Value of determining carbohydrate-deficient transferrin isoforms in the diagnosis of alcoholic liver disease

OBJECTIVE: To determine whether isoform separation of carbohydrate-deficient transferrin (CDT) is of value in the diagnosis of alcoholic liver disease (ALD) and is specific to ALD when compared with other liver diseases. PATIENTS AND METHODS: During 1995 and 1996, 47 patients with ALD were evaluated with CDT at the Mayo Clinic in Rochester, Minn. The diagnosis of ALD was based on biochemical and histological analyses and on a history of drinking that exceeded 5 years with an average alcohol intake of more than 60 g/d. Disease controls included nonalcoholic steatohepatitis (NASH) (n = 26) and other liver disease (n = 22). Normal controls (n = 21) were healthy individuals without liver disease. Transferrin isoforms were quantified by densitometry of Coomassie-stained transferrins after affinity purification and isoelectric focusing. The pentasialo, tetrasialo, trisialo, disialo, monosialo, and asialo isoforms were quantified as percentages of total band densities. RESULTS: Receiver operating characteristic (ROC) curves were constructed for each isoform. The curves for total desialated isoforms (sum of disialo, monosialo, and asialo) displayed the best relationship between sensitivity and specificity with an ROC-area under the curve (AUC) of 0.922. The ROC-AUC values for individual transferrin isoforms in ALD vs NASH for pentasialo, tetrasialo, trisialo, disialo, monosialo, and asialo were 0.806, 0.917, 0.885, 0.933, 0.804, and 0.785, respectively. Only 58% of patients with ALD were detected at a specificity that excluded ALD in 84% of those who did not have it. CONCLUSION: Within alcohol ingestion times reported to us, no associations with recent drinking were observed. Alcohol as a cause of liver disease is not perfectly established by CDT analysis, although a high total CDT value favors ALD over NASH.     

Surgery for cholangiocarcinoma: the role of liver transplantation

Liver transplantation alone for unresectable hilar cholangiocarcinoma (CCA) is fraught with frequent recurrence and poor long-term survival. The Mayo Clinic developed a novel therapeutic protocol combining neoadjuvant chemoradiation and orthotopic liver transplantation (OLT) in 1993 to treat patients with unresectable hilar CCA or CCA arising in the setting of PSC. Aim. We recently reviewed our experience over the past 14 years with the specific aim to evaluate the long-term outcomes of CCA patients treated according to our study protocol. Methods. We analyzed data from all patients enrolled in the Mayo Clinic liver transplant protocol since 1993. Statistical data analysis of recurrence and survival rates was performed using the Kaplan-Meier method. Results. 148 patients were enrolled in the protocol. Of 90 patients who completed neoadjuvant therapy and subsequent OLT, 71 are alive and 19 have died – only 8 due to recurrent CCA. Nineteen patients are awaiting OLT and 39 were removed from the protocol owing to disease progression or death. Overall, 1-, 3-, and 5-year patient survival was 82%, 63%, and 55%, respectively; 1-, 3-, and 5-year survival after OLT was 90%, 80%, and 71%. Conclusions. Neoadjuvant chemoradiation and OLT achieves significantly lower recurrence and higher long-term survival rates than resection, OLT alone, or medical treatment in hilar CCA. Additional experience at independent transplant centers is necessary to confirm these encouraging results, address the role of neoadjuvant therapy and liver transplantation versus conventional resection, determine appropriate inclusion/exclusion criteria, and define the risk of disease progression while awaiting transplantation.     

Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis

Apoptosis has emerged as an important mechanism to reduce numbers of activated stellate cells during the resolution phase of hepatic fibrosis. These observations suggest that activated stellate cells may be more susceptible to apoptotic stimuli than their quiescent counterparts. Because other activated cell types are more sensitive than their quiescent phenotypes to apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined the expression of TRAIL death receptors (DRs) and susceptibility to TRAIL cytotoxicity in stellate cells undergoing progressive activation. A spontaneously immortalized human stellate cell line, LX-2, was analyzed during 14 days of progressive activation following plating, during which time alpha-smooth muscle actin (alpha-SMA) and a beta-crystallin (markers of stellate cell activation) messenger RNA (mRNA) increased 7-fold and 5-fold, respectively. During this same interval, TRAIL-R1/DR4 and TRAIL-R2/DR5 mRNA expression increased 18-fold and 17.6-fold, although TRAIL-R2/DR5 expression was quantitatively 103-fold greater than TRAIL-R1/DR4; parallel changes occurred in TRAIL/DR5 protein expression and cellular susceptibility to TRAIL-mediated apoptosis. Similar findings were observed in primary murine stellate cells undergoing activation on a plastic surface. In conclusion, stellate cells show activation-dependent TRAIL-R2/DR5 expression and TRAIL-mediated apoptosis. Because TRAIL-R2/DR5 is not expressed by hepatocytes, TRAIL/DR5 agonists may be useful in reducing fibrosis by inducing stellate cell apoptosis.     

Lysophosphatidylcholine suppresses apoptotic cell death by inducing cyclooxygenase-2 expression via a Raf-1 dependent mechanism in human cholangiocytes

Purpose The high incidence of biliary tract carcinoma in patients with anomalous pancreaticobiliary ductal junction (APBDJ) implicates that a compositional alteration in bile may contribute to the genesis of this cancer. Lysophosphatidylcholine (LPC) is generated in the bile of these patients. Given the role of cyclooxygenase-2 (COX-2) in biliary tract carcinogenesis, we postulated that LPC induces COX-2 in cholangiocytes.Methods The effect of LPC on COX-2 expression in cholangiocytes was evaluated by immunoblot analysis, real-time PCR and reporter gene assay. Apoptosis was induced by TRAIL treatment, and quantified using DAPI staining.Results Lysophosphatidylcholine increased COX-2 protein expression in cholangiocytes in a concentration- and time-dependent manner. LPC-induced Raf-1 activation was responsible for this COX-2 induction. Accordingly, LPC increased COX-2 mRNA levels in a Raf-1 dependent manner by stabilizing COX-2 mRNA. Finally, LPC attenuated TRAIL-mediated apoptosis through a COX-2/PgE2 dependent mechanism.Conclusions Collectively, these results implicate that LPC inhibits cholangiocyte apoptosis by inducing COX-2 expression via a Raf-1 dependent mechanism. This anti-apoptotic signaling may participate in biliary tract carcinogenesis in APBDJ patients, and therefore, its interruption may be a viable chemopreventative strategy.This work was presented at the European Association for the Study of the Liver in Paris in 2005.