpanzees as a prophylactic/immunotherapeutic vaccination model for HIV-1: prelude to a clinical trial.

Vaccine development strategies have often utilized recombinant envelope glycoproteins which usually generate strong humoral immune responses but which do not generate strong cytotoxic T lymphocytes (CTL). A recent novel experimental vaccination approach involves the technology known as nucleic acid immunization in which DNA plasmids expressing a gene of interest is injected intramuscularly in experimental animals. These expressed proteins then are presented to the immune system with the subsequent development of strong antibody and cellular (particularly CTL) immune responses. These types of immune responses have been elicited in rodents as well as nonhuman primates including chimpanzees. Results from studies on nucleic acid immunization of HIV-1 infected chimpanzees with envelope glycoprotein expressing constructs indicated that this method was able to decrease substantially HIV-1 viral load in these chimpanzees. These data are useful for the development and implementation of human phase I clinical trials with HIV constructs expressing various genes from the HIV-1 genome.

AIDS Vaccines↗
PubMed1997

Detection of latero-cervical metastases from oral cancer.

In squamous cell carcinoma of the oral cavity, the presence of nodal metastases greatly influences prognosis. The evaluation of regional lymph nodal involvement is crucial in the correct management of these neoplasms. The records of 45 patients with oral cancer were reviewed retrospectively to evaluate the accuracy and prognostic value of the techniques used to detect lymph node metastasis in the neck (clinical examination, echo-colour-Doppler, computed tomography and magnetic resonance imaging). Echo-colour-Doppler was the most accurate procedure with a predictive positive value of 95.6% and the lowest false-negative rate. Therefore, a diagnostic preoperative study must include echo-colour-Doppler preferably associated with computed tomography to achieve the greatest diagnostic accuracy.

Adult↗
PubMed1997

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy.

We report here the backbone 1HN, 15N, 13C alpha, 13CO, and 1H alpha NMR assignments for the catalytic domain of human fibroblast collagenase (HFC). Three independent assignment pathways (matching 1H, 13C alpha, and 13CO resonances) were used to establish sequential connections. The connections using 13C alpha resonances were obtained from HNCOCA and HNCA experiments; 13CO connections were obtained from HNCO and HNCACO experiments. The sequential proton assignment pathway was established from a 3D (1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and 15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement with recent X-ray structures of human fibroblast collagenase and human neutrophil collagenase as well as similar to recent NMR structures of a highly homologous protein, stromelysin. All three helices were located; a five-stranded beta-sheet (four parallel strands, one antiparallel strand) was also determined. beta-Sheet regions were identified by cross-strand d alpha N and d NN connections and by strong intraresidue d alpha N correlations, and were corroborated by observing slow amide proton exchange. Chemical shift changes in a selectively 15N-labeled sample suggest that substantial structural changes occur in the active site cleft on the binding of an inhibitor.

Amino Acid Sequence↗
PubMed1997

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

Animals↗
PubMed1996

Rat astroglial P2Z (P2X7) receptors regulate intracellular calcium and purine release.

Treatment of rat astrocyte cultures with 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a P2X7 agonist, but not with adenosine 5-[alpha, beta methylene] triphosphate (alpha, beta meATP), a P2X agonist, increased influx of extracellular Ca2+ and [Ca2+]i. Lucifer yellow, a small molecule which permeates P2X7 receptor-induced pores, entered BzATP-treated but not control astrocytes. BzATP also stimulated efflux of [3H]purine from cultured astrocytes. The P2X7 receptor antagonist oxidized ATP abolished the effects of BzATP on [Ca2+]i, lucifer yellow permeation and [3H]purine release, indicating that these effects were due to P2X7 receptor activation. In neurological diseases or injuries extracellular ATP may activate P2X7 receptors further enhancing [3H]purine release, with important pathophysiological consequences.

Animals↗
PubMed1996

Accuracy in the diagnosis of mandibular involvement by oral cancer.

Cancer of the mandibular region usually has a poor prognosis; this is particularly correlated to invasion of the mandible. To reduce the consequences of poor therapeutic planning, careful preoperative assessment of bone infiltration is necessary. We have examined the records of 50 patients evaluated by clinical examination, conventional radiography, computed tomography (CT), bone scintigraphy with 99mTc and magnetic resonance imaging (MRI). The highest sensitivity (100%) was attained by scintigraphy; the highest values of specificity (96.3%) were reached by CT scan and MRI. A CT scan showed the highest predictive positive value (95.4%) and efficiency (94%), this plus MRI have good values and the associated sensitivity and efficiency are higher than when these techniques are used alone.

Adult↗
PubMed1996

Interaction between A1 adenosine and class II metabotropic glutamate receptors in the regulation of purine and glutamate release from rat hippocampal slices.

Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [14C]glutamate and [3H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-alpha-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N6-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.

Adenosine↗
PubMed1996

In vivo protective anti-HIV immune responses in non-human primates through DNA immunization.

An effective immune response involves the specific recognition of and elimination of an infectious organism at multiple levels. In this context DNA immunization can present functional antigenic proteins to the host for recognition by all arms of the immune system, yet provides the opportunity to delete any genes of the infectious organism which code for antigens or pieces of antigens that may have deleterious effects. Our group has developed the use of nucleic acid immunization as a possible method of vaccination against Human immunodeficiency virus type 1 (HIV-1) [1,2,3,10,11,12]. Sera from non-human primates immunized with DNA vectors that express the envelope proteins from HIV-1 contain antibodies specific to the HIV-1 envelope. These sera also neutralize HIV-1 infection in vitro and inhibit cell to cell infection in tissue culture. Analysis of cellular responses is equally encouraging. T cell proliferation as well as cytotoxic T cell lysis of relevant env expressing target cells were observed. In addition, evidence that DNA vaccines are capable of inducing a protective response against live virus was demonstrated using a chimeric SIV/HIV (SHIV) challenge in vaccinated cynomologous macaques. We found that nucleic acid vaccination induced protection from challenge in one out of four immunized cynomolgus macaques and viral load was lower in the vaccinated group of animals versus the control group of animals. These data encouraged us to analyze this vaccination technique in chimpanzees, the most closely related animal species to man. We observed the induction of both cellular and humoral immune responses with a DNA vaccine in chimpanzees. These studies demonstrate the utility of this technology to induce relevant immune responses in primates which may ultimately lead to effective vaccines.

AIDS Vaccines↗
PubMed1995

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells.

[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca(2+)-free medium + 0.5 mM ethylene glycol-bis(beta-aminoethylether)N,N,N',N'-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 microM modified [3H]purine outflow whereas 0.1 microM omega-conotoxin and 0.03-0.1 microM nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 microM omega-conotoxin + 0.1 microM nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor omega-conotoxin influenced the uptake of 45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the 45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 microM Fura-2/AM. 10-30 microM 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 microM thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC, seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Effects of exogenous ATP and related analogues on the proliferation rate of dissociated primary cultures of rat astrocytes.

The effects of ATP (5-500 microM) were evaluated on the proliferation rate of cultured astrocytes by measuring 3H-thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 16 hours showed that ATP present in the culture medium for the whole period caused a dose-dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferation was increased (always in a dose-dependent manner). A time course study of 3H-thymidine incorporation showed that, in the presence of ATP, 3H-thymidine was incorporated at a slower rate than in controls; the replacement of the culture medium with an ATP-free fresh medium, at the 8th hour, was followed by a 3H-thymidine incorporation occurring at such a fast rate to overshoot the control values. High performance liquid chromatography (HPLC) analysis, carried out to identify purine compounds present in the culture medium during cell exposure to ATP, indicated that more than 95% of the added ATP was metabolized within 1 hr. Conversely, an increase of purine metabolites was measured, this accumulation being greater at the highest concentrations of added ATP. The presence of high levels of extracellular ATP catabolites suggested that these compounds may act on the regulation of cell replication via the different purine receptors. This hypothesis was tested and confirmed by using agonists and antagonists selective for the P1 and the P2 sites. One hundred microM 2methylthio-ATP (2MeSATP), a P2Y agonist metabolized as fast as ATP, reproduced effects very similar to the ATP-induced ones. On the other hand, the nonhydrolisable ATP analogue, adenosine 5'-(beta, gamma-imido)-triphosphate (AMP-PNP) at 100 microM, induced a mitogenic effect as well as the A2 site stimulation. On the contrary, the activation of A1 receptors by 5 microM R-phenyl-isopropyladenosine (R-PIA) inhibited astrocyte proliferation; moreover, 100 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1 site antagonist, reversed the ATP-induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellular breakdown, exerts a dual influence on astrocyte proliferation by the involvement of both P1 and P2Y receptors. These findings might be relevant to such pathological conditions of the central nervous system (CNS), as seizures, hypoxia or ischemia, in which great amounts of purines released in the brain can influence a reactive astrocyte proliferative response to injury.

Adenosine Triphosphate↗
PubMed1994

Interaction between metabotropic receptors and purinergic transmission in rat hippocampal slices.

Inhibition of forskolin-stimulated cAMP formation by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) in rat hippocampal slices was partially obliterated by the adenosine-depleting enzyme, adenosine deaminase, or by the adenosine receptor agonist, 5'-(N-ethylcarboxamido)-adenosine, suggesting that activation of metabotropic glutamate receptors (mGluRs) modulates the release of endogenous adenosine. Consistent with this hypothesis, forskolin stimulated the release of purines from rat hippocampal slices, and this effect was reduced by 1S,3R-ACPD. To establish which transduction pathway is involved in the modulation of forskolin-stimulated purine release, we have tested the novel mGluR2 agonist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), which reduced forskolin-stimulated cAMP formation but, as opposed to 1S,3R-ACPD, did not stimulate polyphosphoinositide hydrolysis. DCG-IV was highly potent and more efficacious than 1S,3R-ACPD in inhibiting forskolin-stimulated purine release. Neither DCG-IV nor 1S,3R-ACPD reduced the release of purines stimulated by depolarizing concentrations of K+, suggesting that their effect was stimulus-specific. These results indicate that, in rat hippocampal slices, activation of mGluR2 receptors attenuates the release of purines induced by forskolin, a process that amplifies the final effect of forskolin on cAMP formation as a result of A2 purinergic receptor activation. Thus, the final effect of mGluR agonists on forskolin-stimulated cAMP formation in hippocampal slices depends on both a direct inhibition of adenylyl cyclase and the inhibition of adenosine release.

Adenosine↗
PubMed1993

Protein kinase C pathway and proliferative responses of aged and young rat vascular smooth muscle cells.

Alterations of vascular smooth muscle cell (VSMC) proliferation have been implicated in the age-dependent susceptibility to atherosclerosis. Although it is known that protein kinase C (PKC) is involved in the mechanism of VSMC proliferation, there are no data on the possible involvement of PKC in disregulating VSMC proliferation in aged vascular cells. We evaluated the proliferative pattern, the PKC responsiveness and the effect of phorbol ester (PMA) treatment on vascular cell growth and cell cycle distribution in VSMCs from young and aged rats. The proliferative response was significantly higher in aged than in young cells after serum stimulation (7.5 vs. 2.8 x 10(4), 18 vs. 12 x 10(4), 26 vs. 22 x 10(4) cells/well, aged vs. young at days 2, 4, 6; P < 0.005). On the contrary, aged cells showed a significant inhibition of DNA synthesis at 48 h incubation with PMA concentrations of 1, 10, 100 nM (-47%, -53%, -58%, respectively) compared with controls (fetal calf serum 0.5%) and cell count (average decrease: -38% from 48 h to 96 h) after treatment with PMA 10 nM. The opposite was observed in young cells on [3H]thymidine incorporation with PMA 1, 10, 100 nM (+52%, +100%, +121%, respectively and cell count (average increase +55% from 48 h to 96 h). In addition, inhibition of the cell cycle from G1 to the S phase and reduction of PKC translocation in aged VSMC were observed. Alterations of PKC function could be involved in the disregulation of aged VSMC proliferation, which seems to characterize the increased susceptibility to atherosclerosis.

Aging↗
PubMed1993

TMB-8 and thapsigargin modulate purine release from dissociated primary cultures of rat brain astrocytes.

In our previous studies, the evoked purine outflow from rat brain cultured astrocytes was reported to be Na+ independent and K+ and [Ca2+]e partially dependent. Thus, the eventual [Ca2+]i influence on purine astrocyte release was investigated in an attempt to better characterize the ionic requirements of this mechanism in cells which serve many complex and still partly unknown functions within the CNS. TMB-8 and Thapsigargin (drugs described as able to inhibit and increase the ion efflux from its internal stores respectively) and BAPTA/AM (able to chelate the cytoplasmic free Ca2+), were used. TMB-8 and BAPTA/AM decreased, whereas Thapsigargin enhanced glial purine outflow. These findings suggest a significant [Ca2+]i dependence of the electrically evoked purine efflux from cultured astrocytes even though further investigations using fluorescent probes are needed.

Adenosine↗