Cytopathology and ultrastructure identification of primary hepatic acinar cell carcinoma: Case report.

INTRODUCTION: A primary acinar cell carcinoma (ACC) of the liver was incidentally diagnosed in a clinically asymptomatic 80-year-old man. This study aimed to delineate critical diagnostic characteristics of an ACC originating uniquely from the liver to improve its future identification. PRESENTATION OF CASE: Enhanced MRI revealed a heterogenous, cystic 7.7 × 11.1 × 10.4 cm tumour occupying hepatic segments II and III. The mass demonstrated mild diffuse enhancement in hepatic arterial phase with minimal portal venous washout in a liver without cirrhotic features. A central stellate T2-hyperintense necrotic scar and outer capsule were apparent. No primary lesion or metastasis outside the liver was discernable. Post-left hepatic lobectomy, the tumour immunophenotype was atypical for presumptive diagnoses of hepatocellular carcinoma (HCC) or cholangiocarcinoma. Extensive morphologic workup on electron microscopy definitively diagnosed primary hepatic ACC by establishing presence of secretory zymogen-like granules, intracytoplasmic microvilli and acinar cell differentiation. Cytopathology revealed cellular lumen expressing PAS-positive diastase-resistant granular cytoplasmic contents. DISCUSSION: This case showcased the novel utility of electron microscopy that was crucial in yielding the definitive diagnosis. The previous literature on hepatic ACC was compiled here in context of the present case. The mechanism of hepatic acinar cell localization was also discussed. CONCLUSION: Primary hepatic ACC may easily be confused for other lesions due to nonspecific imaging patterns. Specifically, the presence of a central scar without risk factors for HCC can favour a diagnosis of benign entities such as focal nodular hyperplasia (FNH). Electron microscopy presents an important tool to identify primary hepatic ACC and may improve future patient outcomes.

Portal vein thrombosis is a potentially preventable complication in clinical islet transplantation.

Percutaneous transhepatic portal access avoids surgery but is rarely associated with bleeding or portal venous thrombosis (PVT). We herein report our large, single-center experience of percutaneous islet implantation and evaluate risk factors of PVT and graft function. Prospective data were collected on 268 intraportal islet transplants (122 subjects). A portal venous Doppler ultrasound was obtained on Days 1 and 7 posttransplant. Therapeutic heparinization, complete ablation of the portal catheter tract with Avitene paste and limiting packed cell volume (PCV) to <5 mL completely prevented any portal thrombosis in the most recent 101 islet transplant procedures over the past 5 years. In the previous cumulative experience, partial thrombosis did not affect islet function. Standard liver volume correlated negatively (r =-0.257, p < 0.001) and PCV correlated positively with portal pressure rise (r = 0.463, p < 0.001). Overall, partial portal thrombosis occurred after 10 procedures (overall incidence 3.7%, most recent 101 patient incidence 0%). There were no cases of complete thrombosis and no patient developed sequelae of portal hypertension. In conclusion, portal thrombosis is a preventable complication in clinical islet transplantation, provided therapeutic anticoagulation is maintained and PCV is limited to <5 mL.

Transplant of Primary Human Hepatocytes Cocultured With Bone Marrow Stromal Cells to SCID Alb-uPA Mice.

Hepatocytes are vulnerable to loss of function and viability in culture. Modified culture methods have been applied to maintain their functional status. Heterotypic interactions between hepatocytes and nonparenchymal neighbors in liver milieu are thought to modulate cell differentiation. Cocultivation of hepatocyte with various cell types has been applied to mimic the hepatic environment. Bone marrow stromal cells (BMSC) are plastic cell lines capable of transforming to other cell types. In this study hepatocyte coculture with BMSCs achieved long-term function of human hepatocytes in culture for 4 weeks. In vitro functional status of human hepatocytes in BMSC coculture was compared with fibroblast coculture and collagen culture by measuring albumin, human-α-1-antitrypsin (hAAT), urea secretion, CYP450 activity, and staining for intracellular albumin and glycogen. After 2 weeks in culture hepatocytes were retrieved and transplanted to severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID Alb-uPA) mice and engraft-ment capacity was analyzed by human hepatic-specific function measured by hAAT levels in mouse serum, and Alu staining of mouse liver for human hepatocytes. Hepatocytes from BMSC coculture had significantly higher albumin, hAAT secretion, urea production, and cytochrome P450 (CYP450) activity than other culture groups. Staining confirmed the higher functional status in BMSC coculture. Transplantation of hepatocytes detached from BMSC cocultures showed significantly higher engraftment function than hepatocytes from other culture groups measured by hAAT levels in mouse serum. In conclusion, BMSC coculture has excellent potential for hepatocyte function preservation in vitro and in vivo after transplant. It is possible to use BMSC hepatocyte coculture as a supply of cell therapy in liver disease.

High risk of sensitization after failed islet transplantation.

Human Leukocyte Antigen (HLA) antibodies posttransplant have been associated with an increased risk of early graft failure in kidney transplants. Whether this also applies to islet transplantation is not clear. To achieve insulin independence after islet transplants multiple donor infusions may be required. Hence, islet transplant recipients are at risk of sensitization after transplantation. Islet transplant recipients were screened for HLA antibodies posttransplant by flow-based methods. A total of 98 patients were studied. Twenty-nine patients (31%) developed de novo donor specific antibodies (DSA) posttransplant. Twenty-three patients developed DSA while on immunosuppression (IS). Among recipients who have discontinued IS, 10/14 (71%) are broadly sensitized with panel reactive antibody (PRA) >or=50%. The risk of becoming broadly sensitized after transplant was 11/69 (16%) if the recipient was unsensitized prior to transplant. The majority of these antibodies have persisted over time. Appearance of HLA antibodies posttransplant is concerning, and the incidence rises abruptly in subjects weaned completely from IS. This may negatively impact the ability of these individuals to undergo further islet, pancreas or kidney transplantation and should be discussed upfront during evaluation of candidates for islet transplantation.

Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution.

Islet transplantation is being offered increasingly for selected patients with unstable type 1 diabetes. Percutaneous transhepatic portal access avoids a need for surgery, but is associated with potential risk of bleeding. Between 1999 and 2005, we performed 132 percutaneous transhepatic islet transplants in 67 patients. We encountered bleeding in 18/132 cases (13.6%). In univariate analysis, the risk of bleeding in the absence of effective track ablation was associated with an increasing number of procedures (2nd and 3rd procedures with an odds ratio (OR) of 9.5 and 20.9, respectively), platelets count <150,000 (OR 4.4), elevated portal pressure (OR 1.1 per mm Hg rise), heparin dose > or =45 U/kg (OR 9.8) and pre-transplant aspirin (81 mg per day) (OR 2.6, p = 0.05). A multivariate analysis further confirmed the cumulative transplant procedure number (p < 0.001) and heparin dose > or =45 U/kg (p = 0.02) as independent risk factors for bleeding. Effective mechanical sealing of the intrahepatic portal catheter tract with thrombostatic coils and tissue fibrin glue completely prevented bleeding in all subsequent procedures (n = 26, p = 0.02). We conclude that bleeding after percutaneous islet implantation is an avoidable complication provided the intraparenchymal liver tract is sealed effectively.

Combination therapy with anti-ICOS and cyclosporine enhances cardiac but not islet allograft survival.

The blockade of costimulatory signals is a powerful strategy to prevent allograft rejection and facilitate transplantation tolerance. In recent years, a series of novel costimulatory molecules have been identified, including an inducible costimulatory molecule (ICOS). To date, little has been uncovered regarding the therapeutic potential of blocking ICOS signaling in the setting of transplantation. In a fully MHC-mismatched mouse model, we studied the effect of blocking ICOS signaling using a specific monoclonal antibody (anti-ICOS mAb) in combination with cyclosporine on cardiac and islet allograft survival. We demonstrated that combined treatment with anti-ICOS mAb and cyclosporine can induce long-term graft acceptance in cardiac but not islet allografts, suggesting that the type of transplanted tissue significantly influences the immunologic patterns of graft acceptance or rejection in this model.

Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Bile leak from duct of Luschka after liver transplantation.

BACKGROUND: We report a case of bile leak from an accessory duct of Luschka during cholecystectomy during liver transplantation. METHODS: Radiological findings suggested that the collection was septated. An intra-operative cholangiogram was obtained by cannulation of the accessory hepatic duct. RESULTS: An infected biloma with Clostridium perfringens was drained surgically. The bile leak that emanated from the gall bladder fossa was found to communicate with an accessory right hepatic duct draining a segmental duct in the right liver lobe. The bile leak resolved completely after direct suture of the accessory duct. CONCLUSIONS: Excessive use of electrocautery to the liver bed during donor cholecystectomy may injure subcapsular ducts in the gallbladder fossa. In liver transplantation, dissection should be kept close to the serosal lining of the gall bladder, preserving the areolar tissue in the gall bladder bed, to avoid injury to the duct of Luschka.

Novel approaches toward early diagnosis of islet allograft rejection.

BACKGROUND: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. METHODS: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred. RESULTS: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%. CONCLUSIONS: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

IFN-gamma alters the pathology of graft rejection: protection from early necrosis.

We studied the effect of host IFN-gamma on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-gamma genes) IFN-gamma genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-gamma (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-gamma to GKO hosts reduced but did not fully prevent the effects of IFN-gamma deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-gamma has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-gamma produced in large quantities in the allograft.

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Interferon-gamma acts directly on rejecting renal allografts to prevent graft necrosis.

In transplant rejection interferon (IFN)-gamma regulates the recipient immune response but also acts directly on IFN-gamma receptors in the graft. We investigated these direct actions by comparing rejecting kidneys from donors lacking IFN-gamma receptors (GRKO mice) or control donors (129Sv/J) in CBA recipients. Beginning day 5, 129Sv/J kidneys displayed high major histocompatibility complex (MHC) expression, progressive infiltration by inflammatory cells, but no thrombosis and little necrosis, even at day 21. GRKO kidneys showed increasing fibrin thrombi in small veins, peritubular capillary congestion, hyaline casts, and patchy parenchymal necrosis, progressing to near total necrosis at day 10. Terminal dUTP nick-end labeling assays were positive only in the interstitial infiltrate, confirming that massive cell death in GRKO transplants was not apoptotic. Paradoxically, GRKO kidneys showed little donor MHC induction and less inflammatory infiltration. Both GRKO and 129Sv/J allografts evoked vigorous host immune responses including alloantibody and mRNA for cytotoxic T cell genes (perforin, granzyme B, Fas ligand), and displayed similar expression of complement inhibitors (CD46, CD55, CD59). GRKO kidneys displayed less mRNA for inducible nitric oxide synthase and monokine inducible by IFN-gamma but increased heme oxygenase-1 mRNA. Thus IFN-gamma acting on IFN-gamma receptors in allografts promotes infiltration and MHC induction but prevents early thrombosis, congestion, and necrosis.

Conservation of phosphorylation state of cardiac phosphofructokinase during in vitro hypothermic hypoxia.

We investigated the metabolic effects of buffering agents alpha-amino-4-imidazole-propionic acid (Histidine), N, N-bis(2-hydroxyethyl)glycine (bicine), N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) on anaerobic energy production (via glycolysis) and conservation of key regulatory enzyme activity, and phosphofructokinase (PFK) throughout prolonged hypothermic hypoxia in porcine hearts. Hearts from 35 to 40 kg pigs were flushed with one of the following five solutions: St. Thomas' Hospital solution (STHS); modified University of Wisconsin (UW) solution; and three solutions containing modified UW plus 90 mM of histidine, bicine, or BES. The hearts were then stored at 4 degrees C for 10 h. After 10 h of hypothermic hypoxia, lactate values were 6.7-12.9 micromol/g higher than control; this reflected an increase in anaerobic end product of 35-67%. The consequences of enhanced anaerobic metabolism were higher ATP, total adenylate, Energy Charge, and ATP/ADP ratios in most of the buffered groups after 4-10 h cold storage; effectiveness of the buffers employed correlated with buffering capacity (BES proved to be the most effective). PFK remained activated throughout most of the 10-h period in hearts stored with buffers and did not undergo the rapid inactivation experienced by hearts stored in STHS. Conservation of PFK integrity with buffering agents was not related to a pH-mediated event; changes in kinetic parameters suggested that this protection was due to an irreversible posttranslational modification, specifically a dephosphorylation event.

Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis.

Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis. Can J Gastroenterol 2000;14(7):637-640. Acute graft-versus-host disease (GVHD) is a common complication after bone marrow transplantation, with characteristic rash and diarrhea being the most common features. After liver transplantation, however, this phenomenon is very rare. Most transplant patients are on a variety of medications, including immunosuppressants; therefore, the differential diagnosis of skin rash or diarrhea is broad. A 37-year-old man who underwent liver transplantation for primary biliary cirrhosis, and developed a rash and watery diarrhea, is presented. Skin and colonic biopsies confirmed acute GVHD. A pulse of intravenous steroids was given. The skin rash improved, but he developed pancytopenia. His course was complicated by central line infection, jugular and subclavian vein thrombosis, pseudomembranous colitis, recurrent bacteremia, cholestasis on total parenteral nutrition and cytomegalovirus infection. After the onset of pleuritic chest pain and clinical sepsis, spiral computed tomography scan of his chest and abdomen revealed septic infarcts in multiple organs. Despite empirical treatment with amphotericin B, he died of multiorgan dysfunction syndrome within 72 h. Autopsy revealed systemic aspergillosis with pancarditis, endocardial vegetations, and septic pulmonary, splenic, hepatic and renal infarcts. The pathogenesis and experience with this rare, but often fatal, complication of liver transplantation are reviewed. In contrast to GVHD after bone marrow transplantation, pancytopenia is common and liver dysfunction is rare. One should have a high level of suspicion in the liver transplant recipient presenting with rash and/or diarrhea.

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.

BACKGROUND: Registry data on patients with type 1 diabetes mellitus who undergo pancreatic islet transplantation indicate that only 8 percent are free of the need for insulin therapy at one year. METHODS: Seven consecutive patients with type 1 diabetes and a history of severe hypoglycemia and metabolic instability underwent islet transplantation in conjunction with a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus, and daclizumab. Islets were isolated by ductal perfusion with cold, purified collagenase, digested and purified in xenoprotein-free medium, and transplanted immediately by means of a percutaneous transhepatic portal embolization. RESULTS: All seven patients quickly attained sustained insulin independence after transplantation of a mean (+/-SD) islet mass of 11,547+/-1604 islet equivalents per kilogram of body weight (median follow-up, 11.9 months; range, 4.4 to 14.9). All recipients required islets from two donor pancreases, and one required a third transplant from two donors to achieve sustained insulin independence. The mean glycosylated hemoglobin values were normal after transplantation in all recipients. The mean amplitude of glycemic excursions (a measure of fluctuations in blood glucose concentrations) was significantly decreased after the attainment of insulin independence (from 198+/-32 mg per deciliter [11.1+/-1.8 mmol per liter] before transplantation to 119+/-37 mg per deciliter [6.7+/-2.1 mmol per liter] after the first transplantation and 51+/-30 mg per deciliter [2.8+/-1.7 mmol per liter] after the attainment of insulin independence; P<0.001). There were no further episodes of hypoglycemic coma. Complications were minor, and there were no significant increases in lipid concentrations during follow-up. CONCLUSIONS: Our observations in patients with type 1 diabetes indicate that islet transplantation can result in insulin independence with excellent metabolic control when glucocorticoid-free immunosuppression is combined with the infusion of an adequate islet mass.

Ogilvie's syndrome associated with acute cytomegaloviral infection after liver transplantation.

Ogilvie's syndrome, or acute colonic pseudo-obstruction, is a rare complication following liver transplantation. We describe two cases in which the onset of Ogilvie's syndrome is strongly temporally associated with acute cytomegaloviral (CMV) infection in immunosuppressed liver transplant recipients. The pseudo-obstruction resolved rapidly in both cases following treatment with intravenous ganciclovir. Acute CMV infection therefore appeared to be causally linked to pathogenesis of Ogilvie's syndrome in these two cases. This association has not been described previously to our knowledge, and should be considered in any transplant patient presenting with Ogilvie's syndrome.

The role of protein kinase A in anaerobic energy production during liver storage.

BACKGROUND/AIM: During cold liver storage in University of Wisconsin solution, glycolysis is inhibited by declining intracellular pH and a reduction in glycogen phosphorylase activity. The current study investigated the effects of a histidine-buffered, modified University of Wisconsin solution with cyclic-AMP analogue plus phosphodiesterase inhibitors to optimize both pH and PK A-mediated limits on glycolytic energy production. METHODS: In an isolated rodent-liver system, dioctanoyl-cAMP was supplemented with each phosphodiesterase inhibitor (isobutylmethylxanthine, papaverine, Ro 20-1724, dipyridamole). Once the most efficacious combination was determined, a separate group of livers was cold-stored for 24 h and then reperfused at 37 degrees C to examine regeneration of high energy adenylates. RESULTS: Lactate accumulation in the histidine-lactobionate-raffinose group was 8.7 micromol/g; net increases were greater with all four phosphodiesterase inhibitors with dioctanoyl-cAMP; dipyridamole resulted in a maximum increase of 16.7 micromol/g. ATP was consistently higher in all treatment groups with phosphodiesterase inhibitors throughout 24 h; even after 10-24 h, levels with dipyridamole-treatment were 250-280% higher than with University of Wisconsin (p<0.05). Assessment of glycogen phosphorylase activity in the dipyridamole-treatment group indicated that increased glycolytic activity over the first 4 h was a direct consequence of elevated enzyme levels. However, between 4-10 h, phosphofructokinase underwent a phosphorylation, leading to an inhibition at this point in glycolysis. Upon reperfusion, the higher ATP/ADP and ADP/ AMP ratios found with phosphodiesterase inhibitor treatment suggested that adenylate regeneration was superior with dipyridamole+dioctanoyl-cAMP. CONCLUSION: Dipyridamole plus dioctanoyl-cAMP treatment achieved increased glycogenolysis throughout 24 h storage by maintaining glycogen phosphorylase in a phosphorylated (active) state; however, a PK A-mediated phosphorylation (inhibition) of phosphofructokinase resulted in decreased glycolytic ATP production between 4-10 h.