Cytokeratin expression in primary epithelial cell culture from bovine conjunctiva.

Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions. Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions. The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER). Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype. Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture. Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions. Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells. These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses.

[The occupational risk in IRCCS of Neurosciences]. / Il rischio professionale negli IRCCS di Neuroscienze.

The results of meta-analysis carried out on all documents for the valuation of the risk, recorded by IRCCS of Neuroscience, has led to identify a risk matrix for the medical personnel of the Scientific Institute of Neuroscience. Such analysis allowed to tackle the day to day risk faced by hospital staff. As a general rule the IRCCS of Neurosciences distinguishes themselves as structures of mainly neurologic typology and as structures of more typical psychiatric interest. In the first case the analogy with the traditional Hospital structures are more noticeable, in the second case instead the kind of patients and the particular relations between the health worker and the patient himself may represent an important differentiation factor. This is, above all, evident on the biological risk; for it's determinism has an important rule the interpersonal relationship between the health worker and the patient. Concerning other risk factors such as the chemical and the allergic one, it is noticeable a closed analogy with the actual Hospital reality. On work organization level, manual handling of patients is often necessary and also the relatives risks. In this kind of structures the stress factor is of the greatest importance, it should therefore have a strong consideration on the valuation of risk by the IRCCS.

[Knowledge of occupational risk in IRCCS of Neurosciences]. / Conoscenza del rischio professionale negli IRCCS di Neuroscienze.

A Professional Multicentric Audit, concerning Staff knowledge level about security on workplaces, was conducted by way of a standardized questionnaire given to 1022 (26.4%) IRCCS professional staff pertaining to Neurobiology. Four were the principal tasks of the questionnaire investigating the "sensibility" and "consciousness" concerning risk prevention on workplaces. It came out that there is a great variability in the knowledge level on this field, even if the "medium value" is "pretty good".

[Phenomenon of occupational accidents of workers of IRCCS of Neurosciences]. / Il fenomeno infortunistico negli operatori degli IRCCS di Neuroscienze.

On the basis of information collected from ten IRCCS, it has emerged altogether that the accident are not much frequent. Accident statistics indices from 1994 to 1998 were respectively: frequency rate 21.36, incidence rate 3.32, severity rate 0.24. In the "Neurology" Institutes we have above all needlestick injuries and cufs to the arms and hands. The workers most exposed are doctors, trained nurses, technicians. In the "Psychic" Institutes we have falling, impact, lifting and physical contact accidents. The injuries are contusions to legs and head. The most exposed workers are therapists and rehabilitation educators.

Heterocyclic aromatic amines efficiently induce mitotic recombination in metabolically competent Saccharomyces cerevisiae strains.

Heterocyclic aromatic amines (HAs) represent a class of potent bacterial mutagens and rodent carcinogens which gain their biological activity upon metabolic conversion by phase I and phase II enzymes. Subsequent to cytochrome P450 (CYP)-dependent hydroxylation, mainly catalyzed by CYP1A2, acetylation mediated by the activity of N-acetyltransferase, NAT2, produces the ultimate electrophilic product that may react with DNA. In addition to point mutations observed in HA-exposed cells as genotoxic endpoint in vitro, loss of heterozygosity (LOH) has often been identified in HA-related rodent tumors as another endpoint in vivo. LOH may reflect a chromosomal deletion, a chromosome loss or a previous mitotic recombination event and it represents a prominent mechanism for the inactivation of tumor suppressor alleles. In this study we have investigated whether LOH observed in several HA-induced rodent tumors is related to a recombinogenic activity of HA compounds, and to address this question we have studied the genotoxic activity of several HAs in metabolically competent Saccharomyces cerevisiae strains. For this purpose expression vectors have been constructed providing simultaneous expression of three human enzymes, CYP1A2, NADPH-cytochrome P450 oxidoreductase and NAT2 in different genotoxicity tester strains. Evidence for functional expression of all three enzymes has been obtained. One strain allowed us to monitor HA-induced gene conversion, another one HA-induced chromosomal translocation. A third strain allowed us to study HA-induced forward mutations in the endogenous URA3 gene. It was found that 2-amino-3-methylimidazo-[4,5-f]quinoline and 2-amino-3, 8-dimethylimidazo-[4,5-f]quinoxaline produced a strong recombinogenic response in either recombination tester strain. The recombinogenic activity was comparable with the mutagenic activity of the compounds. The other HAs, 2-amino-3, 4-dimethyl-imidazo-[4, 5-f]quinoline, 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole, 2-aminodipyrido-[1,2-a:3', 2'-d]imidazole, 3-amino-1-methyl-5H pyrido-[4,3-b]indole and 2-amino-1-methyl-6-phenyl-imidazo-[4, 5-b]pyridine, produced weak or no increases in the genotoxic endpoints of interest. The described strains may provide a suitable tool to characterize the genotoxic potential of HAs in more detail.