Prediction of portal vein thrombosis after hepatectomy for hepatocellular carcinoma.

PURPOSE: Portal vein thrombosis (PVT) following hepatectomy is potentially life-threatening. We aimed to evaluate the incidence of PVT after hepatectomy for hepatocellular carcinoma and identify coagulation and fibrinolytic factors that could predict early-stage postoperative PVT. METHODS: A retrospective analysis was conducted on 65 hepatocellular carcinoma patients who underwent radical hepatectomy. The risk factors for postoperative PVT were identified based on univariate and multivariate analyses, and the levels of coagulation and fibrinolytic factors were measured during the perioperative period. RESULTS: The incidence of PVT after hepatectomy was 20.0%. The patients were divided into two groups: those with PVT (n=13; PVT group) and those without PVT (n=52; no-PVT group). The frequency of the use of the Pringle maneuver during surgery was higher in the PVT group than in the no-PVT group, and the postoperative/preoperative ratios of thrombin-antithrombin III complex (TAT) and of D-dimer were significantly higher in the PVT group. CONCLUSION: A high incidence of PVT was found in hepatocellular carcinoma patients after hepatectomy. The frequency of the Pringle maneuver is a potential risk factor for postoperative PVT, and the postoperative/preoperative TAT and D-dimer ratios may be used as early predictors of PVT after hepatectomy for hepatocellular carcinoma.

Massage of the Hepatoduodenal Ligament Recovers Portal Vein Flow Immediately After the Pringle Maneuver in Hepatectomy.

BACKGROUND: The Pringle maneuver is often used in liver surgery to minimize bleeding during liver transection. Many authors have demonstrated that intermittent use of the Pringle maneuver is safe and effective when performed appropriately. However, some studies have reported that the Pringle maneuver is a significant risk factor for portal vein thrombosis. In this study, we evaluated the effectiveness of portal vein flow after the Pringle maneuver and the impact that massaging the hepatoduodenal ligament after the Pringle maneuver has on portal vein flow. MATERIALS AND METHODS: Patients treated with the Pringle maneuver for hepatectomies performed to treat hepatic disease at our hospital between August 2014 and March 2019 were included in the study (N = 101). We divided these patients into two groups, a massage group and nonmassage group. We measured portal vein blood flow with ultrasonography before and after clamping of the hepatoduodenal ligament. We also evaluated laboratory data after the hepatectomy. RESULTS: Portal vein flow was significantly lower after the Pringle maneuver than before clamping of the hepatoduodenal ligament. The portal vein flow after the Pringle maneuver was improved following massage of the hepatoduodenal ligament. After hepatectomy, serum prothrombin time was significantly higher and serum C-reactive protein was significantly lower in the massage group than in the nonmassage group. CONCLUSION: Massaging the hepatoduodenal ligament after the Pringle maneuver is recommended in order to quickly recover portal vein flow during hepatectomy and to improve coagulability.

[A Case of Survival Following Fourth-Line Outpatient Chemotherapy 19 Months after Surgery for Small Intestinal Cancer with Multiple Liver Metastases in a Patient with Good General Condition].

A 59-year-old woman who complained of melena and lightheadedness visited the outpatient clinic at our hospital.According to her blood test result, she had anemia, and her tumor marker levels were high.Enhanced computed tomography(CT) findings showed small intestinal cancer with multiple liver metastases.Partial resection of the small bowel for the small intestinal cancer was performed.Following the administration of fourth-line outpatient chemotherapy containing S-1 plus irinotecan( IRIS)and IRIS plus bevacizumab(IRIS plus Bev), S-1 plus oxaliplatin plus Bev(SOX plus Bev), and weekly paclitaxel (wPAC), she survived with good condition for 19 months after the surgery.

Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates.

BACKGROUND: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. METHOD AND RESULTS: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). CONCLUSIONS: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.

[A case of gradually progressive, symptomatic, ectopic gastric pancreas].

A 27-year-old man was admitted to a hospital with a complaint of epigastric discomfort. Upper gastrointestinal endoscopy and endoscopic ultrasonography revealed an elevated lesion on the posterior wall of the upper gastric body, and a diagnosis of ectopic gastric pancreas was made. Follow-up endoscopy performed 5 years later revealed an increase in the size of the mass to approximately 5cm in diameter. The location, shape, and clinical course of the mass aroused a suspicion of malignancy; therefore, partial gastrectomy was performed. Histopathologically, the resected mass was diagnosed as ectopic gastric pancreas with chronic inflammation, fibrosis, and bleeding around the acinar cells.

[A successful resected case of advanced esophageal cancer with early gastric cancer responding to neoadjuvant chemotherapy of docetaxel, CDDP and 5-FU].

A 72-year-old male with a chief complaint of dysphagia was admitted to our hospital. Upper gastrointestinal endoscopic examination showed double cancers with thoracic esophageal cancer in the middle esophagus and gastric cancer in the antrum. Pathological examinations of the double cancer revealed the first one to be moderately-differentiated squamous cell carcinoma and the second to be well-differentiated adenocarcinoma. Computed tomography (CT) of the chest and abdomen showed no distant or lymph node metastases. Clinical stagings of the double cancer were stage II (T2N0M0)in esophageal cancer and stage I A (T1N0M0) in gastric cancer. The patient received neoadjuvant chemotherapy using docetaxel, CDDP and 5-FU. After 2 courses of chemotherapy, the adverse event was grade 2 in leucopenia and grade 2 in alopecia. Repeated macroscopic and histological examinations after chemotherapy revealed that the esophageal cancer had significant reductions in the size of tumors, leading to a partial response, and the gastric cancer had disappeared, leading to a complete response. He underwent thoracoscopy-assisted esophagectomy in the prone position, and laparoscopy-assisted gastric tube reconstruction. This neoadjuvant chemotherapy of docetaxel, CDDP and 5-FU might be effective and tolerable as with patients with double cancer of esophageal and gastric cancers.

Two synchronous pseudoaneurysms after bile duct resection for distal cholangiocarcinoma.

Pseudoaneurysm is a potentially life-threatening complication after hepatobiliary pancreatic surgery. Although various measures have been taken to prevent the formation of postoperative pseudoaneurysms, completely avoiding complications can be difficult. An 83-year-old man underwent bile duct resection and systematic regional lymphadenectomies for distal cholangiocarcinoma. Polyethylene glycolic acid mesh with fibrin glue was applied to the pancreas around the distal stump and detached artery to prevent leakage of pancreatic juice and reinforce the arterial wall. Screening contrast-enhanced computed tomography on the 7th postoperative day indicated no pseudoaneurysm. The patient was discharged on the 20th postoperative day after an uneventful course. However, 4 days later, the patient visited the emergency outpatient department with a complaint of fever. Contrast-enhanced computed tomography revealed an abscess formation and a pseudoaneurysm around it. Emergency celiac arteriography revealed two pseudoaneurysms at the left hepatic artery and posterior superior pancreaticoduodenal artery; they were successfully treated with transcatheter arterial embolization using microcoils and covered stent placement. The patient was discharged 9 days after interventional radiology treatment. At the 14 months postoperative follow-up, the patient had no recurrence or stent obstruction. Multiple synchronous pseudoaneurysms are rare; accurately identifying the site by angiography and selecting appropriate treatment for each site is important.

Management of Simple Hepatic Cyst.

Simple hepatic cysts are typically saccular, thin-walled masses with fluid-filled epithelial lined cavities. They arise from aberrant bile duct cells that develop during embryonic development. With the development of diagnostic modalities such as ultrasonography (US), CT, and MRI, simple hepatic cysts are frequently detected in clinical examinations. US is the most useful and noninvasive tool for diagnosis of simple hepatic cysts and can usually differentiate simple hepatic cysts from abscesses, hemangiomas, and malignancies. Cysts with irregular walls, septations, calcifications, or daughter cysts on US should be evaluated with enhanced CT or MRI, to differentiate simple hepatic cysts from cystic neoplasms or hydatid cysts. Growth and compression of hepatic cysts cause abdominal discomfort, pain, distension, and dietary symptoms such as nausea, vomiting, a feeling of fullness, and early satiety. Complications of simple hepatic cysts include infection, spontaneous hemorrhage, rupture, and external compression of biliary tree or major vessels. Asymptomatic simple hepatic cysts do not require treatment. Treatment for symptomatic simple hepatic cysts includes percutaneous aspiration, aspiration followed by sclerotherapy, and surgery. The American College of Gastroenterology clinical guidelines recommend laparoscopic fenestration because of its high success rate and low invasiveness. Percutaneous procedures for treatment of simple hepatic cysts are particularly effective for immediate palliation of patient symptoms; however, they are not generally recommended because of the high rate of recurrence. Management of simple hepatic cysts requires correct differentiation from neoplasms and infections, and selection of a reliable treatment.

Medical Treatment for Hepatocellular Carcinoma in Japan.

Liver cancer, including hepatocellular carcinoma (HCC), is the fifth most common cause of cancer deaths in Japan. The main treatment options for HCC are surgical resection, liver transplantation, radiofrequency ablation (RFA), transarterial chemoembolization (TACE), and systemic chemotherapy. Here, recent medical treatments for HCC, including surgery, percutaneous ablation, transcatheter arterial chemoembolization/transcatheter arterial embolization, and drug therapy, are reviewed with a focus on Japan.

[A case of advanced esophageal carcinoma with nephrotic syndrome completely responding to chemotherapy of docetaxel, nedaplatin and 5-fluorouracil].

A 78-year-old male was admitted to our hospital because of dysphagia. He had been diagnosed as nephritic syndrome at 30 years of age and had been treated with prednisolone 10 mg/day. Blood examination revealed renal dysfunction; BUN 25 mg/dL, Cr 1. 9 mg/dL, and glomerular filtration rate(GFR)47. 4 mL/min. Endoscopy showed a type 2 tumor at the middle thoracic esophagus, and the biopsy specimen revealed moderately differentiated squamous cell carcinoma pathologically. Computed tomography (CT) of the chest and abdomen showed no metastases at distant regions and lymph nodes. Clinical staging was Stage II (cT2cN0cM0). Because of old age and renal function, we chose chemotherapy using docetaxel, nedaplatin and 5-fluorouracil. The adverse event was grade 2 in leucopenia and grade 1 in inappetence, but the renal function did not progress. Repeated endoscopic examinations after chemotherapy revealed that the esophageal cancer was significantly reduced in size, and no cancer cells were pathologically detected by endoscopic biopsy, resulting in a complete response(CR). This chemotherapy of docetaxel, nedaplatin and 5-fluorouracil might be effective and tolerable for patients with renal dys- function due to nephritic syndrome.

Occurrence of specific humoral non-responsiveness to swine antigens following administration of GalT-KO bone marrow to baboons.

BACKGROUND: Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig-to-primate model. We have now utilized swine with knockout of the gene coding for alpha-1,3-galactosyltransferase (GalT-KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells. METHODS: Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti-thymocyte globulin (rATG) and rat anti-primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 x 10(9) cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony-forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation. RESULTS: None of the animals showed detectable pig cells by flow cytometry for more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT-KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre-transplantation titers of anti-non-Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers. CONCLUSIONS: Engraftment and specific non-responsiveness to pig cells has been achieved in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed anti-non-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier.

Huge caudate lobe of the liver due to Budd-Chiari syndrome.

Budd-Chiari syndrome is characterized by hepatic venous outflow obstruction. We describe a patient with a huge caudate lobe of the liver due to Budd-Chiari syndrome. A 49-year-old woman was referred to Nippon Medical School Hospital to receive treatment for enlarged gastric varices. She had been followed up for idiopathic portal hypertension with deformity of the liver for 7 years and had undergone surgery for pharyngeal carcinoma 5 years earlier. Upper gastrointestinal endoscopy revealed solitary gastric varices without esophageal varices. Abdominal computed tomography revealed obstructions and scars of the right, middle, and left hepatic veins. The caudate lobe was enlarged, and the portal vein was displaced ventrally, without thrombus. The drainage vein of the caudate lobe, the short hepatic vein on the left side of the inferior vena cava, was dilated. The portal vein and drainage vein of segment 6 were visualized and showed no atrophy. Venography revealed no obstruction of the inferior vena cava. Budd-Chiari syndrome with solitary gastric varices was diagnosed on the basis of these findings. We performed balloon-occluded retrograde transvenous obliteration and partial splenic embolization to treat the gastric varices. The posttreatment course was uneventful, and the patient was discharged 8 days after embolization. The gastric varices shrank.

[A successful resected case of far-advanced cancer at the esophagogastric junction by chemoradiotherapy with docetaxel, nedaplatin and 5-fluorouracil].

The patient, a 73-year-old male, was admitted to our hospital because of dysphagia. A far-advanced cancer was diagnosed at the esophagogastric junction by upper gastrointestinal endoscopic examination. Pathological biopsy examinations revealed poorly-differentiated adenocarcinoma. Computed tomography (CT) of the chest and abdomen showed invasion to the diaphragm. Clinical Stage was IV in an unresectable far-advanced tumor. He received radiation therapy (40 Gy/total, 2 Gy/day×20 times) in combination with chemotherapy using docetaxel (40 mg/m², day 1), nedaplatin (10mg/body, days 1-5) and 5-fluorouracil (500 mg/body, days 1-5). After this combination chemoradiation therapy (CRT), macroscopic examinations showed significant reductions in the size of tumor, leading a partial response according to the RECIST guidelines. He underwent total gastrectomy, partial resection of the lower esophagus via left thoracotomy, and Roux-en Y reconstruction with jejunostomy. Pathological examination of the resected specimens revealed Stage IV (T3N2P1CY0). The postoperative course was uneventful. He was treated on an outpatient basis without adjuvant therapy, and died 6 months after the operation by liver, spleen and lymph node metastases.

Simultaneous evaluation of portal hemodynamics and liver function by scintiphotosplenoportography in pediatric recipients of living-donor liver transplants.

BACKGROUND/AIMS: The aim of this study was to simultaneously evaluate portal hemodynamics and liver function by scintiphotosplenoportography (SSP) using technetium-99m-diethylenetriaminepentaacetic asid-galactosyl-human serum albumin (99mTc-GSA) in recipients of living-donor liver transplants (LDLT). METHODOLOGY: Three LDLT recipients with biliary atresia who underwent SSP using 99mTc-GSA were studied. The clearance index of 99mTc-GSA (HH15) was calculated by dividing the radioactivity of the heart region of interest (ROI) 15 minutes after injection (H15) by that of the heart ROI 3 minutes after injection (H3), (HH15=H15/H3). The hepatic uptake ratio of 99mTc-GSA (LHL15) was calculated by dividing the radioactivity of the liver ROI at 15 minutes (L15) by H15 plus L15, (LHL15=L15/(H15+L15)). H/L15 (HH15/LHL15). RESULTS: Patient 1: On SSP before transplantation, digital images showed hepatofugal flow, but not the portal vein in early phase (HH15: 0.647, LHL15: 0.861, H/L15: 0.751). On SSP after transplantation, digital images showed the portal vein clearly, and the hepatofugal flow had disappeared (HH15: 0.633, LHL15: 0.971, H/L15: 0.652). Patient 2: On SSP before transplantation, digital images showed hepatofugal flow, and the liver was slightly visible in early phase (HH15: 0.673, LHL15: 0.869, H/L15: 0.774). Patient 3: On SSP before transplantation, digital images showed splenorenal shunt, but not the liver in early phase (HH15: 0.697, LHL15: 0.838, H/L15: 0.832). CONCLUSIONS: SSP using 99mTc-GSA is useful for the simultaneous evaluation of portal hemodynamics and liver function in pediatric recipients of LDLT.

Successful surgical treatment for intrahepatic arterioportal fistula with severe portal hypertension: a case report.

BACKGROUND: Intrahepatic arterioportal fistula (IAPF) is a rare cause of portal hypertension. Interventional radiology (IVR) is generally selected as the first-line therapeutic option. Surgical treatment for IAPF is required in refractory cases of IVR. As the treatment success rate with IVR is high, cases requiring surgical treatment are extremely rare. CASE PRESENTATION: A 54-year-old man was admitted to another hospital complaining of hematemesis due to rupture of the esophageal varices. A computed tomography revealed ascites and arterioportal fistula in the left lobe of the liver. Transcatheter arterial embolization (TAE) was performed to occlude the fistula; however, it could not reach complete occlusion. Thereafter, there were a total of four hematemeses, and six endoscopic variceal ligations were required. The second TAE also failed to reach complete occlusion. He was transferred to our hospital for further treatment. Because liver function was low due to frequent hematemeses and there was also uncontrollable ascites, it was confirmed that hepatectomy could not be performed safely at this time. Therefore, we ligated the left portal branch and ligated and dissected the left gastric vein to decrease portal vein pressure. However, on the 5th day after surgery, the esophageal varices reruptured. As the disappearance of ascites was observed in the postoperative course and the general condition also improved, left hepatectomy was performed to remove IAPF. There was no recurrence of portal hypertension for 1 year and 3 months since hepatectomy. CONCLUSIONS: This case was difficult to treat with IVR and required surgical treatment. Our experience in the present case suggests that hepatectomy to remove arterioportal fistula was considered effective for improving portal hypertension due to IAPF. However, careful treatment selection according to the patient's overall condition and clinical course is necessary for IAPF presenting with severe portal hypertension.

Current Status of Laparoscopic Hepatectomy.

Before the first laparoscopic hepatectomy (LH) was described in 1991, open hepatectomy (OH) was the only choice for surgical treatment of liver tumors. LH indications were initially based solely on tumor location, size, and type. Use of LH has spread rapidly worldwide because it reduces incision size. This review systematically assesses the current status of LH. As compared with OH, LH is significantly less complicated, requires shorter hospital stays, and results in less blood loss. The long-term survival rates of LH and OH are comparable. Development of new techniques and instruments will improve the conversion rate and reduce complications. Furthermore, development of surgical navigation will improve LH safety and efficacy. Laparoscopic major hepatectomy for HCC remains a challenging procedure and should only be performed by experienced surgeons. In the near future, a training system for young surgeons will become mandatory for standardization of LH, and LH will likely become better standardized and have broader applications.

Rare Case of Synchronous Cystic Duct Metastasis from Renal Cell Carcinoma.

INTRODUCTION: The common metastatic sites of renal cell cancer (RCC) are the lung, bone, liver, brain, adrenal glands, and contralateral kidney. Metastasis to the gallbladder is rare, and cystic duct metastasis from RCC has been reported in only one metachronous case. This is the first report of a case of synchronous cystic duct metastasis from RCC. CASE REPORT: A 72-year-old woman presenting with hematuria had a history of Cushing disease approximately 10 years previously. Enhanced computed tomography of the abdomen showed a mass measuring 5.8 × 3.0 cm in the left kidney, which was strongly enhanced in the early phase and washed out in the late phase. A mass measuring 2 cm in diameter was seen in the left adrenal gland, and a 1.0-cm mass was noted in the right adrenal gland. Multiple tiny masses were detected in the cystic duct. Left renal cell carcinoma, cystic duct metastasis, and bilateral adrenal gland metastases were diagnosed. Because the metastatic tumor was close to the common bile duct, we performed left nephrectomy, bilateral adrenalectomy, cholecystectomy, resection of the extrahepatic bile duct, and hepaticojejunostomy. Pathological findings showed that the renal tumor was clear cell carcinoma, as were the bilateral adrenal tumors and cystic duct tumor. The patient died 30 months after the operation.

Conversion Surgery for Metastatic Pancreatic Mucinous Carcinoma Responsive to Systemic Chemotherapy with Modified FOLFIRINOX: A Case Report.

We report a case of metastatic pancreatic-head mucinous carcinoma (with multiple lymph node and bone metastases) and review the relevant literature. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) was useful for diagnosis, and a satisfactory outcome was achieved after systemic chemotherapy with FOLFIRINOX followed by resection of the primary lesion as conversion surgery. The patient was a 55-year-old man. Hematological findings included elevated serum tumor marker levels: CEA 12.7 ng/mL, DUPAN-2 400 U/mL. Findings from several imaging modalities and EUS-FNA confirmed a clinicopathological diagnosis of metastatic pancreatic mucinous carcinoma with multiple bone and lymph node metastases. Five courses of modified FOIFIRINOX (m-FFX) were given as systemic chemotherapy, which had an antitumor effect. Subtotal stomach-preserving pancreaticoduodenectomy and extensive lymph-node dissection were thus performed. Histopathological analysis showed invasive ductal carcinoma, muc (pT3, pN1b, cM1). After surgery, the clinical course was notable for the absence of complications. Tegafur/gimeracil/oteracil (S-1) was started as maintenance adjuvant chemotherapy postoperatively, and no disease progression has been observed at 10 months after surgery.

Role of persistence of antigen and indirect recognition in the maintenance of tolerance to renal allografts.

BACKGROUND: We have previously shown that a 12-day treatment with cyclosporine A (CyA) facilitates induction of tolerance to class-I disparate kidneys, as demonstrated by acceptance of second, donor-matched kidneys without immunosuppression. In the present study, we have examined 1) the duration of tolerance in the absence of donor antigen and 2) the pathway of antigen recognition determining maintenance or loss of tolerance. METHODS: Seventeen miniature swine received class-I mismatched kidneys with 12 days of CyA, and received second donor-matched kidneys without immunosuppression at 0, 1, 3, or 4 months after nephrectomy of the primary graft. Five were sensitized 6 weeks after nephrectomy of the primary graft, three with donor-matched skin grafts, and two with donor class-I peptides to eliminate direct pathway involvement. In addition, two long-term tolerant animals received class-I peptides. RESULTS: Rejection of second grafts required at least a 3 month absence of donor antigen. Although donor-matched skin grafts in animals tolerant to kidneys induced antidonor cytotoxic T lymphocyte responses, second renal transplants revealed no evidence of sensitization. In contrast, immunization of recipients with donor class-I peptides after nephrectomy of the primary graft led to loss of tolerance at both T-cell and B-cell levels, as evidenced by rejection of the second graft in 5 days and development of antidonor immunoglobulin G. Peptide immunization of long-term tolerant in recipients bearing long-term renal grafts did not break tolerance. CONCLUSIONS: These data indicate that the renal allograft is required for the indefinite maintenance of tolerance, that indirect antigen presentation is capable of breaking tolerance, and that in tolerant animals, direct antigen presentation may suppress rejection, allowing tolerance to persist.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays.

UNLABELLED: Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with (3)H for MLR and (51)Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. OBJECTIVE: The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.