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Background Tissue homeostasis is guaranteed by stem proliferating reserve, depending on dynamic changes in gene expression. A high plasticity is shown by the haematopoietic stem cells, potential source for liver regeneration. Aim We aimed to evaluate the gene expression modifications induced by human haematopoietic stem cell therapy after liver injury in rats. Subjects Rats were sorted as follows: (A) human-haematopoietic stem cell injection after allyl alcohol liver damage; (B) only haematopoietic stem cell injection; (C) only allyl alcohol injection; and (D) sacrifice without any treatment. Methods Livers, spleens and bone marrows were analysed with flow-cytometry. Livers were also studied by reverse-transcription PCR, histology, immunohistochemistry and microarray analysis; selected genes were confirmed by real-time PCR. Results In subset A, haematopoietic stem cells were selectively recruited by liver, with respect to the group B, and they improved the liver regeneration process compared to group C. As regards microarrays, haematopoietic stem cell infusion upregulates 265 genes and downregulates 149 genes. Differentially regulated genes belong to a broad range of functional pathways, including proliferation, differentiation, adhesion/migration and transcripts related to oval-cell activation. Real-time PCR validated array results. Conclusions Our study confirmed the capacity of haematopoietic stem cells to contribute to liver regeneration. Moreover, microarray analysis led to the identification of genes whose regulation strongly correlates with a more efficient process of liver repair after haematopoietic stem cell injection.

Background Liver regeneration is a heterogeneous phenomenon involving the proliferation of different cell lineages in response to injury. Under a strong positive selection pressure bone marrow derived stem cells may be involved in this process, by making a contribution to both parenchymal restoration and endothelial cell replacement. We investigate bone marrow stem cell migration to the liver in patients undergoing hepatectomy or with acute on chronic liver failure. Methods We enrolled 6 patients submitted to hepatectomy, 6 patients to cholecystectomy and 8 patients with acute decompensation of liver cirrhosis. Mobilization of CD34+ cells was evaluated by cytofluorimetry on peripheral blood samples at different time points; baseline, 1, 3, 7, 15 and 30 days after surgery and at admission, 1, 7 and discharge among patients with acute on chronic liver failure. 10 healthy subjects undergoing blood donation were also enrolled to evaluated the basal value of CD34+ cells. Results White blood cell counts remained in the normal range (4.1–9.8 × 10 9 /L) in all groups throughout the follow-up. In all patients of Groups 1, 2 and 3, circulating CD34+ failed to show statistically significant differences both as the absolute number and as the percentage at any time point compared to healthy controls. Conclusions Bone marrow derived cell mobilization can not be detected after hepatectomy or during an acute decompensation on a cirrhotic liver. Under these circumstances liver regeneration can probably call upon mature hepatocytes and endogenous progenitor cells. The involvement of extrahepatic progenitors if any, is a rare and limited phenomenon.

Background and Aim Tissue homeostasis and turnover require reserve stem proliferating cells. Several studies performed on immunodeficient animals have suggested a degree of plasticity by the hematopoietic stem cell compartment that may represent source for liver regeneration. We sought to explore the hepatic differentiation potential of hematopoietic stem cells from human cord blood, after toxic liver damage induced by allyl-alcohol in immunocompetent rats. Materials and Methods Wistar rats were divided into groups (A) allyl-alcohol intraperitoneal injection with hematopoietic stem cell intraperitoneal infusion at 1 day and sacrifice 3 days later; (B) stem cell injection and sacrifice 3 days later; (C) allyl-alcohol infusion and sacrifice 4 days later; and (D) sacrifice without any treatment. Livers, spleens, and bone marrows were analysed for human stem cells using flow-cytometry; livers were also tested by histology and immunohistochemistry to study the pattern of hepatic regeneration after damage and human stem cell conversion into hepatocyte-like cells, respectively. Results Flow-cytometry revealed selective recruitment of human hematopoietic stem cells by damaged livers (group A) compared with control group B. In addition, liver damage was reduced in animals treated with stem cells. Immunohistochemistry demonstrated that human stem cells could convert hepatic cells. Conclusions Our study demonstrated that hematopoietic stem cells selectively recruited by injured livers can contribute to hepatic regeneration after acute toxic damage in immunocompetent recipients.

Background and aim Molecular adsorbent recycling system (MARS) treatment is able to remove both hydrosoluble and small- and medium-sized lipophilic toxins. MARS plays an important role in modifying liver failure complications, such as hepatorenal syndrome and hepatic encephalopathy. We sought to evaluate the clinical efficacy and safety of a MARS device in a consecutive series of hepatic failure patients. Materials Twenty patients with acute liver failure, transplantation failure, or acute on chronic liver failure fulfilled the inclusion criteria of total bilirubin ≥10 mg/dL and at least one of the following: hepatic encephalopathy (HE) ≥II grade, hepatorenal syndrome (HRS) for chronic patients or total bilirubin ≥5 mg/dL and HE ≥I grade for acute patients. Results MARS was able to reduce cholestatic parameters and improve neurologic status and renal function parameters in all treated patients. We also observed an improvement in the 3-month survival rate compared to the expected outcome in patients with MELD scores between 20 and 29, as well as 30 and 39. Conclusions Based on these results, we confirm the safety and clinical efficacy of MARS treatment, with the best results in patients with MELD score of 20 to 29. Further studies are necessary to confirm whether this treatment is able to modify patient outcomes and prognosis.

In the last years, a considerable number of studies have been performed on the correlation between Helicobacter pylori infection and ischaemic heart disease. The reason is the supposed role of some chronic infections in the genesis and development of vessel wall injury and atheromatous plaque, as already reported for Chlamydia pneumoniae and herpes viruses. While this association may be theoretically conceivable, it still remains debated from a practical point of view. Epidemiological and animal studies as well as some eradicating trials gave conflicting results, while studies investigating the specific molecular mimicry mechanisms induced by H. pylori strongly support the association. Moreover, none of the studies performed so far did take into account the effect of the genetic susceptibility to develop ischaemic heart disease or to respond to H. pylori infection. In particular, while the exposure to some known risk factor for atherosclerosis should lead to develop ischaemic heart disease, no condition or exposure, either individual or in combination, completely explains the occurrence and the progression of the disease, as many patients develop ischaemic heart disease in the absence of any risk factor. Based on these concepts, can we state that H. pylori infection may cause the same effect in patients with ischaemic heart disease as in healthy subjects? Further studies are needed in order to clarify this issue.

Background. Several studies have demonstrated that bone marrow contains a subpopulation of stem cells capable of participating in the hepatic regenerative process, even if some reports indicate quite a low level of liver repopulation by human stem cells in the normal and transiently injured liver. Aims. In order to overcome the low engraftment levels seen in previous models, we tried the direct intraperitoneal administration of human cord blood stem cells, using a model of hepatic damage induced by allyl alcohol in NOD/SCID mice. Methods. We designed a protocol based on stem cell infusion following liver damage in the absence of irradiation. Flow cytometry, histology, immunohistochemistry and RT-PCR for human hepatic markers were performed to monitor human cell engraftment. Results. Human stem cells were able to transdifferentiate into hepatocytes, to improve liver regeneration after damage and to reduce the mortality rate both in both protocols, even if with qualitative and quantitative differences in the transdifferentiation process. Conclusions. We demonstrated for the first time that the intraperitoneal administration of stem cells can guarantee a rapid liver engraftment. Moreover, the new protocol based on stem cell infusion following liver damage in the absence of irradiation may represent a step forward for the clinical application of stem cell transplantation.

Aims: There is contrasting evidence on the relevance of chronic infection by Helicobacter pylori ( H. pylori ) as a risk factor for stroke. We performed a meta-analysis of case-control studies to assess association of H. pylori infection and more virulent H. pylori strains, bearing the cytotoxin-associated gene-A (CagA) antigen, with different types of stroke. Methods: Outcome measures were: H. pylori and CagA seroprevalence in (1) patients with stroke versus controls, and (2) patients with stroke due to large vessel stroke versus patients with other types of stroke and controls. Results: Seven cross-sectional, case-control studies were included. Odds ratio for individual case-control studies and pooled OR for the association between H. pylori seropositivity and stroke was 1.49 (95% CI 1.24–1.81), for the association between stroke and anti-CagA positivity was 2.23 (95% CI 1.49–3.36). Patients with large vessel stroke had higher odds for H. pylori infection than patients with other types of stroke (odds ratio 1.65; 95% CI 1.12–2.45), and than controls (odds ratio 1.61; 95% CI 1.13–2.32). Conclusions: Association between H. pylori positivity, anti-CagA positivity and stroke is modest and seems higher with stroke due to large vessel disease. This meta-analysis suggests that the role of CagA positive H. pylori strains in different stroke etiologic subclasses should be the target of future prospective investigation.

Background The radial artery has recently been proposed as an alternative arterial conduit for surgical myocardial revascularization. This study was conceived to evaluate the degree of atherosclerotic involvement of the radial artery in patients with coronary artery disease and the eventual influence of a subtle degree of preoperative atherosclerosis on the midterm results of radial artery grafts. Methods and results The intima-media thickness of the radial artery, common carotid artery, and internal thoracic artery was evaluated in 42 coronary artery disease patients and in 26 control patients. All radial arteries were then used for myocardial revascularization; 30 patients submitted to control angiography after 5 years. The mean intima-media thickness was 0.92 ± 0.22 mm for the common carotid artery, 0.54 ± 0.16 mm for the internal thoracic artery, 0.55 ± 0.11 mm for the radial artery in coronary artery disease patients versus 0.79 ± 0.14 mm, 0.52 ± 0.11 mm, and 0.56 ± 0.09 mm, respectively, in control patients ( P = .001 only for the common carotid artery). No correlation was found between the intima-media thickness of the carotid, internal thoracic, and radial artery. No correlation was found between the preoperative intima-media thickness of the radial artery and the midterm patency and endothelial-mediated vasodilating capacity of radial artery grafts. Conclusion In coronary artery disease patients, radial artery atherosclerotic involvement is more frequent than that of the gold standard internal thoracic artery but still by far less severe than that of the common carotid artery. The early atherosclerotic signs often observed in the radial artery do not seem to have the potential to influence radial artery graft patency and endothelial function.

Apolipoprotein E-knockout (apoE KO) mice have peripheral sensory nerve defects, reduced and delayed response to noxious thermal stimuli, abnormal morphology of unmyelinated fibers, and impaired blood-nerve and blood-brain barriers. In this study, we show that, compared to wild-type mice, peripheral nerves of apoE KO mice have impaired ability to respond to ischemia, as demonstrated by measurement of motor and sensory conduction velocity. In addition, mice lacking apoE exhibit a deficit of reinnervation of ischemic epidermis, evaluated by immunofluorescent staining for the pan-neuronal marker PGP 9.5. Also regional nerve blood flow, measured by laser Doppler, and intraneural angiogenesis after ischemia are significantly compromised in apoE-deficient mice. Finally, upregulation of the angiogenic cytokine vascular endothelial growth factor (VEGF), which physiologically occurs after ischemia in the peripheral nerve of wild-type mice, is severely impaired in apoE KO mice. Among the several neural defects that have already been described in mice lacking apoE, this is the first demonstration that functional recovery to ischemia is impaired in the peripheral nerves of these animals. This deficit is mirrored by the inability of upregulating VEGF and mounting an appropriate intraneural angiogenic response following injury. These findings provide new evidence of possible interdependent relationships between VEGF, angiogenesis, and nerve function and regeneration and may provide new important information on the role of apoE in the nervous system.

Background . The hemodynamic significance of patent mammary graft side branches is still controversial. This study was designed to evaluate the potential for flow steal of patent mammary side branches in different hemodynamic conditions. Methods . Echo-Doppler measurement of mammary graft flow was performed at rest and after dipyridamole-induced coronary vasodilatation in 10 patients with angiographic demonstration of evident mammary graft side branches (study group) and in 10 matched control patients (control group). Concomitant thallium-201 myocardial scintigraphy was performed to assess the adequacy of mammary flow to the myocardial oxygen demand. Patients of the study group were also submitted to flow evaluation in condition of selective muscular or combined systemic and coronary relaxation. Results . No difference in mammary flow and adequacy to myocardial oxygen demand was detected between patients of the study and control groups both at rest and after dipyridamole infusion. In patients with patent side branches the systolic-to-diastolic flow ratio was maintained in case of combined coronary and peripheral vasodilatation, whereas selective muscular relaxation led to an increase in the systolic and a reduction in the diastolic flow. Conclusions . Flow steal from patent mammary graft side branches is possible only in case of selective muscular vasodilatation. As this situation is unlikely to occur in the clinical setting, the potential for flow steal of mammary side branches in cardiac surgery patients seems to be minimal.

Reports in the literature have shown plasma fibrinogen and factor XIII (FXIII) levels to be higher in patients with arteriopathy than in healthy subjects. Fibrinogen constitutes the basic matrix for the blood-clotting process. Excessive levels of fibrinogen in the blood result in increased clotting activity and increased aggregation of erythrocytes, leukocytes, and platelets, which can be important factors in thrombotic and atherosclerotic events. FXIII stabilizes fibrin monomers, facilitating the incorporation of alpha 2 -antiplasmin into the stabilized fibrin. In addition, this mechanism promotes the deposit of fibrin on arterial walls and within the arterial lumina; it also can delay activation of the fibrinolytic system and render it incapable of removing fibrin. Therefore, hyperfibrinogenemia and increased FXIII activity can lead to atherosclerosis. The aim of this study was to evaluate the possibility of a pharmacologic reduction of fibrinogen levels and FXIII activity in patients with peripheral arterial occlusive disease. Results of the study demonstrated that acute intravenous administration of high-dose defibrotide reduced both factors in these patients.

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.

Inflammatory processes are considered important in the pathogenesis of Alzheimer's disease (AD). Intercellular adhesion molecule-1 (ICAM-1) is an important mediator of inflammatory response and immune cell activation, is expressed on cerebrovascular endothelium and neuritic plaques in brain of AD patients, and seems to be implicated in the process of neuro-degeneration. A common polymorphism of the ICAM-1 gene (K469E) has been recently reported. In this case-control study, we evaluated the distribution of E/K alleles and genotypes of the ICAM-1 gene in 98 patients affected by sporadic AD and 115 age- and sex-matched controls. The frequency of the EE genotype was significantly higher in AD patients (P

The therapeutic effects of defibrotide, a polydesoxyribonucleotide derivative with antithrombotic, fibrinolytic, and anti-ischemic activity, were explored in 15 patients with occlusion of the central vein (5 cases) or peripheral branches of the retinal venous system (10 cases). Defibrotide was administered by slow intravenous infusion in doses of 2 gm daily for 21 days: at the start and end of the study, all patients underwent ophthalmologic examination with measurements of visual acuity and funduscopy by direct and indirect ophthalmoscopy. Visual acuity improved in 11 patients, while it remained unchanged in four cases; the difference between means was statistically significant ( P P P

L0191 VISUALIZATION OF POST-ISCHEMIC FREE RADICALS FORMATION IN LIVERS ISOLATED FROM RATS CHRONICALLY EXPOSED TO ETHANOL. A Gasbarrini. G Addolorato, M Simoncini, P Pasini, B Nardo, F Franceschi, A De Luca, G Vagni, E Roda, P Pola, M Bemardi, A Roda, G Gasbarrini. Internal Medicine and Gastroenterology Depts, Catholic University of Rome and University of Bologna; Ricerca in Medicina, Bologna; Italy.

Oxydative stress occurs during the reperfusion injury that follows liver transplantation. Ethanol is considered a pro-oxidant agent. In this study, we aimed to assess the effects of chronic ethanol administration on oxydative stress both in basal condition then after reperfusion injury. METHODS: Wistar male rats were exposed to a diet containing sucrose (control) or different amount of ethanol (3, 6, 12, 24 g/d). After 4 weeks, livers were isolated, exposed to warm ischemia for 120 min, then reperfused with an oxygenated medium for 60 min. Adenosine triphosphate (ATP), malondyaldeide (MDA) and reduced gluthatione (GSH) concentration of the liver were determined. RESULTS: in control condition, liver of rats fed with the highest ethanol doses (12 and 24 g/d) showed a significantly lower concentrations of GSH and ATP and a significantly higher concentration of MDA, when compared to the other groups. After 60 min of ischemia, GSH and ATP levels decreased significantly in all groups; newly, however, antioxidant and energy status reached a concentration significantly lower than in controls in livers obtained from rats exposed to 12 and 24 g/ethanol/d. During reperfusion, ATP and GSH recoveedr progressively in all groups at pre-ischemic levels; MDA, however, which increased sharply in all livers, reached significantely greater values in organs from rats treated with 12 and 24 g/ethanol/d. Interestingly, the lowest ethanol concentrations used (3 and 6 g/d) appeared to attenuate MDA tissue generation during reperfusion when compared to livers obtained by control rats. CONCLUSIONS: the chronic administration of high alcohol doses affect liver energy level and antioxidant status and increases the sensitivity of the organs to the oxydative stress caused by ischemia/reperfusion.