Andrew S. Klein

The Role of the Hyaluronan Receptor CD44 in Mesenchymal Stem Cell Migration in the Extracellular Matrix

In a previous investigation, we demonstrated that mesenchymal stem cells (MSCs) actively migrated to cardiac allografts and contributed to graft fibrosis and, to a lesser extent, to myocardial regeneration. The cellular/molecular mechanism responsible for MSC migration, however, is poorly understood. This paper examines the role of CD44‐hyaluronan interaction in MSC migration, using a rat MSC line Ap8c3 and mouse CD44−/− or CD44+/+ bone marrow stromal cells (BMSCs). Platelet‐derived growth factor (PDGF) stimulation of MSC Ap8c3 cells significantly increased the levels of cell surface CD44 detected by flow cytometry. The CD44 standard isoform was predominantly expressed by Ap8c3 cells, accounting for 90% of the CD44 mRNA determined by quantitative real‐time polymerase chain reaction. Mouse CD44−/− BMSCs bonded inefficiently to hyaluronic acid (HA), whereas CD44+/+ BMSC and MSC Ap8c3 adhered strongly to HA. Adhesions of MSC Ap8c3 cells to HA were suppressed by anti‐CD44 antibody and by CD44 small interfering RNA (siRNA). HA coating of the migration chamber significantly promoted passage of CD44+/+ BMSC or Ap8c3 cells, but not CD44−/− BMSCs, through the insert membranes (p < .01). Migration of MSC Ap8c3 was significantly inhibited by anti‐CD44 antibodies (p < .01) and to a lesser extent by CD44 siRNA (p = .05). The data indicate that MSC Ap8c3 cells, in response to PDGF stimulation, express high levels of CD44 standard (CD44s) isoform, which facilitates cell migration through interaction with extracellular HA. Such a migratory mechanism could be critical for recruitment of MSCs into wound sites for the proposition of tissue regeneration, as well as for migration of fibroblast progenitors to allografts in the development of graft fibrosis.

Characterization of Renal Allograft Rejection by Urinary Proteomic Analysis

ObjectiveTo develop a diagnostic method with no morbidity or mortality for the detection of acute renal transplant rejection. Summary Background DataRejection constitutes the major impediment to the success of transplantation. Currently available methods, including clinical presentation and biochemical organ function parameters, often fail to detect rejection until late stages of progression. Renal biopsies have associated morbidity and mortality and provide only a limited sample of the organ. MethodsThirty-four urine samples were collected from 32 renal transplant patients at various stages posttransplantation. Samples were collected from 17 transplant recipients with acute rejection and 15 patients with no rejection. Samples from patients less than 4 days posttransplant were omitted from data analysis due to the presence of excessive inflammatory response proteins. Rejection status was confirmed by kidney biopsy. Specimens were analyzed in triplicate using SELDI mass spectrometry. The obtained spectra were subjected to bioinformatic analysis using ProPeak as well as CART (Classification and Regression Tree) algorithms to identify rejection biomarker candidates. These candidates were identified by their molecular weight and ranked by their ability to distinguish between nonrejection and rejection based on receiver operating characteristic (ROC) analysis. The candidates with the highest area under the ROC curve (AUC) exhibited the best diagnostic performance. ResultsThe best candidate biomarkers demonstrated highly successful diagnostic performance: 6.5 kd (AUC = 0.839, P < .0001), 6.7 kd (AUC = 0.839, P < .0001), 6.6 kd (AUC = 0.807, P < .0001), 7.1 kd (AUC = 0.807, P < .0001), and 13.4 kd (AUC = 0.804, P < .0001). A separate analysis using the CART algorithm in the Ciphergen Biomarker Pattern Software correctly classified 91% of the 34 specimens in the training set, giving a sensitivity of 83% and specificity of 100% using two separate biomarker candidates at 10.0 kd and 3.4 kd. ConclusionsBiomarker candidates exist in urine that have the ability to distinguish between renal transplant patients with no rejection and those with acute rejection. These biomarker candidates are the basis for development of a noninvasive method of diagnosing acute rejection without the morbidity and mortality associated with needle biopsy. The combination of biomarkers into a panel for diagnosis leads to the possibility of enhanced diagnostic performance.

Organ donation and utilization in the United States, 1999-2008: Special feature

Despite the Organ Donation Breakthrough Collaboratives work to engage the transplant community and the suggested positive impact from these efforts, availability of transplanted organs over the past 5 years has declined. Living kidney, liver and lung donations declined from 2004 to 2008. Living liver donors in 2008 dropped to less than 50% of the peak (524) in 2001. There were more living donors that were older and who were unrelated to the recipient. Percentages of living donors from racial minorities remained unchanged over the past 5 years, but percentages of Hispanic/Latino and Asian donors increased, and African American donors decreased. The OPTN/UNOS Living Donor Transplant Committee restructured to enfranchise organ donors and recipients, and to seek their perspectives on living donor transplantation. In 2008, for the first time in OPTN history, deceased donor organs decreased compared to the prior year. Except for lung donors, deceased organ donation fell from 2007 to 2008. Donation after cardiac death (DCD) has accounted for a nearly 10‐fold increase in kidney donors from 1999 to 2008. Use of livers from DCD donors declined in 2008 to 2005 levels. Understanding health risks associated with the transplantation of organs from ‘high‐risk’ donors has received increased scrutiny.

Budd-Chiari Syndrome: Current Management Options

ObjectiveTo assess the outcomes of current treatment strategies for Budd-Chiari syndrome. Summary Background DataBudd-Chiari syndrome, occlusion or obstruction of hepatic venous outflow, is a disease traditionally managed by portal or mesenteric-systemic shunting. The development of other treatment options, such as catheter-directed thrombolysis, transjugular portosystemic shunting (TIPS), and liver transplantation, has expanded the therapeutic algorithm. MethodsThe authors reviewed the medical records of all patients diagnosed with Budd-Chiari syndrome at the Johns Hopkins Hospital during the past 20 years. ResultsA total of 54 patients were identified: 13 (24%) male patients and 41 (76%) female patients, ranging in age from 2 to 76 years (median 33 years). Twenty-one (39%) had polycythemia vera, 3 (5.6%) used estrogens, 11 (20%) had a myeloproliferative or coagulation disorder, and in 7 (13%) the cause remained unknown. Forty-three patients were treated with surgical shunting, 24 mesocaval and 19 mesoatrial. Actuarial survival rates at 1, 3, and 5 years after shunting were 83%, 78%, and 75%, respectively. Of 33 patients surviving more than 4 years, 28 (85%) had relief of clinical symptoms. Five patients required shunt revision and eight had radiologic procedures to maintain shunt patency. Primary and secondary shunt patency rates were 46% and 69% respectively for mesoatrial shunts and 70% and 85% respectively for mesocaval shunts. Clot lysis was successful as primary treatment in seven patients. TIPS was performed in three patients, one after a failed mesocaval shunt. During an average of 4 years of follow-up, these patients required multiple procedures to maintain TIPS patency. Six patients underwent liver transplantation. Of these, three had previous shunt procedures. Five of the transplant recipients are alive with follow-up of 2 to 9 years (median 6). ConclusionsBoth shunting and transplantation can result in a 5-year survival rate of at least 75%, and other treatment modalities may be appropriate for highly selected patients. Optimal management requires that treatment be directed by the predominant clinical symptom (liver failure or portal hypertension) and anatomical considerations and be tempered by careful assessment of surgical risk.

FK506 (tacrolimus) compared with cyclosporine for primary immunosuppression after pediatric liver transplantation: Results from the U.S. multicenter trial

We report on the efficacy and safety of FK506 (tacrolimus) compared with a cyclosporine (CsA)-based immunosuppressive regimen after 1 year of treatment in pediatric liver allograft recipients (< 12 years) participating in a multicenter U.S. randomized trial. Patients received either FK506 or CsA as primary immunosuppression following a first ABO-compatible liver transplant. Intravenous FK506 was initiated at 0.1 mg/kg per day, followed by oral FK506 beginning at 0.3 mg/kg per day. The dose was adjusted to maintain plasma trough levels of 0.5-2.0 ng/ml. The CsA group was treated according to each centers usual protocol. Both groups received the same initial doses of corticosteroids. All rejection episodes were biopsy-proven and a standardized algorithm was adopted for the treatment of rejection. Thirty patients were randomized to the FK506 group and 20 to the CsA group. After twelve months of follow-up 20 patients remained in the FK506 group and 13 in the CsA group. Patient survivals were 80% and graft survival 70% in the FK506 group compared with 81% and 71% respectively, in the CsA group. 48% of the FK506 group remained rejection-free compared with 21% of the CsA group, and 79% of FK506-treated patients did not require OKT3 compared with 68% of CsA treated patients. The cumulative corticosteroid dose was less at each time point throughout the first year in the FK506 group. The incidence of serious and minor infections was similar in both groups. Nephrotoxicity, neurotoxicity, and gastrointestinal disturbances were the major toxicities reported. Differences did not reach statistical significance between the two groups although major neurologic events, diarrhea and dyspepsia were more often reported in the FK506 group. There was no difference in mean serum creatinine at 12 months between the two groups. There was a tendency toward lower mean serum cholesterol in the FK506 group. There was no hirsuitism in the FK506 group compared with a 30% incidence in the CsA group. In conclusion, compared with CsA, there is a trend toward less rejection in FK506-treated pediatric allograft recipients, while both drugs have a similar spectrum of side effects.

Laparoscopic live donor nephrectomy: the recipient

BACKGROUND Laparoscopic live donor nephrectomy offers advantages to the donor in terms of decreased pain and shorter recuperation. Heretofore no detailed analysis of the recipient of laparoscopically procured kidneys has been performed. The purpose of this study was to determine whether laparoscopic donor nephrectomy had any deleterious effect on the recipient. METHODS A retrospective review was conducted of all live donor renal transplantations performed from January 1995 through April 1998. The control group received kidneys procured via a standard flank approach (Open). Rejection was diagnosed histologically. Creatinine clearance was calculated using the Cockroft-Gault formula. RESULTS A total of 110 patients received kidneys from laparoscopic (Lap) and 48 from open donors. One-year recipient (100% vs. 97.0%) and graft (93.5% vs. 91.1%) survival rates were similar for the Open and Lap groups, respectively. A similar incidence of vascular thrombosis (3.4% vs. 2.1%, P=NS) and ureteral complications (9.1% vs. 6.3%, P=NS) were seen in the Lap and Open groups, respectively. The incidence of acute rejection for the first month was 30.1% for the Lap group and 31.9% for the Open group (P=NS). The rate of decline of serum creatinine level in the early posttransplantation period was initially greater in the Open group, but by postoperative day 4 no significant difference existed. No difference was observed in allograft function long-term. The median length of hospital stay was 7.0 days for both groups. CONCLUSIONS Laparoscopic live donor nephrectomy does not adversely effect recipient outcome. The previously demonstrated benefits to the donor, and the increased willingness of individuals to undergo live kidney donation, coupled with the acceptable outcomes experienced by recipients of laparoscopically procured kidneys justifies the continued development and adoption of this operation.

Primary sclerosing cholangitis: resect, dilate, or transplant?

OBJECTIVE The current study examines the results of extrahepatic biliary resection, nonoperative endoscopic biliary dilation with or without percutaneous stenting, and liver transplantation in the management of patients with primary sclerosing cholangitis (PSC). SUMMARY BACKGROUND DATA Primary sclerosing cholangitis is a progressive inflammatory disease leading to secondary biliary cirrhosis. The most effective management of sclerosing cholangitis before the onset of cirrhosis remains unclear. METHODS From 1980 to 1994, 146 patients with PSC were managed with either resection of the extrahepatic bile ducts and long-term transhepatic stenting (50 patients), nonoperative endoscopic biliary dilation with or without percutaneous stenting (54 patients), medical therapy (28 patients), and/or liver transplantation (21 patients). RESULTS Procedure-related morbidity and mortality rates were similar between surgically resected and nonoperatively managed patients. In noncirrhotic patients, the serum bilirubin level was significantly (p < 0.05) reduced from preoperative levels (8.3+/-1.5 mg/dL) 1 (1.7+/-0.4 mg/dL) and 3 (2.7+/-0.9 mg/ dL) years after resection, but not after endoscopic or percutaneous management. For noncirrhotic PSC patients, overall 5-year survival (85% vs. 59%) and survival until death or transplantation (82% vs. 46%) were significantly longer (p < 0.05) after resection than after nonoperative dilation with or without stenting. For cirrhotic patients, survival after liver transplantation was longer than after resection or nonoperative dilation with or without stenting. Five patients developed cholangiocarcinoma, including three (6%) of the nonoperatively managed patients but none of the resected patients. CONCLUSIONS In carefully selected noncirrhotic patients with PSC, resection and long-term stenting remains a good option. Patients with cirrhosis should undergo liver transplantation.

Current management of the Budd-Chiari syndrome.

Twenty-six patients with the Budd-Chiari syndrome were treated surgically at the Johns Hopkins Hospital. Twenty-one of the patients were female and five were male, with a median age at diagnosis of 37 years. Nine patients had polycythemia vera, 6 were receiving estrogen therapy, 5 had a previous hepatitis A or B infection, and 4 had cirrhosis. There was one case each of hepatic malignancy, paroxysmal nocturnal hemoglobinuria, and idiopathic thrombocytopenic purpura. In five cases no etiologic factors or associated disorders were identified. Ascites was the most common presenting feature in this group of patients. Hepatic function at the time of diagnosis, as measured by standard serum chemistries, was only minimally abnormal. The diagnosis of the Budd-Chiari syndrome was confirmed in all 26 patients by hepatic vein catheterization. Inferior vena cavography was also performed and revealed caval occlusion in 4 patients, significant caval obstruction in 13 patients, and a normal vena cava in 9 patients. Interpretation of the vena cavogram was helpful in selecting the appropriate surgical procedure for each patient. Twenty-three of the twenty-six patients underwent percutaneous liver biopsy before operation, with no morbidity or mortality. Four patients had well-established cirrhosis noted on biopsy. Thirty mesenteric-systemic venous shunts were performed on the 26 patients. In 11 patients a mesocaval shunt was performed and in one instance conversion to a mesoatrial shunt was required as a second procedure. In 15 patients a mesoatrial shunt was performed as the initial procedure. Graft thrombosis occurring in 2 of these 15 patients prompted one revision in 1 patient and 2 revisions in the second patient. After mesenteric-systemic venous shunt, eight of the patients (31%) died before discharge from the hospital. The remaining 18 patients in this series were discharged from the hospital alive and well with patent shunts. Patients were followed for a median of 43 months (range, 9 months to 13 years). Five late deaths occurred between 5 and 84 months after the operation. Three- and five-year actuarial survival rates were 65% and 59%, respectively.

Effect of a prior portasystemic shunt on subsequent liver transplantation.

Fifteen patients who had a prior portasystemic shunt underwent orthotopic liver transplantation. Shunt types were portacaval in six patients, H-graft mesocaval in six, distal splenorenal in two, and proximal splenorenal in one. Mean blood loss and hospital stay were highest in the portacaval group. Retransplants (two patients) and deaths (two patients) also were limited to this group. In this report, technical considerations, advantages, and disadvantages of the various shunt types are described. Management of patients with late stages of portal hypertension must include estimation of the effects of a portasystemic shunt on subsequent liver transplantation. It is concluded that portacaval shunts should be avoided in patients who may be considered for transplantation. Distal splenorenal shunts are best performed in younger patients with intractable variceal bleeding who are not expected to require transplantation in the near future. A mesocaval H-graft is the shunt of choice in patients who are current liver transplant candidates.

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut‐derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co‐culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA‐specific CD4+ T‐cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA‐specific T‐cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.