Paolo Bruzzone

Hepatocyte Transplantation: Clinical Experience and Potential for Future Use

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. There are three main areas where the transplantation of isolated hepatocytes has been proposed and used for clinical therapy. Cell transplantation has been used: 1) for temporary metabolic support of patients in end-stage liver failure awaiting whole organ transplantation, 2) as a method to support liver function and facilitate regeneration of the native liver in cases of fulminant hepatic failure, and 3) in a manner similar to gene therapy, as a “cellular therapy” for patients with genetic defects in vital liver functions. We will briefly review the basic research that leads to clinical hepatocyte transplantation, the published clinical experience with this experimental technique, and some possible future uses of hepatocyte transplantation.

Age-related changes in dopamine D2 receptors in rat heart and coronary vessels

1. The distribution of dopamine D2 receptors in rat heart and coronary vessels and the possible age‐related changes in D2 receptor density were studied. The pharmacological characteristics and the anatomical location of dopamine D2‐like receptor sites in rat heart and coronary vessels were investigated using combined binding techniques and light microscopy autoradiography.

Protection from complement‐mediated injury in livers and kidneys of transgenic mice expressing human complement regulators

Abstract: The major problem in the use of phylogenetically distant donors is a fast, strong reaction called hyperacute rejection. This reaction mediated by complement is directed against the vascular endothelia of the transplanted organ. Complement activation is tightly controlled by several regulatory proteins which inhibit the formation and function of different complement components. To verify the hypothesis that organs expressing such inhibitory factors could be spared from complement‐mediated hyperacute rejection, we have generated mice transgenic for the human complement inhibitor membrane cofactor protein (hMCP) and decay accelerating factor (hDAF). Different levels of hMCP and/or hDAF expression, according to the promoter used, were detected by RNA analysis in the major organs, specifically on the organ vascular endothelia, as revealed by immunohistochemical analysis. The development of an in vivo model of human plasma perfusion allowed the characterization of complement‐mediated damage in control animals and the degree of protection due to the presence of hMCP, hDAF, or both in the organs derived from single or double transgenic mice. In this paper we compare the level of expression of complement regulators with the degree of protection in two major organs: liver and kidney.

Giant hemangiomas of the liver: surgical strategies and technical aspects

The incidence of hemangiomas is 2-7% in the general population. We evaluated more than 300 patients with hepatic hemangiomas. Surgical removal of hepatic hemangiomas was performed in 48 cases due to uncertain diagnosis (2 cases), intractable symptoms (26 cases), size increase (18 cases), and liver failure in 2 cases that were treated by hepatic transplantation. In all, 26 patients underwent enucleation of hemangiomas or segmentectomies, while the remaining 20 patients underwent right lobectomies or left lateral segmentectomies. Blood transfusions were required in four cases (including two liver transplants); mean post-resection hospital stay was 6.3 days. We observed no perioperative mortality and only two cases of major morbidity (bile leaks not requiring reoperation). Our experience confirms that, after adequate patient selection, surgical treatment of hepatic hemangiomas is a very effective therapeutic choice with no mortality and low morbidity.

Age-Related Changes in Catecholaminergic Nerve Fibers of Rat Heart and Coronary Vessels

Background: Age-related changes of catecholaminergic nerve fibers supplying the myocardium and the coronary vessels were studied in adult and in old rats. Objective: The purpose of this study was to investigate whether there are quantitative changes in myocardial and/or coronary catecholaminergic nerve fibers and related neurotransmitters. Methods: Samples of myocardium and/or coronary vessels were studied by means of glyoxylic acid induced fluorescence, aldehyde-induced fluorescence, and neuropeptide Y immunoreactivity. All morphological results were submitted to quantitative analysis of images. Biochemical dosage of proteins and of norepinephrine was also performed, and morphological data were compared with biochemical ones. Finally, all data were subjected to statistical analysis. Results: Our findings are indicative of an age-related decrease of all types of fluorescent sympathetic nerve fibers in rat heart and coronary arteries. Moreover, our data show that aging induces a strong decrease of norepinephrine levels in tissue homogenates of rat heart and coronary vessels. The majority of the catecholaminergic nerve fibers that show strong age-related changes are not contiguous with contractile cells of myocardium and/or of vascular wall. In fact, these fibers are located in adventitial (or epicardial) and in endothelial (or endocardial) layers. Conclusion: The decrease of the catecholaminergic innervation of heart and coronary vessels of old rats can be in close relationships to the poor cardiac and/or coronary functions observed in old animals.

Kidneys derived from mice transgenic for human complement blockers are protected in an in vivo model of hyperacute rejection.

PURPOSE The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage. MATERIALS AND METHODS Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 microL./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination. RESULTS Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both hDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage. CONCLUSION A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.

Ethical and Legal Issues in Donation After Cardiac Death in Italy

In Italy death of a human being must be declared either after brain death or after 20 minutes of cardiac arrest, certified by continuous electrocardiography (EKG) recording. It is my personal opinion that in such circumstances after cardiac death (DCD) will allow at best only the retrieval of few marginal kidneys and some tissues, and therefore will not be very helpful for our waiting list patients. I suggest instead modifying first the Italian law in order to be able to declare cardiac death after only 5 minutes of cardiac arrest, certified by continuous EKG recording.

Distribution of Catecholaminergic Neurotransmitters and Related Receptors in Human Bronchus-Associated Lymphoid Tissue

Background: The functions of the bronchus-associated lymphoid tissue (BALT) are under the control of the autonomic nervous system (sympathetic and parasympathetic nerve fibers). Objectives: The relationships between the adrenergic nerve fibers and β-adrenergic receptors were studied in the human BALT with the aim to demonstrate a probable neuromodulation. Methods: Morphological observations (staining with hematoxylin-eosin and scanning electron microscopy images) were carried out on samples of human BALT harvested during autopsies. Moreover, histochemical staining for norepinephrine (adrenaline = adrenergic nerve fibers) as well as for other catecholamines was performed. Finally, β-adrenergic receptors were stained by means of a β-blocking, radiolabeled drug (pindolol 125I). All our data were submitted to morphometric analysis (quantitative analysis of images and statistical analysis of data). Results: Our results provide direct evidence of the presence and distribution of catecholaminergic nerve fibers and related β-adrenergic receptors in BALT. β-Adrenergic receptors are present above all in the most richly innervated part of the BALT, and are, therefore, in close relationship with their related adrenergic nerve fibers. Conclusions: Studies on the distribution of adrenergicneurotransmitters and related β-adrenergic receptors in the human BALT are the first step for the demonstration of a probable neuromodulation of BALT.

Age-related changes in the human thymus studied with scanning electron microscopy

This investigation describes some morphological, age‐related changes in different compartments and cells of the human thymus. Scanning electron microscopic observations were done on human thymus samples obtained from four young and eight elderly patients during thoracic surgery and/or diagnostic biopsy of the thymus, after receiving authorization from the Ethical Committee of our university. The morphological data were submitted to quantitative image analysis so as to obtain quantitative results. Subsequently, the related values were used for statistical analysis. Our findings demonstrate that (1) all thymus compartments (subcapsular spaces, cortical, medullar, thymus microenvironment) contain numerous thymocytes even after the thymus has aged. (2) In older humans, residual thymus lymphoid islets contain, in addition to fatty cells and/or fibrous cells, also the same types of resident and nonresident cells (permanent and moving cells) that are found in young and adult subjects. (3) Endothelial cells of thymus microvessels contain numerous gaps. These gaps are tight in young subjects and become loose with age. (4) Thymocytes, in older subjects, are always found near these loose endothelial gaps of thymus microvessels. (5) While thymus cortical microvessels are provided with pericytes and/or periarteriolar spaces, microvessels of the thymus medullar are free of such spaces. Our morphological and quantitative results lead us to consider the possibility that the thymus fraction of resident and permanent cells (including thymocytes and reticular epithelial cells) is larger in younger subjects compared with older ones. The endothelial loose gaps of thymus microvessels, in older subjects, can allow the bidirectional transit of thymocytes through the wall of the said microvessels. Microsc. Res. Tech., 2008.

Catecholaminergic nerve fibers and β-adrenergic receptors in the human heart and coronary vessels

Abstract. The relationship between catecholaminergic nerve fibers and β-adrenergic receptors in the heart and in the coronary vessels was studied in humans. Only living tissues, harvested from brain-dead heart donors, were used in this experiment. Morphological observations and histochemical staining for norepinephrine and other catecholamines were carried out and β-adrenergic receptors were stained by means of a β-blocking fluorescent drug. All morphological data underwent quantitative analysis of images and statistical evaluation. This study provided direct evidence for the distribution of catecholaminergic nerve fibers and of β-adrenergic receptors in the myocardium and in the coronary arteries. Catecholaminergic nerve fibers are located in the periadventitial tissue, in the adventitia and in the transitional zone between the intima and media. The β-adrenergic receptors are mainly located in the more innervated part of the heart and coronary arteries but there is a major, although incomplete, overlap with related catecholaminergic nerve fibers. Moreover, using specific substrates and/or inhibitors, two types of β-adrenergic receptors, β1 and β2, were demonstrated. Owing to the scarcity of available material (we used only four hearts), and objective difficulties in enrolling other patients, we are at this moment unable to draw general conclusions.