Stefano Gatti

Isolation and Characterization of a Stem Cell Population from Adult Human Liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin‐19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, α‐fetoprotein, and in a small percentage of cells, cytokeratin‐8 and cytokeratin‐18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated α‐fetoprotein and expressed cytokeratin‐8 and cytokeratin‐18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin‐producing islet‐like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe‐combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.

Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats

Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4‐integrin‐dependent mechanism. However, MVs pre‐treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA‐dependent effect. Microarray analysis and quantitative RT‐PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre‐treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.

Effects of continuous negative extra-abdominal pressure on cardiorespiratory function during abdominal hypertension: an experimental study

ObjectiveTo investigate whether negative extra-abdominal pressure (NEXAP) improves respiratory function and induces a blood shift from the intrathoracic compartment and to assess whether these effects are influenced by abdominal pressure.Design and settingProspective, randomized, controlled trial in the animal laboratory of a university hospital.SubjectsEight sedated and paralyzed pigs (19.6±3.4xa0kg).InterventionsApplication of NEXAP (−20xa0cmH2O).Measurements and resultsAirway, esophageal, gastric and central venous pressures were recorded simultaneously. Intrathoracic blood volume was assessed by PiCCO. The effects of NEXAP were assessed with and without abdominal hypertension by intraperitoneal insufflation of helium. NEXAP caused a lasting drop of gastric (1.97±2.26xa0mmHg) and esophageal (1.21±0.67xa0mmHg) pressures, while end-expiratory airway pressure was similar, hence transpulmonary pressure increased. Intrathoracic blood volume dropped from 358±47 to 314±47xa0ml. The fall was associated with a decrease in central venous pressure (R2=0.820). When peritoneal pressure was raised (24.7±5.5xa0mmHg), the effects were less marked. However, the difference between negative pressure around the abdomen and the pressure inside the abdomen (effective NEXAP) was correlated with the proportional changes in intrathoracic blood volume (R2=0.648), being greater with more negative effective NEXAP. NEXAP improved chest wall elastance during abdominal hypertension (from 0.067±0.023 to 0.056±0.021xa0cmH2O/ml).ConclusionsNEXAP increases lung volume and causes a shift of blood from the intrathoracic compartment. It needs to be tailored against abdominal pressure to be effective

A blunt complex abdominal trauma: total hepatectomy and liver transplantation

Abstract A victim of a motor accident was referred to the Emergency Department. He was unconscious, breathing was rapid and shallow, and had a mean arterial pressure of 60xa0mmHg with a distended abdomen. A peritoneal lavage was positive for blood. An immediate laparotomy showed a massive hepatic injury with bilobar disruptions. After an unsuccessful hepatorrhaphy an extensive perhepatic gauze packing was done. During the operation the patient was massively transfused and high doses of dopamine were used. At the end a relative stability was reached, but a few hours later, due to the high risk of sepsis and abdominal compartment syndrome, a second laparotomy was performed. Despite efforts to reach a good hemostasis, it was decided to perform a total hepatectomy and portocaval shunt. The patient was put on the waiting list for a compatible liver. After 36xa0h in this anhepatic state, the patient received the new graft. The graft showed immediate recovery. In cases of severe and complex liver trauma, performing a total hepatectomy followed by liver transplantation is justified.

Extracorporeal Lung Perfusion and Ventilation to Improve Donor Lung Function and Increase the Number of Organs Available for Transplantation

INTRODUCTIONnEx vivo lung perfusion (EVLP) has been validated as a valuable technique to increase the pool of organs available for lung transplantation.nnnMATERIAL AND METHODSnAfter a preclinical experience, we obtained permission from the Ethics Committee of our institution to transplant lungs after EVLP reconditioning. ABO compatibility, size match, and donor arterial oxygen pressure (PaO(2))/fraction of inspired oxygen (FiO(2)) ≤ 300 mm Hg were considered to be inclusion criteria, whereas the presence of chest trauma and lung contusion, evidence of gastric content aspiration, pneumonia, sepsis, or systemic disease were exclusion criteria. We only considered subjects on an extra corporeal membrane oxygenation (ECMO) bridge to transplantation with rapid functional deterioration. Using Steen solution with packed red blood cells oxygenated with 21% O(2), 5% to 7% CO(2) was delivered, targeted with a blood flow of approximately 40% predicted cardiac output. Once normothermic, the lungs were ventilated with a tidal volume of 7 mL/kg a PEEP of 5 cmH(2)O and a respiratory rate of 7 bpm. Lungs were considered to be suitable for transplantation if well oxygenated [P(v-a) O(2) > 350 mm Hg on FiO(2) 100%], in the absence of deterioration of pulmonary vascular resistance and lung mechanics over the perfusion time.nnnRESULTSnFrom March to September 2011, six lung transplantations were performed, including two with EVLP. The functional outcomes were similar between groups: at T72 posttransplantation, the median PaO(2)/FiO(2) were 306 mm Hg (range, 282 to 331 mm Hg) and 323 mm Hg (range, 270 to 396 mm Hg) (P = 1, EVLP versus conventional). Intensive care unit ICU and hospital length of stay were similar (P = .533 and P = .663, respectively) with no mortality at 60 days in both groups. EVLP donors were older (49 ± 6 y versus 21 ± 7 y, P < .05), less well oxygenated (184 ± 6 mm Hg versus 570 ± 30, P < .05), displaying higher Oto scores (9.5 ± 0.7 versus 1.7 ± 1.5, P < .05).nnnCONCLUSIONSnThe first 6 months of the EVLP program allowed us to increase the number of organs available for transplantation with short-term outcomes comparable to conventional transplantations.

Metabolic syndrome after liver transplantation : short-term prevalence and pre- and post-operative risk factors

BACKGROUNDnThe metabolic syndrome is a common condition among liver transplanted patients and contributes to morbidity and mortality by favouring the development of cardiovascular diseases.nnnAIMSnThis prospective study assessed the prevalence of metabolic syndrome in the first year after orthotopic liver transplantation, the associated pre-operative and post-operative risk factors and the influence of nutritional factors.nnnMETHODSn84 cirrhotic patients (75% male, mean age 53.9±9.3 years) were evaluated at baseline and after liver transplantation. Metabolic syndrome was defined according to 2004 Adult Treatment Panel-III criteria. Nutritional habits were assessed using 3-day food records.nnnRESULTSnPrevalence of metabolic syndrome before orthotopic liver transplantation was 14/84 (16.6%); at 3, 6 and 12 months post-orthotopic liver transplantation it was 27/84 (32.1%), 30/84 (35.7%), and 32/81 (39.5%), respectively. Diabetes, family history of diabetes, and excess body weight at baseline independently correlated with incidence of metabolic syndrome. After orthotopic liver transplantation, patients with metabolic syndrome showed a higher increase in the intake of total energy and saturated fats and a higher prevalence of complications, especially cardiovascular events, than subjects without metabolic syndrome.nnnCONCLUSIONnOccurrence of metabolic syndrome is an early phenomenon after liver transplantation. Pre-operative and post-operative factors predispose patients to metabolic syndrome, which may be reduced by controlling modifiable risk factors, such as body weight and dietary intake.

Pharmacokinetics of FK506 and mycophenolic acid in experimental and clinical intestinal transplantation.

Abstract Rejection control remains the crucial problem of intestinal transplantation. Currently, this type of transplantation requires prolonged administration of high-dose immunosuppressive treatment via parenteral route, resulting in a high incidence of toxicity.1−3 The aim of our study is to investigate the pharmacokinetics of both FK506 and mycophenolic acid (MPA) after oral administration in swine and human intestinal transplantation and to verify whether administration via oral route only is sufficient to reach and subsequently to maintain target drug levels.

Sodium bicarbonate treatment during transient or sustained lactic acidemia in normoxic and normotensive rats.

Introduction Lactic acidosis is a frequent cause of poor outcome in the intensive care settings. We set up an experimental model of lactic acid infusion in normoxic and normotensive rats to investigate the systemic effects of lactic acidemia per se without the confounding factor of an underlying organic cause of acidosis. Methodology Sprague Dawley rats underwent a primed endovenous infusion of L(+) lactic acid during general anesthesia. Normoxic and normotensive animals were then randomized to the following study groups (nu200a=u200a8 per group): S) sustained infusion of lactic acid, S+B) sustained infusion+sodium bicarbonate, T) transient infusion, T+B transient infusion+sodium bicarbonate. Hemodynamic, respiratory and acid-base parameters were measured over time. Lactate pharmacokinetics and muscle phosphofructokinase enzymes activity were also measured. Principal Findings Following lactic acid infusion blood lactate rose (P<0.05), pH (P<0.05) and strong ion difference (P<0.05) drop. Some rats developed hemodynamic instability during the primed infusion of lactic acid. In the normoxic and normotensive animals bicarbonate treatment normalized pH during sustained infusion of lactic acid (from 7.22±0.02 to 7.36±0.04, P<0.05) while overshoot to alkalemic values when the infusion was transient (from 7.24±0.01 to 7.53±0.03, P<0.05). When acid load was interrupted bicarbonate infusion affected lactate wash-out kinetics (P<0.05) so that blood lactate was higher (2.9±1 mmol/l vs. 1.0±0.2, P<0.05, group T vs. T+B respectively). The activity of phosphofructokinase enzyme was correlated with blood pH (R2u200a=u200a0.475, P<0.05). Conclusions pH decreased with acid infusion and rose with bicarbonate administration but the effects of bicarbonate infusion on pH differed under a persistent or transient acid load. Alkalization affected the rate of lactate disposal during the transient acid load.

Evidence that SMS 201-995 enhances the immunosuppressive effect of FK506

Somatostatin (SS) was originally described as a growth hormone release inhibiting factor synthesised in the hypothalamus. Recently, SS and its receptor (SSTR) have been demonstrated in lymphoid tissues and seem to play a regulatory, largely inhibitory, role in immune responses. The aim of the present study was to check the immunosuppressive effect of a SS derived peptide, the octreotide (SMS 201-995) and to verify whether this molecule acted synergistically with FK506. An immunosuppressive effect of SMS was observed on the proliferation of rat spleen cells induced in vitro, either by polyclonal mitogens such as PHA or by alloantigens. With PHA stimulation, 10(-14) M SMS significantly enhanced the immunosuppressive action of 0.00001 microg/ml FK506. The addition of SMS in MLR (10(-11)-10(-9)M) increased the antiproliferative effect of both 0.0001 microg/ml and 0.00001 microg/ml FK506. In consideration of the extremely low concentration of both drugs that was required to obtain a good immunosuppression in vitro, we verified the association of FK506 and SMS in vivo in an allogeneic skin graft model that used Lewis (Lew) rats as donors and Brown Norway (BN) rats as recipients. BN treated with 0.1 mg/kg FK506 and 0.5-10 microg/kg SMS showed a significant increase in mean skin allograft survival time when compared to either a monotherapy or control group. None of the animals died or showed signs of drug-related toxicity. In conclusion, a combined therapy of SMS and FK506, administered at lower dosages than those that are considered therapeutic, led to an effective immunosuppression without any undesirable side effects.

Magnetic Resonance (MR) Imaging and MR Spectroscopy of Nerve Regeneration and Target Muscle Energy Metabolism in a Model of Prosthesis-Guided Reinnervation in Rats

RATIONALE AND OBJECTIVESnWe monitored the regeneration of the rat sciatic nerve after its transection and the concomitant alteration in the high-energy phosphates content in the target tibialis anterior muscle.nnnMETHODSnRat sciatic nerve was resected and the gap connected with a prosthesis of polytetrafluoroethylene. Progress of reinnervation was monitored by 1H MR imaging, whereas muscular energy metabolism was evaluated by localized 31P MR spectroscopy.nnnRESULTSnReconstitution between the nerve stumps was resumed 8-12 weeks postoperatively. The ratio of phosphocreatine to inorganic phosphate reached a plateau at 46% of the initial level approximately 8 weeks after the operation and recovered thereafter. Immediately after the surgery, muscular pH became slightly alkaline and returned to normal with the progress of reinnervation.nnnCONCLUSIONnRecovery of the muscular energy metabolism began after the reconnection of the severed nerve stumps. The combination of MR imaging and MR spectroscopy followed noninvasively the progress of reinnervation and muscular energy metabolism of the prosthesis-guided nerve regeneration.