Patrick P. McHugh

An in vitro study of liposomal curcumin: Stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells

Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.

Predictors of relapse to alcohol and illicit drugs after liver transplantation for alcoholic liver disease.

Background. Alcoholic liver disease (ALD) is a common indication for transplantation worldwide. This study identifies factors predicting posttransplant recidivism. Methods. Clinical and laboratory data were reviewed. Uni- and multivariate analyses for survival and relapse to alcohol and illicit drugs were performed. Result. Between July 1995 and November 2007, 387 patients underwent liver transplantation at our institution. Of these, 147 patients (38%) were found to have ALD. Five patients (3.4%) were excluded because of perioperative mortality. Overall survival was 96.2%, 89.6%, and 84.4% at 1, 3, and 5 years, respectively, with a median follow-up of 41.2 months. Twenty-seven patients (19%) returned to alcohol after transplantation. By univariate analysis, depression was the only significant factor affecting survival (P=0.01), whereas posttransplant relapse to alcohol trended toward significance (P=0.059). Multivariate analysis showed both factors to be independently associated with poor survival (P=0.008 and 0.017, respectively). Factors associated with relapse included less than 12 months of abstinence before transplant (P=0.019) and participation in rehabilitation (P=0.026). Multivariate analysis showed pretransplant abstinence less than 12 months as the only independent factor (P=0.037) associated with alcohol relapse after transplantation. Twenty-five patients (17.2%) had documented drug use after transplantation. Drug abuse before transplantation was the only independent predictor of drug abuse after transplantation (P=0.017). Conclusions. Excellent results can be obtained in patients undergoing liver transplantation for ALD, though depression and recidivism adversely impact survival. In our series, abstinence less than 12 months was associated with relapse to alcohol. Similarly, those with prior drug abuse are more likely to continue drug use after transplantation.

Obesity, diabetes, and smoking are important determinants of resource utilization in liver resection: a multicenter analysis of 1029 patients.

Objective:To investigate independent contributions of obesity, diabetes, and smoking to resource utilization in patients following liver resection. Summary Background Data:Despite being highly resource-intensive, liver resections are performed with increasing frequency. This study evaluates how potentially modifiable factors affect measures of resource utilization after hepatectomy. Methods:The American College of Surgeons’ National Surgical Quality Improvement Program (ACS NSQIP) public-use database was queried for patients undergoing liver resection. Resource variables were operative time (OT), intraoperative transfusion, length of stay (LOS), ventilator support at 48 hours, and reoperation. Bivariable and multivariable linear and logistic regressions were performed. Results:There were 1029 patients identified. Most resections involved less than a hemiliver (599 patients, 58.2%). Mean BMI was 28.0 ± 6.0. Mean OT was 253 ± 122 minutes (range, 27 to 794) but varied by procedure (P < 0.001). Mean LOS was 8.7 ± 10.7 days (range, 0 to 202). Morbid obesity added 48 minutes to OT (P = 0.018), 1.1 units to transfusions (P = 0.049), 2.2 days to LOS (P < 0.001), and accounted for delayed ventilator weaning (odds ratio, 4.5; P = 0.022). Underweight patients had shorter OT, but stayed 3.3 days longer than normal weight patients (P < 0.001). Insulin-treated patients with diabetes had longer OT (P < 0.001), increased transfusions (P < 0.001), and delayed ventilator weaning (odds ratio, 6.7; P < 0.001), while orally-treated patients with diabetes showed opposite trends. Smokers stayed 1.9 days longer (P < 0.001), with increased risk of prolonged ventilation (odds ratio, 3.3; P = 0.002) and reoperation (odds ratio, 2.3; P = 0.015). Conclusion:Obesity, diabetes, and smoking are each associated with important components of healthcare expenditure. Education and prevention programs are needed to limit their impact on overall resource utilization.

Alpha-fetoprotein and tumour size are associated with microvascular invasion in explanted livers of patients undergoing transplantation with hepatocellular carcinoma

BACKGROUND To determine factors associated with outcomes and microvascular invasion (MVI) in patients undergoing liver transplantation (LT) for hepatocellular carcinoma (HCC). METHODS Between July 1996 and August 2008 at the Universities of Kentucky or Tennessee, LT recipients were retrospectively analysed. RESULTS One hundred and one patients had HCC in the explanted liver; one patient was excluded because of fibrolamellar histology. Seventy-nine (79%) were male and 81 (81%) were older than 50. HCC was incidental in 32 patients (32%). Median follow-up was 31 months. Ten patients (10%) developed recurrence, which was associated with poor survival (P= 0.006). Overall 1-, 3-, and 5-year survival rates were 87%, 69% and 62%, respectively. Excluding patients with lymph node metastasis (LNM) or MVI yielded 91%, 81% and 75% survival at the same time points. MVI was independently associated with recurrence (OR 28.40, 95% CI 1.77-456.48, P= 0.018) and decreased survival (OR 4.70, 95% CI 1.24-17.80, P= 0.023), and LNM with decreased survival (OR 6.05, 95% CI 1.23-29.71, P= 0.027). Tumour size (OR 4.1, 95% CI 1.2-13.5, P= 0.013) and alpha-fetoprotein (AFP) > 100 (OR 5.0, 95% CI 1.4-18.1, P= 0.006) were associated with MVI. CONCLUSIONS MVI greatly increases the risk of recurrence and death after LT for HCC, and is strongly associated with tumour size and AFP > 100.

Prevalent immunosuppressive strategies in liver transplantation for hepatitis C: results of a multi‐center international survey

To determine current immunosuppression regimens and strategies for acute cellular rejection in hepatitis C virus (HCV) patients after liver transplantation (LT), questionnaires were sent to 264 LT programs worldwide. Surveys from 81 programs were reviewed. In 27 centers (33.8%) the immunosuppression protocol used in HCV differed from non‐HCV patients. Tacrolimus‐based immunosuppression is utilized in 70 centers (86.42%). Triple therapy using tacrolimus, mycophenolate mofetil and steroids is the most common regimen (41%). Six programs (7.4%) use steroid‐free protocols. In nine centers (11%) steroids are discontinued within a week, 56% within 3 months, and 98% within the first year. At 75% of centers, mild rejection is treated by increasing baseline immunosuppression. Moderate rejection is treated by increasing baseline immunosuppression in 38% of centers, steroid bolus in 44%, and either in 16%. For severe rejection, 46% of centers give bolus steroid, and 16% administer antibodies. Among respondents, non‐US programs use significantly more cyclosporine than US programs (35.6% vs. 2.8%, P < 0.001). Duration of steroid therapy is significantly shorter in US programs than non‐US (10.8 vs. 29.4 weeks, P < 0.001). There is no consensus regarding the best immunosuppressive regimen and rejection treatments in HCV patients after LT. Our results reveal the most prevalent management practices in this difficult group of patients.

Cyclosporine Promotes Epstein-Barr Virus-Infected Human B-Cell Transformation Assayed by Three Correlated Assay Methods

We have reported that cyclosporine (CsA) has direct effect to promote Epstein-Barr virus (EBV) transformation of human peripheral blood B lymphocytes. In this article, we have reported that CsA promoted EBV-infected, human B-cell transformation as assayed by three methods of colony number counting, cell number counting, and (3)H-thymidine incorporation. At first, we sought to correlate the three methods in EBV-infected human B-cell transformation, observing that they are convenient correlate with each other, and only vary in the degree when transformed cells are compared to the controls. Based on these pilot experiments, the three assay methods were then applied to CsA-treated and nontreated, EBV-infected human B cells to investigate whether CsA treatment promoted EBV-infected human B-cell transformation. We observed that CsA treatment increased colony formation above the control value of 28 +/- 4.5/well to 49 +/- 4.3 (colonies/well; n = 5; P < .05). CsA treatment increased the cell number from the control of 33,025 +/- 1900 to 50,925 +/- 4194 (cells/well; n = 5; P < .05). CsA treatment increased (3)H-thymidine incorporation from the control result of 12,481 +/- 1341 to 26,514 +/- 5464 (CPM/well; n = 5; P < .05). In conclusion, CsA promoted EBV-B-cell transformation in three correlated assay methods in vitro using a model of posttransplant lymphoproliferative disorder.

Vaginal varices with massive hemorrhage in a patient with nonalcoholic steatohepatitis and portal hypertension: Successful treatment with liver transplantation.

We present a case of massive bleeding secondary to vaginal varices complicating portal hypertension in a patient awaiting orthotopic liver transplantation (OLT). The vagina is among the rarest of locations reported for portal hypertensive varices causing hemorrhage; in fact, our patient represents only the seventh such case since the first report by Kreek in 1967 and the only case in which the initial and definitive management was OLT. A 58-year-old white female, gravida 3, para 3, was referred to our transplant center for liver transplant evaluation for nonalcoholic steatohepatitis. Her past medical history included diabetes mellitus type 2 and nephrolithiasis, and her surgical history was notable for a total abdominal hysterectomy performed 17 years earlier for endometriosis as well as open cholecystectomy, bilateral tubal ligation, and renal lithotripsy. On physical examination, she had mild ascites but was otherwise unremarkable. Upon completion of the transplant evaluation, she was activated on the transplant waiting list. Approximately 1 year after she was listed, the patient underwent routine computed tomography imaging, which showed a small, cirrhotic liver without ascites, and patent vasculature. Three months later, she presented with a sudden onset of significant vaginal bleeding. Vaginal varices were identified by direct visualization, and after a 3-unit transfusion of packed red blood cells, she underwent emergent suture ligation of the varices with cessation of bleeding. However, bleeding recurred after an asymptomatic interval of 2 weeks. Shortly after her return to the hospital, the patient became hypotensive and was discovered to have a hematocrit of 22%. She was aggressively resuscitated and taken emergently to the operating room for examination under anesthesia. Actively bleeding varices were again seen at the upper aspect of the vaginal cuff; these were suture-ligated and packed, with cessation of bleeding. In all, she received 4 units of packed red blood cells, 1 unit of fresh frozen plasma, and a six-pack of platelets during this admission, with return of hemodynamic stability and no additional hemorrhage. During this time, placement of a transjugular intrahepatic portosystemic shunt (TIPS) was considered, but in light of the patient’s recent stability, this was not performed, and she was discharged to the care of her primary physician. Ten days later, the patient again developed hemodynamically significant vaginal hemorrhage. At this time, she was evaluated with reconsideration of TIPS placement. Computed tomography imaging showed a patent splenic vein but also the new finding of portal vein thrombosis, which was then confirmed by carbon dioxide angiogram; TIPS placement was therefore precluded. Pelvic varices were appreciated adjacent to the bladder and vagina (Fig. 1). Of note,

Surgical treatment of a rare primary renal carcinoid tumor with liver metastasis

BackgroundCarcinoid tumors are characteristically low grade malignant neoplasms with neuroendocrine differentiation that arise in various body sites, most commonly the lung and gastrointestinal tract, but less frequently the kidneys, breasts, ovaries, testes, prostate and other locations. We report a case of a carcinoid of renal origin with synchronous single liver metastases on radiological studies.Case presentationA 45 year-old patient who presented with abdominal pain was found on CT scan to have lesions in the right ovary, right kidney, and left hepatic lobe. CA-125, CEA, and CA 19-9 were within normal limits, as were preoperative liver function tests and renal function. Biopsy of the liver mass demonstrated metastatic neuroendocrine tumor. At laparotomy, the patient underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy, radical right nephrectomy with lymphadenectomy, and left hepatectomy. Pathology evaluation reported a right ovarian borderline serous tumor, well-differentiated neuroendocrine carcinoma of the kidney (carcinoid) with 2 positive retroperitoneal lymph nodes, and a single liver metastasis. Immunohistochemistry revealed that this lesion was positive for synaptophysin and CD56, but negative for chromogranin as well as CD10, CD7, and CD20, consistent with a well-differentiated neuroendocrine tumor. She is doing well one year after her initial surgery, with no evidence of tumor recurrence.ConclusionEarly surgical intervention, together with careful surveillance and follow-up, can achieve successful long-term outcomes in patients with this rare malignancy.

Cyclosporin A-Induced Lipid and Protein Oxidation in Human B-Cells and in Epstein-Barr Virus-Infected B-Cells is Prevented by Antioxidants

The incidence of post-transplant lymphoproliferative disorder (PTLD) has increased since cyclosporin A (CsA) became the mainstay of transplant immunosuppression. We have previously shown that, in addition to its potent immunosuppressive property, CsA-induced oxidative stress plays an important role in Epstein-Barr virus (EBV)-related PTLD. Using lipid hydroperoxide and malondialdehyde as markers of lipid oxidation, and protein carbonyls as markers of protein oxidation, we further investigated the in vitro effect of CsA on human B cells and EBV-infected human B cells. We found that CsA at 500 ng/ml, a relatively safe and effective blood concentration in organ transplant recipients, induced the highest lipid hydroperoxide and malondialdehyde after 10 min of treatment in time- and concentration-related kinetic studies. We also found that treatment with CsA at 500 ng/ml for 10 min increased the EBV-infected B cell protein carbonyl formation as assayed by immunoblot method. CsA-induced lipid and protein oxidation could be inhibited by vitamin E, N-acetyl cysteine, and pyrollidine dithiocarbamate. CsA significantly promoted the EBV-B cell transformation as assayed by colony counting, cell counting, and 3H-thymidine incorporation. Our recent study provides further evidence to support the hypothesis that CsA exerts direct oxidative stress in EBV-infected as well as non-EBV-infected human B cells. A greater understanding of these cellular and molecular mechanisms may benefit the clinical practice and prevention of PTLD.

Cyclosporin A Up-Regulates and Activates Protein Kinase C-ζ in EBV-Infected and EBV-Transformed Human B-Cells

BACKGROUND Protein Kinase C (PKC) is a family of enzymes that plays a key role in cell signaling pathways leading to cellular activation and proliferation. Conventional PKC (cPKC) is dependent on calcium for activation. We have proposed that cyclosporin A (CsA), despite being a calcineurin inhibitor, will activate PKC in B cells, thus promoting Epstein-Barr virus (EBV)-induced transformation. Here we show that CsA promoted atypical PKC isoform PKC-zeta in B cells. MATERIALS AND METHODS Western-blot was used to assay PKC-zeta protein level in EBV-B cells. Confocal microscopy was used to assay PKC-zeta translocation from cytosol to cell membrane, a known process of PKC activation. RESULTS CsA (500 ng/mL) time dependently increased PKC-zeta from control of 7055 units to 7145, 10,805, 10,914, and 12,705 units, respectively, after 15 min, 1 h, 12 h, and 24 h of incubation in EBV-transformed human B-cell line (LCL). CsA increased PKC-zeta expression was inhibited 50% by Vit.E (40 microM) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H(2)O(2) (0.1 mM) treatment, PKC-zeta protein level in LCL cells increased 124%, 257%, 349%, and 359% after 15 min, 1 h, 12 h, and 24 h of culture compared with control. Addition of Vit.E (40 microM) in H(2)O(2) (0.1 mM) treatment and then with Vit.E in the culture decreased PKC-zeta level in LCL cells 26%, 20%, 41%, and 60% after 15 min, 1 h, 12 h, and 24 h of culture. In confocal microscopy in Jurkat T cell line, phorbol 12-myristate 13-acetate (PMA) activated cPKC isoform PKCalpha after 30 min treatment and activated PKC-zeta after 60 min treatment. CsA inhibited PMA activation of PKC-alpha, but not PKC-zeta. CsA alone did not activate PKC-alpha or PKC-zeta in Jurkat T cells. In LCL and in EBV-infected human B-cells, PMA stimulated PKC-alpha activation after 30 min treatment and stimulated PKC-zeta activation after 60 min treatment. CsA inhibited PMA activation of PKC-alpha, but not PKC-zeta. In addition, CsA activated PKC-zeta in the EBV-transformed and EBV-infected human B cells. CONCLUSION These experiments show that CsA-induced oxidative stress caused PKC-zeta up-regulation in LCL cells, and show the differential effect of CsA in the PKC signaling pathways in T cells versus B cells. CsA-induced PKC-zeta activation may be an important signaling step in EBV-induced post-transplant lymphoproliferative disorders.