Giuseppe Cavallari

Mesenchymal Stem Cells and Islet Cotransplantation in Diabetic Rats: Improved Islet Graft Revascularization and Function by Human Adipose Tissue-Derived Stem Cells Preconditioned With Natural Molecules

Hypoxia plays an important role in limiting the engraftment, survival, and function of intrahepatically transplanted islets. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection but also to promote revascularization. Among different possible origins, adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose tissue-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs (hMSCs). Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF) as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

Urological Complications After Kidney Transplantation: Experience of More Than 1000 Transplantations

OBJECTIVE Urinary fistulas and stenoses are the most common complications that may require surgical revision after kidney transplantation. The aim of this study was to retrospectively assess the incidence of and risk factors for early (within 30 days) or late major urological complications (stenoses and fistulas) after kidney transplantation. PATIENTS AND METHODS The study population comprised 1142 consecutive patients who underwent kidney transplantation between January 1990 and September 2007. Endpoints were early and late urological complications (stenoses and fistulas). The variables considered upon multivariate and univariate analyses were: recipient age, sex, etiology of renal failure, number (first/second) and type (single/double/combined with other organs) of kidney transplantations, cold ischemia time, type of urinary reconstruction, stent positioning, as well as donor cause of death, sex, age, and serum creatinine and clearance. We also examined the presence of graft polar arteries, acute rejection episodes, and postoperative graft function. RESULTS Among 1142 transplantation performed at our center, 100 patients (8.7%) experienced 107 urological complications: 85 (79.4%) were early (56 fistulas, 29 stenoses) and 22 (20.5%) late (7 fistulas and 15 stenoses). Multivariate analysis for all complications revealed significant associations with male recipient sex (P = .00, HR = 2), while first kidney transplantation was protective (P = .00, HR = .4). Male gender both of the recipient and of the donor was significantly associated with early fistulas (P = .01, HR = 2.5 and P = .02, HR = 2, respectively). First (versus second) kidney transplantation had a protective effect on early stenoses (P = .01, HR = .27). Late fistulas were associated with anastomotic stenting (P = .03) in univariate but not multivariate analysis. Multivariate analysis for late stenoses did not demonstrate any significant association with the considered variables; however, the late stenosis cases showed significantly higher recipient and donor ages (P < .05) and a lower donor creatinine clearance (P < .05). The type of urinary anastomosis, stenting, cold ischemia time, presence of polar arterial branches, and type of transplantation did not influence the incidence of urinary fistulas or stenoses. CONCLUSIONS Our data confirmed that older recipients and organs from older donors, especially of male gender, and retransplantations are to be considered risk factors for urological complications. The present analysis cannot suggest any modification of the actual surgical strategy that would prevent urological complications in kidney transplantation.

Increased generation of reactive oxygen species in isolated rat fatty liver during postischemic reoxygenation.

Background. Whether fatty infiltration of the liver influences the generation of reactive oxygen species (ROS) during reperfusion is unclear. Thus, this study aimed to compare the ROS formation that occurs during postanoxic reoxygenation in isolated normal and fatty livers. Methods. Isolated livers from fed Sprague-Dawley rats with normal or fatty livers induced by a choline-deficient diet were reperfused at 37°C for 60 min with an oxygenated medium containing 10 &mgr;M of lucigenin after 1 hr of warm ischemia. Superoxide anion generation was assessed by the chemiluminescence (CLS) signal emitted from the organ surface. The hepatic content of malondialdehyde (MDA) and glutathione was determined at the end of reperfusion. Tissue injury was evaluated by the liver histology and the alanine aminotransferase (ALT) release in the perfusate. Results. CLS started rapidly with reoxygenation and it diffused to the whole organ in both groups. However, CLS emission was significantly higher in fatty liver (after 10 min: 812.425±39.898 vs. 294.525±21.068 photons/cm2/sec;P <0.01). A greater concentration of MDA was measured at the end of reoxygenation in fatty liver. Finally, the liver histology and the ALT release indicated a greater injury in steatotic than normal liver. Conclusions. The CLS technique allows a direct visualization and comparison of ROS generation from the organ surface. Fatty infiltration increases ROS generation in the liver during postischemic reoxygenation, likely leading to the greater lipid peroxidation observed in these experiments. The increased oxidative stress may contribute to the reduced tolerance of steatotic livers to ischemia-reperfusion injury.

Randomized clinical study comparing UW and celsior solution in liver preservation for transplantation : Preliminary results

ALTHOUGH University of Wisconsin (UW) solution remains the most commonly used for all intrabdominal organs, Celsior solution, widely utilized in clinical setting for the preservation of intrathoracic organs, has been recently proposed as a cold-storage solution for abdominal organs. One of the reasons for focusing on abdominal preservation with Celsior is to proceed with one solution for the preservation of all organs. Experimental studies have shown excellent results in kidney, liver, and tissue preservation in alternative to the U W. Celsior, due to its peculiar physical properties (osmotic pressure), is able to limit the edema that develops rapidly after cold ischemia and able to prevent free-radical mediated tissue injury by its biochemical characteristics. Its clinical use in these organs is now under investigation in different centers. The only report about Celsior solution for human liver preservation experiences in a small, uncontrolled series, and the study suggested that this solution is appropriate for clinical use in liver preservation. A prospective, randomized, multicenter open clinical trial was instituted to compare preservation properties of the two solutions in liver transplantation, and a detailed report of the completed study will be made at a later date. This preliminary report contains the data collected from a single center to prove the equivalence of both solutions with regard to initial functioning and shortand long-term graft and patient survival.

Analysis of 80 dual-kidney transplantations: a multicenter experience.

INTRODUCTION The use of kidneys from expanded criteria donors (ECD) is an attractive strategy to enlarge the pool of organs available for transplantation. Considering the fact that ECD organs have a reduced nephron mass, they are preferentially allocated for dual-kidney transplantation (DKT). Authors have reported excellent results of DKT when pretransplant ECD organs are evaluated for histological scores. The aim of this study was to evaluate DKT donor and recipient characteristics for comparison with DKT posttransplant outcomes versus those of recipients of single-kidney transplantations from expanded criteria (edSKT) and ideal donors (idSKT). We analyzed the potential prognostic factors involved in DKT among a population derived from three transplant centers. MATERIALS AND METHODS Between 2001 and 2007, DKT (n = 80) were performed based upon the ECD kidney allocation assessed by biopsy. RESULTS The average donor ages for the DKT, edSKT, and idSKT groups were 68.8 ± 7.8, 65.3 ± 7.2, and 40.1 ± 13.8 years, respectively (P < .001). The number of human leukocyte antigen mismatches was greater in the DKT group (3.1 ± 1.2, P < .05). Patient and graft 5-year survival rates were similar among DKT, edSKT, and idSKT recipients, namely, 97.5% versus 95.8% versus 96.9% and 93.7% versus 87.4% versus 86.9%, respectively. Mean serum creatinine values at discharge were lower in the DKT and idSKT recipients (1.5 ± 0.9 and 1.6 ± 0.7 mg/dL; P < .05) compared with the edSKT group (1.9 ± 0.7 mg/dL). Correlations between supposed prognostic factors and survival among the DKT group noted worse outcomes in reoperation cases (P < .05). CONCLUSION We confirmed that DKT produced successful outcomes. An accurate surgical procedure is particularly important to try to avoid reoperations. In our experience, the use of a biopsy as an absolute criterion to allocate ECD kidneys may be too protective.

Aortoiliac surgery concomitant with kidney transplantation: a single center experience

Abstract:  Introduction:  Aortoiliac pathology in kidney allograft recipients is not rare but treatment timing is controversial. As most publications on this topic are case reports it’s difficult to evaluate long‐term outcomes of those clinical challenges. Herein we report long‐term results of these procedures.

Multicenter study on double kidney transplantation.

BACKGROUND Marginal organs not suitable for single kidney transplantation are considered for double kidney transplantation (DKT). Herein we have reviewed short and long-term outcomes of DKT over a 7-year experience. PATIENTS AND METHODS Between 2001 and 2007, 80 DKT were performed in the transplant centers of Bologna, Parma, and Modena, Italy. Recipient mean age was 61+/-5 years. The main indications were glomerular nephropathy (n=33) and hypertensive nephroangiosclerosis (n=14). Mean HLA A, B, and DR mismatches were 3.1+/-1.2. Donor mean age was 69+/-8 years and mean creatinine clearance was 75+/-27 mL/min. Almost all kidneys were perfused with Celsior solution. Mean cold ischemia time was 17+/-4 hours and mean warm ischemia time was 41+/-17 minutes. Mean biopsy score was 4.4. Immunosuppression was based on tacrolimus (n=52) or cyclosporine (n=26). RESULTS Fifty (62.5%) patients displayed good postoperative renal function. Thirty (37.5%) experienced acute tubular necrosis and required postoperative dialysis treatment; 8 acute rejections occurred. Urinary complications were 13.7% with 8/11 requiring surgical revision. There were 6 surgical reexplorations: intestinal perforation (n=2), bleeding (n=3), and lymphocele (n=1). Two patients lost both grafts due to vascular and infectious complications at 7 or 58 days after transplantation. Two patients underwent intraoperative transplantectomy due to massive vascular thrombosis. Four (5%) patients underwent transplantectomy of a single graft due to vascular complications (n=2), bleeding (n=1), or infectious complications (n=1). Graft and patient survivals were 95% and 100% versus 93% and 97% at 3 versus 36 months, respectively. CONCLUSIONS DKT is a safe approach for organ shortage. The score used in this study is useful to determine whether a kidney should be refused or accepted.

Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets.

Previously we demonstrated the superiority of small islets vs large islets in terms of function and survival after transplantation, and we generated reaggregated rat islets (pseudo‐islets) of standardized small dimensions by the hanging‐drop culture method (HDCM). The aim of this study was to generate human pseudo‐islets by HDCM and to evaluate and compare the physiological properties of rat and human pseudo‐islets. Isolated rat and human islets were dissociated into single cells and incubated for 6–14 days by HDCM. Newly formed pseudo‐islets were analysed for dimensions, morphology, glucose‐stimulated insulin secretion (GSIS) and total insulin content. The morphology of reaggregated human islets was similar to that of native islets, while rat pseudo‐islets had a reduced content of α and δ cells. GSIS of small rat and human pseudo‐islets (250 cells) was increased up to 4.0‐fold (p < 0.01) and 2.5‐fold (p < 0.001), respectively, when compared to their native counterparts. Human pseudo‐islets showed a more pronounced first‐phase insulin secretion as compared to intact islets. GSIS was inversely correlated to islet size, and small islets (250 cells) contained up to six‐fold more insulin/cell than large islets (1500 cells). Tissue loss with this new technology could be reduced to 49.2 ± 1.5% in rat islets, as compared to the starting amount. With HDCM, pseudo‐islets of standardized size with similar cellular composition and improved biological function can be generated, which compensates for tissue loss during production. Transplantation of small pseudo‐islets may represent an attractive strategy to improve graft survival and function, due to better oxygen and nutrient supply during the phase of revascularization. Copyright

Extracorporeal Portal Vein Oxygenation Improves Outcome of Acute Liver Failure in Swine

BACKGROUND Portal vein arterialization (PVA) has shown efficacy to treat acute liver failure (ALF) in preclinical studies. The next step is to perform large animal studies that propose a clinically acceptable method of PVA. In this study, we assessed the efficacy of PVA using an extracorporeal device to treat 2 ALF models in swine. MATERIALS AND METHODS The 2 ALF swine models were carbon tetrachloride toxic ALF and subtotal hepatectomy using 8 animals per group. PVA was performed with an extracorporeal device that may be suitable for future clinical studies. Arterial blood was drawn from the iliac artery and delivered into the portal vein for a 6-hour treatment. We analyzed biochemical, blood gas, and histological parameters as well as 1-week survival rates. RESULTS In both models, ALF was successfully achieved. Control group animals deteriorated biochemically, dropping their prothrombin times and increasing the liver enzymes. In contrast, treated animals improved with a survival rate of 75% at 7 days compared with 0% for the former group. CONCLUSIONS PVA using an extracorporeal device was feasible and effective to treat both toxic and resective ALF in swine.

Effect of Colic Vein Ligature in Rats with Loperamide-Induced Constipation

Introduction. Medical treatment in chronic constipation is not always successful. Surgery is indicated in unresponsive selected severe cases. This study presents the distal venous colic ligation in rat as a novel surgical approach. Materials and Methods. 16 rats (study group) were evaluated in 3 phases of 6 days each: A (normal conditions), B (loperamide-induced constipation), and C (colic vein legation) and compared with rats treated in phase C with PEG 4,000 (control group). Blood biochemical and physiological parameters, daily fecal water content (FWC), and histological analysis were performed in all study phases. Results. No biochemical and physiological parameters changes were observed. FWC decreased in phase B and increased in phase C in both groups with a grow up to 2.3-fold in study group compared to control (P < 0.0001). Moreover, in study group, a high number of colonic goblet cells were detected (phase C versus phase B: P < 0.001) while no differences were registered in control. Conclusion. By ligature of the colic vein in constipated rats, an increase in FWC and goblet cells higher than in PEG treated rats was detected. The described surgical procedure appeared effective, simple, and safe; further studies in animal models, however, are necessary to assess its clinical applicability.