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For a number of years, the laboratory has been collaborating with the pharmaceutical house Bracco Spa to investigate the transport mechanisms which regulate the elimination of bile and certain contrast agents for MRI.

To date, the studies conducted demonstrate that certain contrast agents already in clinical use or in advanced phases of experimentation are substrates for the MRP2 canalicular transporter, which therefore seems to be the principal factor in their biliary elimination (Lorusso V. et al. 2002, Biochem. Biophys. Res. Comm. 293-1:100-5).

On the other hand it is not yet entirely clear which is the transporter which regulates the entrance of these compounds into the hepatic cell. Much indirect evidence indicates that the class of contrast agents comprising gadolinium complexes and with a chemical structure derived from DTPA (diethylene-triamino-pentacetic acid) is the substrate for a family of transporters known as OATP’s (organic anion transporting polypeptides). At the moment there are no conclusive demonstrations as to which protein of the family is effectively involved, and even less is known of the pharmacological significance of this transport. From the experimentation in progress, it can be deduced that there are many transporters of the OATP family (and perhaps not only them) which can react to the hepatic accumulation of gadolinium complexes.

Recently an absolute quantitative system has been developed for gene expression at the RNA level of 13 passive (secondarily active) human transporters for organic anions, cations and small peptides. These transporters are: OATP-A, OATP-B, OATP-C, OATP-D, OATP-8, OATP-D, OATP-E, NTCP, OAT2, OAT3, OCT1, PEPT1, PEPT2.

These transporters, chosen for their importance at the hepatic level are also expressed in other tissues, especially in those which are involved in the metabolism and detoxification of pharmaceuticals. They are in fact those which are mainly involved in the cellular accumulation of the majority of pharmaceuticals in common use (Lorusso V. et al. 2005, Curr Pharm Des, review in press), except for the molecules which reach the transporters by means of nutrients, nucleotides and various canals.

The evaluation system used is based on quantitative RT-PCR analysis which becomes absolute thanks to the use of a calibration curves constructed with specific amplifications inserted in plasmids. To date, expression of 13 transporters has been investigated in hepatic cells in line (Chang liver and HepG2) and in a sample of normal liver. The analysis has validated the hypothesis of the OATP protein intervention in the hepatic transport of the B22956 contrast agent, demonstrating above all a great affinity on the part of the OATP-8 transporter. (Libra A. et al. 2005, FEBS Journal 272 (s1): abstract C3-045P).

This protein is defined as hepatospecific because of its almost exclusive presence in healthy liver, and it is interesting from a diagnostic point of view that it is almost completely absent in the progress of hepatic carcinoma.

The importance of evaluating the expression of the above mentioned transporters lies in the fact that it allows a better interpretation of possible transport studies and bio-availability of molecules of interest, as well as guiding studies of drug targeting for healthy and pathological tissue.

It is proposed to us the described experimental system to evaluate the presence and importance of the above mentioned genes in different cellular systems and extracts of human tissue, among which intestinal cells (Caco2, HT29), renal cells (podocytes and tubular cells), pathological tissues and other cellular models.

Another aspect of the research into contrast agents for MRI is the investigation into the intracellular destiny of these molecules, to highlight possible areas which may interfere with or increase the measurement of signal imaging. A polyclonal antibody capable of recognising the Gd-DTPA molecule is in preparation, as are consequently the majority of the contrast agents derived from this nucleon. The utilisation of the molecule allows diverse in vitro analyses appropriate for investigation into the cellular and tissue metabolisms of these compounds.

Microscopic X-ray fluorescence having the same aims will be conducted in collaboration with the LILLIT-TASC group of the Elettra Synchrotron.

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In the pathologies involving the terminal ileum, there exists the elevated risk of the appearance of biliary lithiasis whose physio-pathological mechanisms are still not understood. In general, the biliary calculi comprise cholesterol, calcium bilirubinates or other calcium salts. A greater abundance of pigmented calculi (bilirubinates) are registered in the pathologies of the ilium and above all following a resection or bypass of the same. The bilirubin which is produced at the level of the endothelial reticular system is normally metabolised by the liver which sees to  its conjugation with glucuronic acid, to then transfer it in this form in the biliary canals. While the most part is excreted with the faeces, a certain amount of the bilirubin is deconjugated in the colon by the intestinal flora and reabsorbed to return to the liver.

During the course of ileal pathology, the bile composition and the state of the bacterial flora become altered. An objective of our investigation is to throw some light on the mechanisms which regulate the enterohepatic circulation of non-conjugated bilirubin in these particular pathologic conditions, with the hypothesis that a changed/increased re-absorption of the bilirubin may be the basis for the formation for the formation of pigmented calculi in the ilium pathologies. To this end, experiments will be conducted in animal models with ileal resection and studies will be carried out on cellular lines (e.g.Caco2), derived from the colon. In these cells, the possible presence of diffusion mechanisms will occur, carrier mechanisms mediated passive or active. In the final phase it will be attempted to associate the levels of bilirubin transporter levels and/or biliary acids to a normal or altered enterohepatic circulation of the non-conjugated bilirubin.

It is expected that the investigations will show that in the absence of  a functional ileum, the reabsorption of non-conjugated bilirubin is increased, as has been hypothesised in a variety of other studies (Mendez-Sanchez N. et al. 2001 Eur J Clin Invest 31(9):773-80). The hypothesis is that, for the presence of biliary acids in the colon and/or for an altered intestinal transit time of the bile components, the bilirubin is more deconjugated and hence reabsorbed by the colon epithelium. Such an increase in re-circulation would be the origin of the pigmented lithiasis associated with ileal pathologies.
One may hence shed light upon the molecular mechanisms involved in this often not considered but important phase of bilirubin metabolism.
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Numerous epidemiological observations have revealed a significant inverse correlation between the incidence of ischaemic cardiovascular illness and the bilirubin serum levels. (summarised in Rigato I et al. TTM 2005; 11:277-283). In certain cases, the bilirubinemia value is actually identifiable as an independent risk factor of illness (Hopkins PN. Arterioscler Thromb Vasc. 1996; 16: 250). Analogous observations have been documented in the prevalence of peripheral athero-sclerotic vascular illness (Krijgsman B.Int Angiol. 2002; 21:44).

Cardiovascular disease is the most important cause of death and disability in the western world. (The World Health Report 2002.) and among the cardiovascular diseases, the athero-sclerotic vascular disease is the highest proportion.

The athero-sclerotic lesions form mainly in the medium and large calibre arteries with elastic muscular walls and may cause ischaemia in the tissues downstream from the lesion itself. The principal organs which are mainly affected by these effects are the heart, the central nervous system, the kidneys and the extremities (Ross R. NEJM. 1999; 340:115). The first event which occurs at the appearance of an athero-sclerotic lesion is the occurrence of endothelial disfunction (PanzaJA, NEJM 323, 1990 – RossR NEJM 340, 1999) which determines an increase of the expression of the adhesion molecules (VCMA-1, ECAM-1) at the endothelium which would favour the adhesion of lympho/monocytes and the subsequent migration  in the internal layer.

Endothelial disfunction means a succession of pathological conditions which involve the vascular endothelium determined by a reduced bio-availability of NO. This reduction may be caused by a variety of mechanisms; reduced expression of eNOS (endothelial NO Synthase), absence of substrate or co-factors for eNOS or accelerated consumption of NO on the part of oxygen free radicals (ROS) (Cai H, Circ Res 2000; 87: 840).

This research project has as its objective the study of possible effects of non-conjugated bilirubin in the appearance of endothelial disfunction using cellular models of altered endothelial functionality in vitro.
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The Ferrodoxin NADP(H) oxidoreductases (FNRs EC 1.18.1.2) are flavoenzymes ubiquitously distributed in animals, plants and procariot cells, in which they catalyse the reversible transfer of electrons between two  electron-tied transporter molecules (ferrodoxin, flavodoxin) and a single molecule of NADP(H). It has been shown that the  FNRs protect from oxidative stress in protobacteria and cyanobacteria. The isoforms found in plants are 200-500 times more active than those in animals or procariots, a characteristic which may explain their notable capacity as antioxidants in experimental bacterial models. The antioxidant properties of FNR may be particularly important in avoiding and counterbalancing a change in the redox state. The flavodoxin of Anabaena is capable of protecting transgenic tobacco plants from photo-oxidative stress and from water deprivation. The flavodoxins are redox proteins which have the flavina-mononucleotide as a co-factor and are part of various systems of electron transport, functionally analogous to the ferrodoxin. Even if the flavodoxins are found only in bacteria and algae, these proteins exhibit similarities to protein families of procariotic and also eucariotic origin. From the moment that these proteins demonstrated protection against oxidative stress in both bacteria and higher plants, the objective of this project has been to study their protective role when they are expressed in eucaristic systems such as mammalian cells. The experiments are carried out on cellular lines derived from Cos-7 (ECACC, No. 87021302) which express FNR and flavodoxin subjected to a variety of oxidative insults in vitro. After the treatment, various vitality tests are performed as well as the determination of early and late cellular oxidation parameters which are compared with those of the non-sectioned cells.
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The work involves the investigation into the molecular mechanisms of bilirubin metabolism in animal models, in particular the possible involvement in bilirubin transport on the part of certain membrane proteins, known to be organic anion transporters. In particular, the study concentrates on the the MRPI protein, a transporter which belongs to the super-family of ABC proteins.

The research involves the induction of acute toxic damage from bilirubin in Gunn rats, a branch of mutant rats with a congenital defect of bilirubin metabolism. The induction of hyperbilirubinemia in neonatal Gunn rats casues a toxic damage which results in the neurological syndrome BIND (Bilirubin Induced Neurologic Disfunction) whose most serious aspect is kernicterus. Kernicterus may affect neonatal babies and causes a deficit in the motory capacity of the child, deafness, mental retardation and eventual death. Different studies have demonstrated that the central nervous system is not uniformly compromised by the toxic effect of bilirubin, but that only certain cerebral areas are destroyed. The toxic damage is particularly evident in the base nuclei, in the inferior colliculus, in the nuclei of the cerebellum and in the hippocampus. The fact that certain cerebral areas are compromised while others remain intact indicates that there exists a diverse expression of the bilirubin transport proteins. The objective of the study will be to evaluate the expression of MRPI and/or other membrane transporters in different cerebral areas of the Gunn rat and to evaluate the protective role of these transporters with respect to the neurons.

The second part of the study is to follow the evolution of the neurological damage from bilirubin "in vivo" using  magnetic resonance imaging (MRI). MRI is used as a diagnostic tool in the diagnosis of Kernicterus in man, but the images obtained have all been when the disease is already established. Until now, MRI has not been used to follow the evolution of Kernicterus from the asymptomatic phase to established disease, as also the correlation between the induction of an acute bilirubinemia acuta and the compromise of the cerebral structures visible in MRI. A stimulus for neurological damage in neonatal Gunn rats will be induced by means of the acute induction of a hyperbilirubinemia, after which the affected cerebral structures will be displayed and studied by means of a 7 Tesla MRI machine adapted for rats and mice by  Bracco Imaging at Ivrea. Hide details.