[Soft tissue pathologies of the oral cavity]. / Le patologie dei tessuti molli del cavo orale.

The most frequent form of neoplasia in the oral cavity is the squamous cell carcinoma (about 90% of cases) representing the 3-5% of all malignant tumors with about 56% of mortality rate, at 5 years from the diagnosis. In general, the neoplastic disease is now unanimly considered as a multifactorial and multiphasic pathology. Multiphasic since the carcinogenic process consists in the cellular capacity to acquire oncological potentialities through several stages such as: moltiplication (a), transmission (b) of malignity caracteristics to progenic cells, invasivity (c), capacity to give metastasis (d) and also resistance to chemiotherapy. Multifactorial since in the onset of the disease intrinsic and extrinsic factors are certainly involved. In the carcinogenic process of CCS a high percentage has been noticed of loss of heterozygosity (LOH) in the short arm (P) of cromosoms 3 and 9, which contains the tumor-suppressor genes p53 and DDC (Deleted in colon rectal cancer). In the onset of VADS carcinoma and in particular of oral CCS, it has also been formulated the hypothesis of an intrinsic genetic factor (Llewellyn et al., 2001) between patients, also young, who present the neoplasia even trough they have never been exposed to extrinsic risk factors such as smoke and alcohol. Since part of patients with oral CCS do not always refer a common risk factors history as possible extrinsic neoplasia causes, it has been formulated the hypothesis that some viral infections, for their oncogenic capacity, could be the main ethiological factors predisposing to this neoplasia. The HPV are responsible, either in the oral cavity or on the epidermis, for benign proliferations such as: Verruca Vulgaris, Condyloma Acuminatum, Focal Epithelial Hyperplasia, Squamous Cell Papillomas, but also lesions that are potentially or certainly malignant such as CCS and Verrucous Carcinoma. The molecular analysis performed show that proteins produced from E6 and E7 portions of viral genoma (HPV 16-18) interfer and degrade proteins p53 and pRb produced by tumor suppressor genes (TSg). Recently, thanks to new molecular biology techniques, several authors are studying potentially neoplastic lesions, in order to better understand the association with HPV.

Proliferative activity and motoneurone recruitment persist at the spinal cord central canal during larval and some postlarval stages in the rainbow trout (Oncorhynchus mykiss).

We previously found a linear relationship between the cross sectional myotomal area and the motoneurone number in the growing trout during postlarval stages. These neurones increased in number until a fish length of 150 mm, which prompted us to examine how motor neurones are recruited afterwards to meet the growth of their target myotomal muscle. Young adult (260 mm in length), fingerlings (F, 120-170 mm), fry (Fr, 70 mm) and eleutherembryos (Es, 20-30 mm) of rainbow trout (Oncorhyncus mykiss) were employed in this study. PCNA immunohistochemistry was used for monitoring the proliferative activity in the epithelium of the spinal cord central canal. This activity was quantified as the number of PCNA labelled cells for each spinal cord section. In Es and Fry, a mean value of 3-5 labelled cells for each section was found with a sharp decrease in young F (120 mm long). After this fish length, it was not possible to quantitatively evaluate the proliferative activity at the central canal. However, labelled cells were seldom found in the spinal cord sections until a fish length of 260 mm. From these data it is possible to conclude that motoneurone recruitment in the trout spinal cord is down-regulated at the F stage. Afterwards, we found that motoneurones increase in size to meet the growth of their target myotomal muscle.

Acetylcholine/dopamine interaction in planaria.

Planaria represents the most primitive example of centralization and cephalization of nervous system. Previous reports indicate that planaria shows specific behavioral patterns, analogous to mammalian stereotypes, in response to drugs acting on acetylcholine or dopamine transmission. Here we further characterized these responses, and investigated the interactions between cholinergic and dopaminergic systems by means of behavioral methods. Exposure to cholinergic agonists physostigmine or nicotine produced hypokinesia with 'bridge-like' and 'walnut' positions, respectively. Blockade of muscarinic receptors by atropine produced 'screw-like' hyperkinesia. Exposure to dopamine agonists (nomifensine, apomorphine) produced marked hyperkinesia with 'screw-like' movements. Finally, exposure to dopamine antagonists produced immobility or 'bridge-like' position. Pre-exposure to physostigmine blocked the behavioral effects of nomifensine and reduced and markedly delayed the behavioral effects of apomorphine. Pre-exposure to apomorphine slightly reduced and delayed the behavioral changes by physostigmine. Finally, planaria exposed to atropine after either SCH23388 or sulpiride showed 'C-like' or 'screw-like' hyperkinesia, respectively. Thus, reduction of cholinergic transmission seems to play a pivotal role in determining hyperkinesia in planaria. Under these conditions, different patterns of hyperkinetic activities occur, according to the subpopulation of dopamine receptors stimulated by drugs. These findings suggest that interactions between cholinergic and dopaminergic systems occur very early in animal phylogeny.

Opioid-dopamine interaction in planaria: a behavioral study.

The behavioral response of planaria to the exposure to selective opioid agonists was studied. The mu agonist [d-ala2, N-methyl-Phe4,Gly5-ol]enkephalin (DAMGO) and the 6 agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) failed to alter motor activity at all doses tested. Low doses of the selective kappa agonist (+/-)-trans-U-50-trans-3,4-dichloro-N-methyl-N[2-(1-pyrrodinyl)-cyclohexyl]benzene acetamide methasulphonate (U50, 488) and bremazocine-HCl increased motor activity leading to C-like position (CLP) and screw-like hyperkinesia (SLH). These changes were identical to those seen previously with the exposure to D2 or D1 dopamine receptor agonists, respectively. Higher doses of kappa agonists produced the enhancement of CLP and SLH together with robust snake-like movements (SLM). This latter response, that was typical of stimulation of kappa opioid receptors, was blocked by co-exposure to naloxone or the selective kappa antagonist Nor-binaltorphimine (Nor-BNI). Finally, co-exposure to sulpiride or SH-23390 respectively blocked the CLP or SLH response produced by U50,488 or bremazocine. Our data indicate the presence of kappa opioid receptors in planaria and suggest the functional interaction between the opioid and dopamine system in this simple animal model.

PCNA positivity in the telencephalic matrix areas in the adult of a lizard, Podarcis sicula.

In the present paper a reappraisal is made of the persistence and size of telencephalic matrix areas in normal adult specimens of a lizard, Podarcis sicula, using a recent in situ marker of cells in the S phase, the Proliferating Cell Nuclear Antigen (PCNA), belonging to the cyclin family, proteins that regulate the cell cycle and that can be immunocytochemically evidenced using monoclonal antibodies. Only a small number of positive PCNA elements, randomly arranged in the dorsal (medial and lateral) matrix areas, were found in the telencephalic ependymal tissue. These elements were found in larger numbers and distributed more uniformly in the ventral matrix areas, both opposite the ventricular lumen and outside the ependyma.

Treatment with 6-hydroxydopamine in planaria (Dugesia gonocephala s.l.): morphological and behavioral study.

Morpho-functional and behavioral effects of exposure to 6-hydroxydopamine (OHDA)-HCI (24 microg/ml per day for 24 h and 7 days) were studied in planarias (Dugesia gonocephala s.l.). Exposure to 6-OHDA-HC1 for 24 h produced hypokinesia of the specimens. These behavioral changes were more pronounced, leading to complete immobility, after 7 days of exposure to the neurotoxin. Moreover, specimens exposed to 6-OHDA-HCI for 24 h and 7 days failed to show any behavioral response to nomifensine, thus furnishing evidence of the damage of presynaptic dopamine terminals. Exposure to 6-OHDA-HCl for 24 h significantly reduced cathecolamine content in neuropil region, as demonstrated by histochemistry, and electron-dense presynaptic vesicles, as observed on electron microscopy examination. All these alterations were significantly more pronounced and were accompanied by swelling and strong increase of electron-density in cytoplasm of numerous neurons after exposure to the neurotoxin for 7 days. This appears to be the first demonstration of the neurotoxic effects of 6-OHDA-HCI in flatworms.

Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.

PCNA positivity in the telencephalic matrix areas in the adult of a newt, Triturus carnifex.

The Proliferating Cell Nuclear Antigen (PCNA) is an auxiliary protein of the DNA polymerase delta, belonging to the cyclin family, which attains appreciable levels only in those phases of the cell cycle in which DNA synthesis occurs. Using an immunocytochemical method that exploits this proliferative marker, we observed a certain PCNA positivity in the telencephalon of normal adult individuals of Triturus carnifex. The elements that display this peculiarity occupy an ependymal and/or sub-ependymal site. They are located in the anterior portion of the telencephalon only at the dorsal level and tend to decrease in number until they disappear temporarily as the intermediate portion is approached. In the posterior portion of the telencephalon, the dorsal labelling patterns, the size of which is however smaller than that observed more anteriorly, reappear and in the ventral region a large population of labelled cells appears. Then, again proceeding in a caudal direction, while the PCNA-positive elements lying dorsally diminish and disappear, they persist in the ventral region and gradually taper off until they final disappear where the telencephalic ventricles come together. This immunocytochemical picture supports the findings of other authors in adult Urodeles under both normal and experimental conditions (with classical and autoradiographic histological techniques) concerning the persistence of the telencephalon in dorsal and ventral germinative areas responsible for physiological and plastic neurogenetic events.

Effects of cocaine treatment on the nervous system of planaria (Dugesia gonocephala s. l.). Histochemical and ultrastructural observations.

Acute high dose treatment with cocaine in planaria has been shown to produce hyperkinesia followed by immobilization, thus suggesting progressive neuronal dopamine (DA) depletion. On the contrary, treatment with low doses of cocaine inhibits motor activity in planaria, without producing hyperkinesias. Here we investigated the morpho-functional changes of the DA presynaptic terminals following cocaine treatment in planaria (acute high dose and chronic low dose). Neuronal DA content was determined by means of histochemical methods, and nerve cell ultrastructure was examined by electron microscopy. The effects of cocaine were compared to those of L-dopa, reserpine (used as positive and negative controls, respectively) and normal untreated specimens. Presynaptic vesicles and DA content were significantly reduced by chronic low-dose cocaine treatment. These effects were even more robust when the drug was acutely administered at high dose. Thus, depletion of DA vesicles is produced by cocaine in planaria, as well as in mammals. The behavioral effects of chronic low-dose treatment with cocaine, however, suggest that the drug acts not only as a DA reuptake blocker, but also as a direct agonist on presynaptic DA receptors. Acute high-dose administration of cocaine also produced signs of neuronal suffering, thus providing evidence for a direct neurotoxic effect of the drug.

A pharmacological study of cocaine activity in planaria.

Planaria has been proposed as a suitable research model in neurobiology because of its relatively simple organization. Dopaminergic agonists induce in this flatworm typical hyperkinesias that can be antagonized by dopaminergic blocking agents. The neurochemical basis of the effects of cocaine in vertebrates has not been fully elucidated, but the inhibition of catecholamine reuptake at a presynaptic level seems to play an important role. In this study we analyzed the involvement of the dopaminergic system in the mechanism of action of cocaine in planaria. The dose-related effects of cocaine on planaria motility and the response to cocaine treatment associated with the administration of specific D1 or D2 dopamine agonists and antagonists were investigated. The effects of reuptake inhibitors on cocaine activity were also studied. Planaria specimens treated with low doses of cocaine become motionless, whereas high doses induce a typical behavioural response, identical to the response induced by specific D2 agonists. This response is inhibited by a D2 selective blocking agent. Nomifensine, a specific dopamine reuptake inhibitor, induces a mixed D1/D2 response. The results of these experiments are discussed, also in relation with the conservation of dopaminergic receptors during evolution.

Medullary and gangliar regeneration after unilateral removal of a segment of spinal cord of the trunk and corresponding ganglion in adult newts.

Regeneration of the spinal cord, segmental nerves and sensory ganglia takes place after tail amputation in the newt. Many histological and immunocytochemical observations provide evidence that the ependymal tube is the source not only of new neurons and glial cells in the spinal cord, but also of some cells that go on to participate in the formation of the spinal ganglia of the regenerating tail. In previous experiments involving the removal of the spinal ganglia of the trunk, no regeneration was observed and it is thought that the trunk region differs from the tail region with regard to the ability to regenerate sensory ganglia. However, in these experiments the spinal cord of the trunk was not damaged. In the present work involving adult newts (Triturus carnifex Laur.), unilateral ablation of a segment of the spinal cord of the trunk in addition to removal of a corresponding spinal ganglion was performed. In these experimental conditions, regeneration of a rudimentary spinal ganglion near the regenerated side of the spinal cord segment was observed in several cases. Histological observations carried out 2, 4, 6 and 13 months after the operation support the view that some cells migrating from the lateroventral part of the regenerating side of the spinal cord via the developing ventral root could participate in the formation of the rudimentary spinal ganglion.

Persistence of a high degree of structural organization of the telencephalon in brain homotransplants in adult Triturus carnifex (Urodele Amphibians).

Brains of adult Urodele Amphibians (Triturus carnifex Laur.) were homoplastically transplanted in conditions of complete morpho-functional isolation and fixed 100 and 365 days after the operation. Results show that in the post-telencephalic regions structural organization disappear in all cases, while, in some cases the telencephalon retain a fairly complex structural organization even after one year. The heterogeneous nature of the histological patterns observed may be attributed to the different size of the matrix areas, besides the different degree of immune response of the host to the graft. In the telencephalic district, where the matrix areas are particularly well developed, undifferentiated elements of these areas would be capable of opposing the degenerative phenomena in the transplants by neoformation of neurons. However, in post-telencephalic districts, where the matrix areas are less developed or absent, it would not be possible to make up for neuronal degeneration and these regions disappear or are formed only by some undifferentiated elements outside the surviving ependyma portions.

Degenerative and regenerative phenomena in brain heterotopic homoplastic transplants of adult Triturus carnifex (Urodele Amphibians).

The brains of adult Urodele Amphibians (Triturus carnifex Laur.) were homoplastically transplanted in conditions of complete morpho-functional isolation. Despite the heterogeneous nature of the histological aspects observed, the results point to the following general pattern for the fate of the brain transplants: 1) rooting phase (about day 5 after the operation) as indicated by degenerative patterns affecting the various encephalic districts and mainly involving the more peripheral neurons and the nerve fibers, and to a lesser extent the periventricular gray and the ependyma; 2) phase of rejection by host (from day 10 until end of experiment on day 30) as indicated by lymphocytic infiltration, the presence of macrophages and pyknoses, and the formation of a connective tissue capsule surrounding the transplant; 3) regenerative phase (from about day 10) as indicated by the onset of mitotic activity affecting elements of the ependymal epithelium and the periventricular gray, particularly in the telencephalic district. By the end of the experiment, in most of the transplants examined, degenerative processes were found to have prevailed over the regenerative phenomena. However, in some cases, and particularly in one of them, a distinctly higher degree of structural organization of neurons and nerve fibers can be observed at the telencephalic level. It may be postulated that, whenever a greater degree of compatibility exists between the host and the transplanted organ, it is possible, even in conditions of complete morpho-functional isolation, for the brain to express regenerative power through the ependyma and the matrix zones which have already been identified by some Authors in telencephalic periventricular areas or scattered through the mesencephalic gray matter.

Transient expression of glial-fibrillary acidic protein (GFAP) in the ependyma of the regenerating spinal cord in adult newts.

In the homeotherms the presence of glial-fibrillary acidic protein (GFAP) and that of neurofilaments (NF) are used as specific molecular markers associated with intermediate filaments (IF) of the glial and neuronal elements respectively. On this basis a comparison was made between trends in the immunohistochemical response to these markers of the ependyma of a both a normal and a regenerating spinal cord in Triturus carnifex (Urodele Amphibian) adults, whose high spinal cord regenerative capacity is due to the ependymal epithelium cells. Under normal conditions, the ependymal cells are vimentin positive and negative for GFAP and the IF, respectively, while GFAP and NF positivity is found in the grey and white matter. During regeneration, by about day 10, vimentin positivity begins to decrease in the ependymal epithelium of the caudal stump and GFAP positive elements appear (subsequently, this was observed also in the ependymal epithelium of the cephalic stump). High GFAP positivity is found also in the newly formed apical ampullae. From day 20 to after day 30 these patterns develop both in the stumps and in the regenerating spinal cord. NF positivity is always restricted to the grey matter alone and to the axonal processes of the white matter. At about day 60, by which time the regenerative processes may be considered as having terminated and the spinal cord has virtually regained its original structure, the immunohistochemical features typical of the rest state are restored in the ependymal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Phylogenetic evolution of intermediate filament associated proteins in ependymal cells of several adult poikilotherm vertebrates.

The cytoskeletal proteins are phylogenetically well preserved in Vertebrates. However, unlike the case of Mammals, comparatively little is known about the immunocytochemical characterization of the intermediate filaments (IF) of the ependymal epithelium cells in the lower Vertebrates. The immunohistochemical response of the ependymal epithelium of the spinal cord has been examined in several poikilotherm Vertebrates. The latter were selected on the basis of the high degree of variation in adult spinal cord plasticity, a property due to the ependymal cells and probably related to different IF-associated proteins contained in them. In Triturus (Urodele Amphibian) the presence of cells having an exclusively vimentine positive cytoskeleton, apparently not organized into filamentous structures, was observed throughout ependymal epithelium. In Rana (Anuran Amphibian) the ependymal epithelium cells were found to be comparatively positive with respect to both vimentine, which was structured into cytoskeletal filaments, and glial fibrillary acidic protein (GFAP), detected using monoclonal anti-chicken-GFAP antibodies. In Lacerta (Scaly Reptile) the ependymal epithelium cells only rarely had a vimentine positive cytoskeleton and mostly appeared to stain intensely with polyclonal anti-bovine-GFAP. The different immunocytochemical response of the cytoskeleton of the ependymal epithelium cells in the poikilotherm Vertebrates examined can be explained in terms of the varying plasticity of the spinal cord. In adult animals, this plasticity is still high in the Urodeles, lower in the Anurans, much weaker in the Reptiles and disappears completely in the homeotherm Vertebrates.

A pharmacological study of dopaminergic receptors in planaria.

The dopaminergic receptors of planaria have been studied with pharmacological and biochemical criteria. Dopamine D1 selective agonists (CY 208243 (10 micrograms/ml) and SKF 38393 (10 micrograms/ml] induced in planaria typical screw-like hyperkinesias, that were inhibited by a D1 antagonist (SCH 23390 (10 micrograms/ml], but not by a D2 antagonist (sulpiride (1000 micrograms/ml]. Dopamine D2 selective agonists (PHNO (5 micrograms/ml), lisuride (5 micrograms/ml] on the contrary induced a typical "C" like curling, that was inhibited by pretreatment with D2 selective blocking agents, but not by D1 selective blocking agents. With agonists with a D1/D2 mixed action (apomorphine 60 micrograms/ml) or with amphetamine (100 micrograms/ml), the D1 type movements appeared to be more evident. Dopamine D1-selective agonists, mixed action agonists or D2-selective agonists, all induced a significant increase in levels of cAMP, that was prevented by pretreatment with the specific DA blocking agent.

Central axonal regeneration and autoimmunity in adult birds.

After traumatic lesion adult cock spinal cord displays lively axonal regenerative activity. After 22 days, morphological regeneration regresses; functional regeneration is never observed. Before and after spinal lesion in the cocks, IgG decoration of myelin sheaths is never observed. The sera of both injured and intact animals tested both by immunohistochemical methods on intact spinal cord sections and by immunoelectrophoresis on a protein extract of homologous spinal cord are always negative. The authors suggest that the absence in Birds of any autoimmune response against c.n.s. antigens after surgical exposition, as observed in Mammals, is possibly related to the capacity of the central axons to regenerate in Birds, as observed by the Authors in Amphibia and Reptilia (Triturus and Lacerta).

Autoimmunity and central nervous system regeneration in urodele amphibians.

Spinal cord lesion in newt is followed by complete cell and fibre regeneration. Previous observations showed that the presence of a granuloma due to a foreign substance (talcum) in the vicinity of the cut spinal cord slows down and/or prevents regeneration. The present experiments, while confirming previous evidence, show that, in animals with a paraspinal granuloma or a subcutaneous granuloma containing an autoplastic and homoplastic spinal cord implant, immunocomplexes appear on the cut ends as has been observed by the same authors in animals in which spinal cord regeneration does not occur (Mammals). The authors discuss the results in view of their theory of the autoimmune nature of the absence of axonal regeneration.

Preliminary histochemical and electron microscopic observations on the nervous system of hydra.

The nervous system of a primitive Coelenterate (Chlorohydra viridissima) has been studied using ultrastructural and histochemical techniques. The authors confirm the ultrastructural pattern of nerve cells described by Lentz and coworkers. Reserpine treatment fails to induce any reduction of catechol- and indoleamine content visible to histochemical observation. In vivo treatment with tetanus toxin does not induce behavioural changes and no specific binding of toxin is revealed by immunocytological analysis. This suggests that neuron tetanus toxin receptor sites are absent in hydra. Hydra nerve cells must therefore be considered as extremely primitive elements, which the Authors consider to support the hypothesis of neurons having originated as a gradual differentiation of myoepithelial cells, as proposed by Pantin (1956) and by Passano (1963).