Successful Treatment of Fibrosing Cholestatic Hepatitis With Daclatasvir and Asunaprevir After Liver Transplantation: A Case Report.

BACKGROUND: Fibrosing cholestatic hepatitis (FCH) is an aggressive form of hepatitis C virus (HCV) recurrence after liver transplantation (LT). Most FCH cases are fatal, occurring as a secondary disease following rapidly progressive liver dysfunction and graft failure. We report a case of early-onset FCH after LT that was successfully treated using daclatasvir and asunaprevir. CASE REPORT: A 59-year-old woman underwent living donor LT for HCV-related liver cirrhosis. However, liver function was not improved after LT and gradually worsened. A liver biopsy was performed at 30 and 47 days after the living donor LT to identify the cause of the liver dysfunction. The first biopsy result showed no specific finding. However, combined treatment with pegylated interferon and ribavirin was started because of a high HCV viral load (> 8.0 log IU/mL). Nevertheless, liver function and HCV viral load deteriorated, and the second biopsy performed on postoperative day 47 revealed FCH. We converted the antiviral agents into daclatasvir and asunaprevir and performed plasmapheresis twice. Since then, the liver dysfunction and HCV viral load gradually improved, and HCV RNA clearance occurred at week 11 after treatment. The patient achieved a sustained virologic response at week 24 after completion of the treatment. CONCLUSION: Daclatasvir combined with asunaprevir can be a useful treatment option in potentially fatal FCH after LT.

Urgent Living-Donor Liver Transplantation in a Patient With Concurrent Active Tuberculosis: A Case Report.

BACKGROUND: Although active tuberculosis (TB) is considered a contraindication for liver transplantation (LT), this is the only treatment in patients with liver failure and concurrent active TB. We report a case with successful urgent living-donor LT for irreversible liver failure in the presence of active TB. CASE PRESENTATION: A 48-year-old man, with a history of decompensated alcoholic liver cirrhosis, was presented with stupor. At admission, his consciousness had deteriorated to semi-coma, and his renal function also rapidly deteriorated to hepatorenal syndrome. A preoperative computed tomography scan of the chest revealed several small cavitary lesions in both upper lobes, and acid-fast bacillus stain from his sputum was graded 2+. Adenosine deaminase levels from ascites were elevated, suggesting TB peritonitis. A first-line anti-TB drug regimen was started immediately (rifampin, isoniazid, levofloxacin, and amikacin). An urgent living-donor LT was performed 2 days later. After LT, the regimen was changed to second-line anti-TB drugs (amikacin, levofloxacin, cycloserine, and pyridoxine). The sputum acid-fast bacillus stain tested negative on postoperative day 10. His liver function remained well preserved, even after the reversion to first-line anti-TB treatment. The patient recovered without any anti-TB medication-related complications and was discharged. CONCLUSIONS: LT can be prudently performed as a life-saving option, particularly for patients with liver failure and concurrent active TB.

Liver transplantation in Korea: past, present, and future.

PURPOSE: This study reviewed the past and present status of liver transplantation (LT) and outlooks for the future of LT in Korea. METHOD: The first LT in Korea was successfully performed using a deceased donor graft in 1988. Pediatric and adult living donor liver transplantations (LDLTs) were initiated in 1994 and 1997, respectively. From 1988 to 2013, 10,581 LTs were performed at 40 centers, whereas LDLT accounted for 76.5% of all LTs. RESULTS: In the early 1990s, the deceased organ donation rate was less than 1.5 per million population (PMP) per year, but it increased to 5 PMP beginning in 2008. Despite the increasing number of deceased donor liver transplantations (DDLTs), high prevalence of hepatitis B virus (HBV)-induced cirrhosis and hepatocellular carcinoma (HCC) has provoked persistent performance of adult LDLT with technical advancement including middle hepatic vein (MHV) reconstruction of right lobe graft and dual graft LDLT with 1 nationwide donor mortality. CONCLUSION: The number of LTs in Korea in 2010 was 23.2 PMP (1042 LTs/45 million population), lower than 23.5 PMP of Spain, but higher than 20 PMP of the United States. However, future LT numbers may decrease because of lowering the HBV carrier rate (neonatal HBV universal vaccination began in 1992), new potent anti-HBV agents, and lowest birth rate (1.22 children per family) with a decrease of potential live donors.

Long-term survival analysis of liver transplantation for hepatocellular carcinoma with bile duct tumor thrombus.

BACKGROUND: Long-term prognosis of liver transplantation (LT) for hepatocellular carcinoma (HCC) with macroscopic bile duct tumor thrombus (BDTT) has not been well assessed. This study intended to analyze the post-transplantation outcomes in patients who had HCC with macroscopic BDTT. METHODS: A retrospective study was performed with 14 patients who underwent LT for HCC with BDTT (0.7%) after selection from an institutional database of 2052 adult LT cases. RESULTS: Types of LT were living donor LT in 13 and deceased donor LT in 1. The extents of BDTT were Ueda type 1 in 4, type 2 in 3, and type 3 in 7. Milan criteria were met in 8 (57.1%). Concurrent bile duct resection was performed in 7 (50%). Mean model for end-stage liver disease score was 18.7 ± 4.9. Mean graft-recipient weight ratio was 1.2 ± 0.3. There was one case of perioperative mortality and one case of HCC-unrelated late mortality. Cumulative HCC recurrence rates were 15.4% at 1 year, 46.2% at 3 years, and 46.2% at 5 years. Overall patient survival rates were 92.9% at 1 year, 57.1% at 3 years, and 50% at 5 years. Univariate risk factor analyses revealed that only macrovascular invasion was a significant risk factor for HCC recurrence (P = .019). CONCLUSIONS: The results of this study revealed that LT for HCC with macroscopic BDTT has a high risk of post-transplantation HCC recurrence; therefore, further large-volume studies are necessary to elucidate the risk factors.

Complications analysis of polytetrafluoroethylene grafts used for middle hepatic vein reconstruction in living-donor liver transplantation.

BACKGROUND: We previously showed that ringed polytetrafluoroethylene (PTFE) grafts combined with small allograft patches showed high patency rates similar to those of iliac vein grafts and therefore that they can be used for middle hepatic vein (MHV) reconstruction. Although such use of PTFE graft showed high patency rates, its long-term safety regarding infection and other types of complications were not presented. In this study, we investigated the actual risk of complications directly associated with PTFE graft interposition for MHV reconstruction. METHODS: During the study period of 30 months, we performed 215 cases of adult living-donor liver transplantation with modified right lobe graft and PTFE grafts. We classified the potential complications directly associated with PTFE graft interposition as infectious and surgical complications. The medical records of study patients were retrospectively reviewed. RESULTS: MHV graft patency rate was 76.3% at 6 months and 36.7% at 12 months. Their 1-year graft and patient survival rates were 92.6% and 93.5%, respectively. The 1-year actual incidences of infectious complication and surgical complication were near zero and 1 case (0.5%), respectively. In 1 recipient, the PTFE graft penetrated into the stomach wall 6 months after transplantation, but the patient did not complain of any specific symptoms. The PTFE graft was removed with the use of laparotomy, and the patient recovered uneventfully. CONCLUSIONS: Although the incidence of PTFE graft-associated complication rate is very low, we suggest that it is necessary to closely monitor the PTFE graft, because unexpected complications can happen during long-term follow-up.

Role of hand-assisted laparoscopic surgery in living-donor right liver harvest.

OBJECTIVE: The cosmetic aspects of abdominal skin incisions are a matter of concern for both live liver donors and surgeons. We performed a prospective comparative study on the use of minilaparotomy to perform right liver graft harvests with and without hand-assisted laparoscopic surgery (HALS). METHODS: Young donors were indicated for surgery using minilaparotomy with or without HALS. In the non-HALS group (n = 20), a 10-12-cm-long right subcostal incision was used for right liver graft harvest. In the HALS group (n = 20), an 8-cm-sized right subcostal incision was used for hand assistance and 3 laparoscopic holes made for manipulation. The retrohepatic inferior vena cava (IVC) was initially laparoscopically dissected while using air inflation. The skin incision was extended to 10-12 cm, and then hilar dissection and hepatic transection were performed through the skin incision. RESULTS: In all 40 donors in the study cohort, safe uneventful harvesting of the right liver grafts was successfully achieved through the minilaparotomy incisions. The HALS group required an additional 30 minutes for laparoscopic preparation and dissection compared with the non-HALS group. HALS facilitated retrohepatic IVC dissection, and the remaining part of the surgery was the same as that for minimal-incision surgery. The minimal skin incision for the delivery of the liver from the abdomen was an average 10 cm for grafts <500 g and 12 cm for grafts ≥700 g. Compared with the patient profiles, there were no differences regarding donor age, body mass index, graft weight, intraoperative blood loss, postoperative increase in peak liver enzymes, total hospital stay, and incidence of postoperative complications. CONCLUSIONS: HALS facilitates the performance of donor hepatectomy with the use of a minimal incision, which probably allows for a wider selection of living donors.

Survival rates among patients awaiting deceased donor liver transplants at a single high-volume Korean center.

BACKGROUND: Since the establishment of the Korean Network for Organ Sharing (KONOS) in 2000, thousands of patients have been enrolled on the waiting list, but only a small proportion have received a deceased donor liver transplantation. This report on waiting list mortality in Korea based on data from a single institution. METHODS: The 1772 patients enrolled on the waiting list between February 2000 and December 2011 either have not yet received at the time of analysis or have died before receiving an organ. Survival information was obtained in February 2012 by reviewing medical records or by telephone. We excluded patients who died immediately after enrollment or after retransplantation. RESULTS: Primary diagnoses of those awaiting transplantation were hepatitis B virus-associated cirrhosis (63.7%), alcoholic liver disease (14.3%), hepatitis C virus-associated cirrhosis (13.8%), and acute liver failure due to other causes (8.1%). The priority status of patients on the waiting list was KONOS status 1 (highest priority) in 3.8%, status 2A in 3.9%, status 2B in 41.9%, status 3 to 7 (lowest priority) in 50.5%. Their median survival periods were 1, 1, 18, and 59 months, respectively. The mean Child-Pugh score was 8.5 ± 2.5 and Model for End-stage Liver Disease (MELD) score 18.1 ± 9.8. CONCLUSIONS: Patients with high MELD scores or hepatocellular carcinoma succumbed soon after being entered on to the waiting list. By increasing organ donation rates and developing a risk-based allocation system, it should be possible to reduce mortality among patients on organ waiting lists.

Unification venoplasty to cope with recipient portal vein anomaly during living donor liver transplantation.

PURPOSE: To cope with recipient portal vein (PV) anomalies, such as early branching of the right posterior section (RPS), during living donor liver transplantation (LDLT) surgery, we performed a simulation study to standardize the surgical technique for unification portal venoplasty. METHODS: This study included an observational analysis of conventional methods utilizing RPS PV, simulation-based design of a new surgical technique, and clinical application of this new technique. RESULTS: In a case encountering RPS PV, a mild anastomotic PV stenosis was persistent over 6 months postsurgery, indicating the need for technical refinement. After computational simulation analysis, we found that simple suturing of the PV branch patch automatically resulted in a funnel-shaped elongation. A prospective recipient study (n = 30) indicated that usual PV reconstruction via the PV bifurcation method is feasible in the absence of unusual donor or recipient PV anomaly. Retrospective living donor PV anatomy analysis (n = 20) revealed that 20-mm-long limbs of the first-order PV branches are necessary to make a 10- to l5-mm-long funneled PV stump. This technique of unification venoplasty for an anomalous recipient PV was applied to an adult patient undergoing LDLT with a right liver graft, for which it was shown to be technically feasible and effective. CONCLUSIONS: A simplified unification venoplasty technique was developed to cope with a recipient PV anomaly in adult LDLT.

Long-term survival outcomes for living donor liver transplant recipients with pathologically nonviable hepatocellular carcinoma.

PURPOSE: Complete necrosis of hepatocellular carcinoma (HCC) lesions has occasionally been found by explant pathology after pretransplant neoadjuvant treatment. This study sought to investigate the long-term prognostic effect of loss of tumor viability after HCC treatment in living donor liver transplant (LDLT) recipients. METHODS: We reviewed retrospectively the 5-year records of 37 patients who demonstrated nonviable HCC on explant pathology. RESULTS: The most common primary disease was hepatitis-B-virus-associated liver cirrhosis (n = 34). Single explant tumors were found in 29 patients; the mean maximal tumor size was 2.1 ± 0.9 cm (range: 0.8-4.0). No patients showed microvascular invasion. The median level of alpha-fetoprotein was 12 ng/mL (range: 1-1160). The 1 patient who showed a recurrence at 20 months remains alive more than 6 years after adrenalectomy and repeated pulmonary metastasectomy. The 5-year HCC recurrence rate was thus 2.1%. There were 2 late mortalities, each due to graft failure and recurrent gastric cancer. The overall patient survival rate was 97.3% at 5 and 92.7% at 10 years. CONCLUSIONS: The results of this study revealed that the loss of tumor viability induced by pretransplant neoadjuvant treatment definitely decreased the risk of post-transplant HCC recurrence. Therefore, patients with nonviable HCC can be regarded as members of a superselect group with minimal risk for HCC recurrence, and may be exempted from routine HCC screening.

Long-term outcome of liver transplantation for combined hepatocellular carcinoma and cholangiocarcinoma.

PURPOSE: Combined hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) is a rare pair of intrahepatic malignancies. Differential diagnosis among combined HCC-CCC, HCC, or CCC can be difficult; thus malignancies other than ordinary HCC are occasionally encountered unexpectedly in explanted liver specimens. The present study analyzed the long-term outcomes of liver transplantation (OLT) among patients with HCC-CCC. METHODS: Between January 1999 and December 2009, we performed 2137 adult OLT at our institution including 15 cases of pathologically confirmed HCC-CCC, who all underwent OLT with a pretransplant diagnosis of HCC. We reviewed retrospectively the medical records of these 15 patients. RESULTS: Their mean age was 58.9 ± 7.2 years. The median preoperative alpha-fetoprotein level was 32.6 ng/mL. Fourteen patients underwent living donor and one deceased donor OLT. The Milan criteria were met in 12 cases. A single tumor was identified in 8 and multiple lesions in 7 patients. The maximal tumor diameter was 2.9 ± 1.7 cm. Seven patients experienced tumor recurrences: including 6 within the first 12 months. All of the patients who experienced recurrences died at a median 4 months after that diagnosis. The overall patient survival rates were 66.7% at 1 year and 60.0% at 3 and 5 years. Disease-free patient survival rates were 60.0% at 1 year and 53.3% at 3 and 5 years. CONCLUSIONS: Patients with combined HCC-CCC showed a high rate of early recurrences, particularly within the first year.

Real-time prediction of respiratory motion based on a local dynamic model in an augmented space.

Motion-adaptive radiotherapy aims to deliver ablative radiation dose to the tumor target with minimal normal tissue exposure, by accounting for real-time target movement. In practice, prediction is usually necessary to compensate for system latency induced by measurement, communication and control. This work focuses on predicting respiratory motion, which is most dominant for thoracic and abdominal tumors. We develop and investigate the use of a local dynamic model in an augmented space, motivated by the observation that respiratory movement exhibits a locally circular pattern in a plane augmented with a delayed axis. By including the angular velocity as part of the system state, the proposed dynamic model effectively captures the natural evolution of respiratory motion. The first-order extended Kalman filter is used to propagate and update the state estimate. The target location is predicted by evaluating the local dynamic model equations at the required prediction length. This method is complementary to existing work in that (1) the local circular motion model characterizes 'turning', overcoming the limitation of linear motion models; (2) it uses a natural state representation including the local angular velocity and updates the state estimate systematically, offering explicit physical interpretations; (3) it relies on a parametric model and is much less data-satiate than the typical adaptive semiparametric or nonparametric method. We tested the performance of the proposed method with ten RPM traces, using the normalized root mean squared difference between the predicted value and the retrospective observation as the error metric. Its performance was compared with predictors based on the linear model, the interacting multiple linear models and the kernel density estimator for various combinations of prediction lengths and observation rates. The local dynamic model based approach provides the best performance for short to medium prediction lengths under relatively low observation rate. Sensitivity analysis indicates its robustness toward the choice of parameters. Its simplicity, robustness and low computation cost makes the proposed local dynamic model an attractive tool for real-time prediction with system latencies below 0.4 s.

Endogenous urinary steroids in premenopausal women with uterine leiomyomas.

OBJECTIVES: To study the effect of endogenous steroids on the presence of uterine leiomyomas. METHODS: Urine samples of 27 premenopausal women with leiomyomas and 25 age-matched healthy premenopausal women were collected. The concentration of estrogens and androgens in the urine samples of the two groups were determined using a gas chromatography mass spectrometer and the two groups were compared. To study metabolic changes in patients indirectly, the concentration ratios of precursor metabolite to product metabolite of the two groups were also compared. RESULTS: Urinary concentrations of 17beta-estradiol, 5-androstene-3beta, 16beta, 17beta, triol, 11-keto-ethiocholanolone, 11beta-hydroxy-androsterone, 11beta-hydroxy-etiocholanolone, THS, THA, THE, alpha-cortol and beta-cortol were significantly higher in patients than in controls. The concentration ratios of 17beta-estradiol/estrone and 11/beta-hydroxy-ethiocholanolone/11beta-hydroxy-androsterone increased in patients. CONCLUSIONS: The presence of uterine leiomyomas correlates with an increase in urinary concentrations of estrogens and androgens, and it appears to be caused by a decrease in patients' metabolism of steroids.

Relationship between urinary profile of the endogenous steroids and postmenopausal women with stress urinary incontinence.

AIMS: The aims of this study were to investigate whether endogenous steroid hormones are (1) related to pathogenesis of stress urinary incontinence after menopause, (2) are related to severity of stress urinary incontinence, and (3) are related to prognostic parameters of stress urinary incontinence. METHODS: Twenty post-partum women with clinically diagnosed stress urinary incontinence and 20 age-matched postmenopausal women without stress urinary incontinence (control group) were evaluated. We compared urinary profile of the endogenous steroid hormones patients with stress urinary incontinence and controls, and between grade I and grade II of stress urinary incontinence. We also investigated the relationship between urinary profile of the endogenous steroid hormones and prognostic parameters of stress urinary incontinence (maximal urethral closure pressure, functional urethral length, Valsalva leak point pressure, cough leak point pressure, posterior urethrovesical angle, bladder neck descent, and stress urethral axis). The ages of the patients and those in the control group were 64.3 +/- 5.6 and 57.5 +/- 3.8 years old and the body mass indexes were 24.96 +/- 3.14 and 22.11 +/- 2.73 kg/m2 in patients and in normal subjects, respectively. Nine patients were grade I and 11 were grade II. Estrone and 17beta-estradiol only were detected in all subjects, regardless of control or patient group. It is noteworthy that there were no significant differences (P > 0.05) in the levels of estrone and 17beta-estradiol in the urine of postmenopausal normal subjects compared with in the urine of postmenopausal patients with urinary incontinence. E2/E1 ratio was not different between the two groups (P > 0.05). Among the objective steroids, DHEA, Delta4-dione, Delta5-diol, Te, DHT, 16alpha-DHT, 11-keto An, THDOC, and THB were not detected either in the urine of normal subjects and nor in the urine of the patients. After comparing androgen levels between normal subjects and patients, no significant differences (P>0.05) were detected, except for 5alpha-THB and 5alpha-THF. Neither 5alpha-THB or 5alpha-THF were detected in the patients' urine. Et/An (11beta-OH Et/11beta-OH An) concentration ratios were not significantly different between the two groups, either (P > 0.05). There were not significant differences of concentrations (micromol/g creatinine) of urinary steroids between grade I and grade II of stress urinary incontinence. Pregnanediol was significantly related to bladder neck descent in supine and sitting positions (R = 0.79, P = 0.01, and R = 0.73, P = 0.03, respectively), and pregnanetriol was significantly related to maximal urethral closure pressure and functional urethral length (R = 0.68, P = 0.04, and R = -0.79, P = 0.01, respectively). Androsterone was significantly related to bladder neck descent in supine and sitting positions (R = 0.68, P = 0.04, and R = 0.78, P = 0.01, respectively). 5-AT was significantly related to bladder neck descent in sitting position and stress urethral axis (R = 0.72, P = 0.03, and R = -0.71, P = 0.03). 11-keto Et was significantly related to bladder neck descent in supine and sitting positions and related to stress urethral axis (R = 0.82, P = 0.01, and R = 0.81, P = 0.01, R = -0.67, P = 0.04, respectively). THS was significantly related to bladder neck descent in supine and sitting positions and related to stress urethral axis (R = 0.76, P = 0.02, and R = 0.74, P = 0.02, R = -0.68, P = 0.04, respectively). THE was significantly related to bladder neck descent in sitting position (R = 0.67, P = 0.04).beta-Tetrahydrocortisol/alpha-tetrahydrocortisol (beta-THF/alpha-THF) and alpha-cortol were significantly related to maximal urethral closure pressure and functional urethral length (R = 0.74, P = 0.02, and R = -0.92, P = 0.01; R = 0.71, P = 0.36, and R = -0.87, P = 0.000, respectively). 17beta-estradiol (E2) was significantly related to bladder neck descent in supine position (R = -0.62, P = 0.04) and 17beta-estradiol/estrone (E2/E1) was significantly related to cough leak point pressure (R = 0.79, P = 0.01). In conclusion, the urinary concentrations of endogenous steroid metabolites in postmenopausal patients with stress urinary incontinence were not significantly different from normal patients and were not significantly different between grade I and grade II patients with stress urinary incontinence. Some endogenous steroid metabolites were positively or negatively significantly related to prognostic parameters of stress urinary incontinence.

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells.

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-gamma (IFN-gamma) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-gamma, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-gamma-differentiated U937 cells. Western blot analysis revealed that, in IFN-gamma-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-gamma-differentiation, compared to that of the control. These results suggest that the IFN-gamma-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.

Urinary profile of endogenous steroids in postmenopausal women with stress urinary incontinence.

OBJECTIVE: To predict the role of estrogen in prevention of and therapy for stress urinary incontinence by comparing the urinary levels of estrogens and androgens and, to indirectly evaluate metabolism of estrogens and androgens by comparing the concentration ratios of precursor metabolites with those in controls (normal subjects). STUDY DESIGN: Urine samples collected for 24 hours were obtained from postmenopausal women with stress urinary incontinence (n = 20) and from age-matched, postmenopausal, normal female subjects (n = 14). The urinary levels of 20 estrogens and 25 androgens were analyzed by gas chromatography/mass spectrometry. RESULTS: The urinary levels of androgens were significantly higher in patients with stress urinary incontinence than normal subjects, and the urinary levels of estrogens were somewhat higher in patients than normal subjects. However, there were no significant differences between the groups, nor were there significant differences in the metabolism of estrogens and androgens between two groups. CONCLUSION: The urinary levels of endogenous steroids were rather higher in patients with stress urinary incontinence than in normal subjects, so it appears that estrogen should not play a significant role in prevention of and therapy for stress urinary incontinence.

Activation of c-Jun N-terminal kinase 1 (JNK1) in mistletoe lectin II-induced apoptosis of human myeloleukemic U937 cells.

Extracts of mistletoe (Viscum album var. coloratum) have been used for several decades as an anticancer immunomodulating agent in clinical fields. However, the mechanism by which the plant extracts kill tumor cells has remained elusive. We investigated the direct effects of beta-galactoside- and N-acetyl-d-galactosamine-specific mistletoe lectin II in inducing apoptotic death of U937 cells. Three distinct components of mistletoe, including beta-galactoside- and N-acetyl-D-galactosamine-specific lectin II (60 kDa), polysaccharides, and viscotoxin (5 kDa), induced apoptotic cell death, characterized by DNA ladder pattern fragmentation of U937 cells at 12 hr after treatment. Consistent with apoptosis of the cells, mistletoe extracts markedly increased the phosphotransferase activity of c-Jun N-terminal kinase 1 (JNK1)/stress-activated protein kinase (SAPK) in U937 cells. Among the three components, lectin II was the most potent in inducing apoptosis as well as JNK1 activation of U937 cells in a dose- and time-dependent manner. Catalytic activation of JNK1 induced by mistletoe lectin II was inhibited by the addition of peptide aC-DEVD-CHO, but not by aC-YVAD-CHO. In addition, mistletoe lectin II induced apoptosis in a variety of cell types including Jurkat T cells, RAW 264.7 cells, HL-60 cells, DLD-1 cells, and primary acute myelocytic leukemic cells.

Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells.

This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP-dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid.

Prolonged delivery of nicotine in rats via nasal administration of proliposomes.

In order to achieve a prolonged delivery of nicotine to the systemic circulation, proliposomes containing nicotine base (NB-proliposomes) or nicotine hydrogen tartarate salt (NS-proliposomes) and a mixture of powdered nicotine hydrogen tartarate salt and sorbitol (1:9 mixture, MP) were administered intranasally to rats at a nicotine dose of 1 mg/kg. Proliposomes, lipid-sorbitol mixtures that form liposomes upon contact with water, were prepared according to previously established methods, and the mixture (MP) was prepared by mixing NS powder with sorbitol particles (105-350 micrometer in size). Nasal absorption of nicotine from these formulations was very rapid (i.e. less than 10 min was required to reach plasma peaks) and showed substantially sustained plasma nicotine levels compared to saline solutions of NB and NS, and previously reported nasal nicotine sprays. The AUC values from the proliposomes and MP were comparable to those from the saline solutions of NB and NS. However, the mean residence time (MRT) and plasma half-life (T(1/2beta)) of nicotine in the present study were much larger than those from the saline solutions. Thus, a prolonged delivery of nicotine to systemic circulation via the application of proliposomes or MP intranasally appears feasible. NB-proliposomes exhibited the best characteristics in terms of the area under the plasma concentration (AUC), MRT and T(1/2beta) of nicotine, which was followed by NS-proliposomes and MP. Retarded conversion of proliposomes and MP to liposomal emulsions and solution in the nasal cavity seems responsible, in part, for the sustained plasma nicotine concentrations, since the emulsions and solution yielded very short MRT and T(1/2beta) of nicotine. In addition, reduced metabolism to cotinine from the proliposomes and MP was apparently responsible for the sustained plasma nicotine levels. These dosage forms of nicotine appear to circumvent some of the shortcomings of transdermal patches (i.e. long T(max)) and nasal sprays (i.e. short T(1/2beta) and physicochemical instability).

LPS induces direct death of IFN-gamma primed murine embryonic hepatocyte, BNL CL2 cells in a TNF-alpha independent manner.

Although it has been well known that the role of LPS on liver damage is mediated through TNF-alpha, the mechanism by which LPS modulates the cytotoxicity of IFN-gamma on hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that IFN-gamma mediated apoptosis in murine embryonic hepatocyte BNL CL2 cells is potentiated by the addition of LPS (0.5 microg/ml). Consistently, LPS markedly increases the catalytic activity of caspase 3-like protease but not caspase 1-like protease in IFN-gamma treated cells. In addition, TNF-alpha alone does not affect cell viability but rather it potentiates the cytotoxic effect of IFN-gamma on BNL CL2 cells. However, the cell viability of IFN-gamma/LPS treated cells is affected by the addition of polymyxin B but not by TNF binding protein I (TNF-BPI). These data suggest that the lipid moiety of LPS may mediate direct cytotoxicity of BNL CL2 cells in a TNF-alpha independent manner.

Activation of caspase cascades in Korean mistletoe (Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells.

Mistletoe lectins are of high biological activity and exert cytotoxic effects. We have previously shown that Korean mistletoe, Viscum album var. coloratum, lectin-II specifically induces apoptotic cell death in cancer cells, not normal lymphocytes. The destructive mechanism by mistletoe lectins on tumor cells was mediated by activation of c-JUN N-terminal kinase (JNK)/stress-activated protein kinase. Herein, we investigated the involvement of caspase cascade and its proteolytic cleavage effects on biosubstrates of human myeloleukemic U937 cells by D-galactoside and N-acetyl-galactosamine-specific Korean mistletoe lectin-II. Mistletoe lectin-II induced ladder pattern DNA fragmentation and activation of caspase-3, -8, and -9 of U937 cells, but not caspase-1 protease, in a time- and dose-dependent manner. Consistent with catalytic activation of protease, both poly(ADP-ribose) polymerase (PARP) and protein kinase C-delta (PKC-delta) are also cleaved in mistletoe lectin-II-treated U937 cells. An inhibitor of caspase-3-like protease, DEVD-CHO peptide, significantly inhibited mistletoe lectin-II-induced apoptosis, PARP cleavage, and fragmentation of DNA. These results provide the evidence that Korean mistletoe lectin-II induces apoptotic death of U937 cells via activation of caspase cascades.