Epidemiological data suggest that non-steroidal anti-inflammatory drugs prevent colon cancer. The evidence for other types of tumour is less conclusive, though animal and in vitro studies indicate that they may be effective against mammary cancer cells. We assessed the effect of dietary acetylsalicylic and salicylic acid against dimethylbenzanthracene-induced rat tumours. Tumour angiogenesis was also investigated to explore the mechanism responsible for salicylate effect. Mammary tumours were induced in female Sprague-Dawley rats fed with different amounts of acetylsalicylic and salicylic acid. Serum vascular endothelial growth factor concentrations were measured and vascularization of basement membrane proteins injected in vivo (Matrigel) was determined by evaluation of haemoglobin content to assess the extent to which angiogenesis was inhibited. Dimethylbenzanthracene-induced carcinogenesis was inhibited by both acids and there was a log-dose/response correlation between the tumour diameter and salicylate concentration. Salicylic acid seems more effective than acetylsalicylic acid. Vascular endothelial growth factor was less concentrated in treated animals than in the controls and so was Matrigel haemoglobin. The mechanism involved, however, is still uncertain, though concomitant inhibition of tumour angiogenesis may be an important component. The documented salicylate serum VEGF modulation is interesting also for presence of the flk-1 receptor in mammary tumour cells of our model. Although misoprostol is a prostaglandin analogous its concomitant administration did not compromise the salicylate anti-tumour effect.

Abnormalities in ΔNp63 expression have been regarded as early indicators of carcinogenesis of squamous origin, including oral carcinomas. In our hypothesis, the α isoform of ΔNp63 might represent an early marker for identifying neoplastic tissues left behind after surgical ablation of an oral carcinoma. To demonstrate this hypothesis, we are prospectively screening ΔNp63α overexpression in surgical margins. In one of our analysis we found a correlation between ΔNp63α overexpression and the presence of tumoral cells subsequently confirmed by histology. This observation supports our hypothesis while waiting for the main study to be completed.

In an in vivo rat model of liver ischemia followed by reperfusion a consistent appearance of necrosis and activation of biochemical pathways of apoptosis was reproduced and monitored after 30 minutes reperfusion. Preconditioning by application of a short cycle of ischemia-reperfusion (10 minutes + 10 minutes) positively conditioned recovery of the organ at reperfusion, attenuating both necrotic and apoptotic events. Preconditioning at least halved cell oxidative damage occurring early at reperfusion, and as a major consequence, the increase of cytolysis and apoptosis occurring at reperfusion was about 50% less. The attenuation of both pathways of cell death by preconditioning appeared at least partly related to its modulate action on H(2)O(2) and 4-hydroxy-2,3-trans-nonenal production. The overall data point to a marked diminished oxidant generation and oxidative reactions as one major possible mechanism through which ischemic preconditioning exerts protection against necrotic and apoptotic insult to the postischemic liver.

Unscheduled DNA synthesis refers to DNA synthesis not followed by cell division. Previous studies have suggested that this phenomenon may occur in neurons from peripheral myenteric ganglia in conditions of functional hyperstimulation. In order to verify these observations, we have carried on an immunohistochemical study on myenteric neurons from the hypertrophic intestinal loops upstream from a partial obstruction (an experimental condition that induces a relevant increase of the neuronal workload) after labelling with two different markers: the proliferating cell nuclear antigen (PCNA), that is specifically expressed in cell nuclei during the S-phase, and the protein gene product 9.5 (PGP 9.5), a specific marker of nerve cells. While no myenteric neuron immunopositive for the anti-PCNA antibody was found in the control intestine, in the hypertrophic myenteric ganglia some neurons were positive for PCNA. These results provide an unequivocal evidence on the existence of unscheduled DNA synthesis in myenteric neurons from the hypertrophic intestine.

Squamous cell carcinoma of the oral cavity continues to be a major clinical problem, with about 100,000 new deaths each year worldwide. There is therefore a need to search for new tools to aid oral cancer treatment. We tested the inhibitory activity on chemical carcinogenesis of the three principal protein fractions of about 50, 14, and 8.5 kDa of the mixture UK101 derived from goat liver. These are composed principally of a glycoprotein rich in mannose residues, a protein with analogy to the heat shock protein family, and ubiquitin, respectively. The animal model employed was dimethylbenzanthracene-induced hamster cheek pouch carcinoma. Number of tumours per animal, tumour mass per animal, and proliferating cell nuclear antigen (PCNA) in non-tumour mucosa were quantified: the 14-kDa fraction was the most active; this was also confirmed by testing its corresponding recombinant material. The 50-kDa fraction was inactive, while the ubiquitin showed only low inhibitory activity. It is possible that the technique described and the results obtained could lead to an interesting clinical approach to the treatment of oral cancer.

Silver impregnation studies in chick embryos have shown that, by the 2nd day of incubation, the earliest neurofibrillar differentiation occurred in neuroblasts located at the diencephalic-mesencephalic junction and in the rhombencephalon; some of these neuroblasts were believed to become reticular neurons. Since calretinin, a cytosolic calcium-binding protein of the "E-F hand" family, occurs in reticular neurons, the present study investigated immunohistochemically whether the early differentiating reticular neurons are also the first neurons to express this marker during chick embryo development. The first calretinin-immunoreactive neuroblasts appeared at stage 11 (40-45 h of incubation according to the series of Hamburger and Hamilton), and were located in the basal plate of the diencephalic-mesencephalic junction and of rhombomeres adjacent to the otic placode and in the alar plate and intermediate zone of the cervical spinal cord. In bromodeoxyuridine-injected embryos, these earliest calretinin-immunoreactive neurons were shown to express the calcium-binding protein 11-16 h after their last mitosis. By stage 11 up to the 14th day of incubation (stage 40), the calretinin-immunostained neurons increased in number and ultimately formed a chemically defined subset of neurons belonging to the tegmental reticular formation and raphe region of the brainstem. In the meantime, early calretinin-immunostained nerve processes were shown to form two conspicuous longitudinal bundles which run in the ventral and lateral margins of the brainstem and spinal cord.

Liver ischemia-reperfusion is characterized by an increased oxygen-dependent free radical chain-reaction rate and an increased steady-state concentration of reactive oxygen species. The aim of this study was to evaluate the in situ generation of reactive oxygen species and its relationship with phagocyte activation and recruitment in reperfused rat liver. Rat livers were subjected to 2 hours of selective lobular ischemia and reperfusion for up to 12 hours. The following parameters were determined: in situ liver chemiluminescence, understood to reflect the tissue steady-state concentration of singlet oxygen (1O2); myeloperoxidase tissue activity; the number of neutrophils; and the degree of necrosis. An early chemiluminescence burst was measured after 30 minutes of blood reflow (early phase of oxidative stress), followed by a relapse and a further increase after 4 to 12 hours of reperfusion (late phase of oxidative stress). Both early and late phases were modified by pretreatment with gadolinium chloride (GdCl3), pointing to a key role of the Kupffer cells. Neutrophils infiltrated into the liver, myeloperoxidase activity, in situ chemiluminescence, and necrosis were found to be strongly correlated over the 4- to 12-hour reperfusion period (r = .960; average of the 4 correlation coefficients). Together with resident phagocytes, neutrophil recruitment and activation appear to provide a major contribution to the increase of oxygen-dependent free-radical reactions and amplification of liver reperfusion damage. Surface chemiluminescence appears to properly describe the in situ and in vivo progressive organization of the acute inflammatory response with phagocyte-mediated liver injury.

A morphological analysis of types and sub-types of neurons from dorsal root ganglia at different spinal levels was carried out by combined light and electron microscopy in Podarcis sicula. Two neuron types were recognized: small dark cells (type D) and large light cells (type L). Type L cells were further sub-divided into three sub-types (L1, L2, L3) on the basis of entity and distribution of neurofilaments. Percentage distribution of neuron types did not vary in relation to the spinal level. ON the contrary, differences were found in the percentage of the three type L neuron sub-types; a higher percentage of cells very rich in neurofilaments (L2,L3) was found in dorsal root ganglia from the cervical and the lumbar spinal levels. Results are discussed in relation to the spinal-level-related extent of innervation territory of dorsal root ganglia.

Chemically induced Syrian hamster cheek-pouch squamous cell carcinoma is very similar to the corresponding human tumour. This paper describes a blind study in which inhibition of dimethylbenzanthracene-induced cheek-pouch tumours by a goat liver extract denominated UK101 was investigated. Less than 40% of animals treated with UK101 developed tumours compared with 100% of the controls. Intermediate results (80%) were noted in a positive control group treated with Calmette-Guerin bacillus. Immunocytochemical testing of cheek-pouch mucosa by Mib5 showed significantly less proliferating cells in UK101 animals than in the controls. The effect of UK101 was completely reversed when dexamethasone was added in a third control group. A significant difference in complement-mediated cytotoxicity was noted in the sera of UK101-tested and control animals. These findings suggest that an immune mechanism is responsible for the inhibition of hamster cheek-pouch carcinoma by UK101.

The ontogeny of two calcium-binding proteins (calbindin-D28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.

To define the type of cell death occurring in notochordal tissue, the cytological features of degenerating notochord were studied by transmission electron-microscope in thirty chick embryos from the 20th hour to the 15th incubation day. During the first two days the notochordal cells show nuclei with large nucleoli and cytoplasm with yolk granules, lipid droplets, phagolysosomes and deposits of glycogen. From the 3rd to the 5th incubation day, besides the peculiar vacuolization, disaggregation of the endoplasmic reticulum, transformation of the mitochondrial morphology, and disintegration of the cell membrane are detectable. Nuclei are normal up to advanced stages of cytoplasmic degeneration. On the 6th day a large number of cells are dying and, later on, the tissue disintegrates at the level of the vertebral bodies. Cell death in the notochord does not seem to be classifiable as one of the types of developmental cell death described in literature: the comparison with similar cytological features referred by pathologists as a consequence of metabolic damage, suggests that the degeneration of the notochord may be related to its morphological isolation and thus to trophic deprivation.

The significance and relevance of the term "chordoid tissue" are discussed on the ground of some ultrastructural features of the notochordal cells, observed in 15 chick embryos from stage 5HH to hatching. The characteristics of the cytoplasmic organelles and of the nucleus, the membrane alterations and in particular the vacuolization show the notochordal cells undergo a process of necrobiosis rather than of apoptosis. The degeneration of the notochord, that not casually starts in the notochordal core, seems to be due to a progressive metabolic isolation, related to the lack of blood vessels and to the formation of a thick perichordal sheath. These findings suggest that the notochordal tissue shows the same cytologic features of any tissue degenerating owing to a metabolic damage. The term "chordoid tissue", in the meaning of a tissue with peculiar structure and function, does not seem hence to be relevant, also on the ground of the fact that in the species in which a nucleous pulposus arises from notochord remnants, the notochordal cells show the same ultrastructural characteristics detectable in chick embryos.

The cytology of the notochord was investigated by means of L.M. and T.E.M. in fifty chick embryos from the 7th HH stage to hatching, at different levels of the longitudinal axis and at different levels in each somite. The cytodifferentiation of the notochord was compared with the one of the surrounding axial and paraxial structures (neuro-epithelium and somites). Close segmental relations among notochord, neural groove and sclerotome, i.e. reciprocal contacts of cell processes and filopodia, where observed in 7-9 HH stages embryos. During the following stages in the notochordal cells signs of secretory activity are detectable, but at the same time degenerative changes are evident and they will increase up to the total regression of the notochord. These morphological observations seem to suggest the presence of reciprocal morphogenetic influences among notochord, neuro-epithelium and sclerotome, only before the 9th stage, rather than a determinant role of the notochord in the surrounding structures at later developmental stages.

Previous ultrastructural studies on the developing spine induced the Authors to analyse the presence of Ca-binding proteins in the vertebral anlagen of the chick embryo, by immunostaining for calretinin. At 7 incubation days, intense positivity for calretinin at level of the intervertebral region is detectable. From the 7th day the positivity is localized in particular to the intervertebral menisci and it appears also in the degenerating chondrocytes of the chondrifying vertebrae and at level of the peripheral zones of vascular erosion in the ossifying vertebrae. The intervertebral positivity for calretinin can be related to the presence at this level of fibroblast-like cells, that for such immunoreactivity are distinguishable from true fibroblasts. Since other Authors showed presence of S-100 protein in fibroblast-like cells during histogenesis of white adipose tissue, the significance of the expression of some Ca-binding proteins in the differentiation of these cell types can be supposed. The meaning of the immunoreactivity in the vertebral bodies is still uncertain and needs further immunohistochemical studies.

The authors focused their attention on the cytology of the degenerating notochord at the level of the vertebral anlagen and compared it with data of the literature on the histopathology of the spinal chordomas. The purpose of the research was to investigate the notochordal histological features justifying the fact that the remnants of a structure destined to atrophy during prenatal life, maintain a proliferative potential in postnatal life. Therefore, from the earliest stages (24th incubation hour) to the terms of development, the notochord was studied by electron microscope in chick embryo. The data obtained show that, in most notochordal cells, signs of metabolic damage and progressive degeneration coexist with signs of secretory and mitotic activity, from which the proliferative potential of these cells seems to derive. Vacuolar degeneration is also likely due to the progressive metabolic segregation of the notochord, owing to the absence of blood-vessels and the appearance of perichordal sheath.

Since in literature the question of the spatio-temporal sequence of the cartilage maturation in the developing vertebra is still controversial, the authors studied by light and electron microscope, the chondrification of the vertebral body in chick embryo from the 6th to the 13th incubation days, in order to define the correlations between morphology and distribution of the cartilage cells in this phase of vertebral development. The results show that the chondrogenesis follows spatio-temporal gradients, starting at about the 8th incubation day from a zone located between notochord and neural tube, slightly cranially to the midvertebral level. From this starting point the chondrification proceeds with dorso-lateral and radial progression, and at the same time extends towards the cranial and caudal plates of the developing vertebra. These data are compared to the findings obtained by other authors on the ultrastructural and biochemical aspects of the vertebral development.