Bile duct proliferation associated with bile salt-induced hypercholeresis in Mdr2 P-glycoprotein-deficient mice.

BACKGROUND/AIMS: Bile flow consists of bile salt-dependent bile flow (BSDF), generated by canalicular secretion of bile salts, and bile salt-independent flow (BSIF), probably of combined canalicular and ductular origin. Bile salt transport proteins have been identified in cholangiocytes, suggesting a role in control of BSDF and/or in control of bile salt synthesis through cholehepatic shunting. METHODS: We studied effects of bile duct proliferation under non-cholestatic conditions in multidrug resistance-2 P-glycoprotein (Abcb4)-deficient multidrug resistance gene-2 (Mdr2(-/-)) mice. BSDF and BSIF were determined in wild-type and Mdr2(-/-) mice during infusion of step-wise increasing dosages of tauroursodeoxycholate (TUDC). Cholate synthesis rate was determined by 2H4-cholate dilution. Results were related to expression of transport proteins in liver and intestine. RESULTS: During TUDC infusion, BSDF was increased by approximately 50% and BSIF by approximately 100% in Mdr2(-/-) mice compared with controls. Cholate synthesis rate was unaffected in Mdr2(-/-) mice. Hepatic expression of the apical sodium-dependent bile salt transporter (Asbt), its truncated form (tAsbt) and the multidrug resistance-related protein 3 were upregulated in Mdr2(-/-) mice. CONCLUSIONS: Bile duct proliferation in Mdr2(-/-) mice enhances cholehepatic shunting of bile salts, which is associated with a disproportionally high bile flow but does not affect bile salt synthesis.     

Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38- haematopoietic stem cells

Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34+CD38- cells from purified CD34+ cells upon stimulation with Flt3-ligand, stem cell factor and thrombopoietin. Over a 100-fold (range 80 to 128-fold) expansion of CD34+CD38- cells was observed with bone marrow and cord blood (CB). The expanded CD34+CD38- cells remained negative for lineage-specific markers and could be induced to differentiate into granulocytes, monocytes, megakaryocytes, erythrocytes, and T and B-lymphocytes in vitro. Lineage differentiation assays with single CD34+CD38- cells showed no loss of multilineage potential of expanded cells after ex vivo culture. We also demonstrated that the increase in frequency of CD34+CD38- cells was not as a result of the downregulation of CD38 expression during the culture. Quantitative analysis showed that the number of 6 week cobblestone area forming cells (CAFCwk6), a measure of proliferating HSC, in cytokine-stimulated CD34+ cells were increased by 20-fold. Expanded CD34+CD38- cells could be transduced efficiently with retroviruses encoding the low affinity nerve growth factor receptor (LNGFR) marker gene (17% to 44%, mean 27%), resulting in long-lasting expression of retroviral-encoded genes in progeny HSC and differentiated progenitors. We conclude that the combination Flt3-ligand (FL), stem cell factor and thrombopoietin (TPO) induced strong ex vivo proliferation of CD34+CD38- cells and that the absolute number of expanded cells with stem cell activity increased substantially in this population.     

Diagnosis of functional disorders of defecation causing the solitary rectal ulcer syndrome

The solitary rectal ulcer and colitis cystica profunda are different manifestations of the solitary rectal ulcer syndrome. The cause of solitary rectal ulcer syndrome remains unknown. Since defecation disorders are common among patients with solitary rectal ulcer syndrome, defecography is indicated. Defecography was performed on 19 patients with solitary rectal ulcer syndrome. In five patients, the spastic pelvic floor syndrome had occurred. Twelve patients had internal intussusception of the rectum, and one patient had an anterior rectal wall prolapse. In one patient, no abnormalities could be detected. These abnormalities led to severe straining, which can damage the anterior rectal wall. Findings strongly support the hypothesis that solitary rectal ulcers are traumatic lesions caused by straining. Defecography is a suitable procedure for detecting the causative disorder of defecation and for selecting patients for treatment. Read at the meeting of the American Society of Colon and Rectal Surgeons, San Diego, California, May 5 to 10, 1985. No reprints are available.     

Mapping of monoclonal antibodies to human factor IX

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF- like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.     

Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.     

Effect of statin therapy on mortality in patients with peripheral arterial disease and comparison of those with versus without associated chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) and peripheral arterial disease (PAD) are both inflammatory conditions. Statins are commonly used in patients with PAD and have anti-inflammatory properties, which may have beneficial effects in patients with COPD. The relation between statin use and mortality was investigated in patients with PAD with and without COPD. From 1990 to 2006, we studied 3,371 vascular surgery patients. Statin use was noted at baseline and, if prescribed, converted to <25% (low dose) and > or =25% (intensified dose) of the maximum recommended therapeutic dose. The diagnosis of COPD was based on the Global Initiative for Chronic Obstructive Lung Disease guidelines using pulmonary function test. End points were short- (30-day) and long-term (10-year) mortality. A total of 330 patients with COPD (25%) used statins, and 480 patients (23%) without COPD. Statin use was independently associated with improved short- and long-term survival in patients with COPD (odds ratio 0.48, 95% confidence interval [CI] 0.23 to 1.00; hazard ratio 0.67, 95% CI 0.52 to 0.86, respectively). In patients without COPD, statins were also associated with improved short- and long-term survival (odds ratio 0.42, 95% CI 0.20 to 0.87; hazard ratio 0.76, 95% CI 0.60 to 0.95, respectively). In patients with COPD, only an intensified dose of statins was associated with improved short-term survival. However, for the long term, both low-dose and intensive statin therapy were beneficial. In conclusion, statin use was associated with improved short- and long-term survival in patients with PAD with and without COPD. Patients with COPD should be treated with an intensified dose of statins to achieve an optimal effect on both the short and long term.     

Increasing pancreatic cancer is not paralleled by pancreaticoduodenectomy volumes in Brazil: A time trend analysis

Background

Currently, surgical resection represents the only curative treatment for pancreatic cancer (PC), however, the majority of tumors are no longer resectable by the time of diagnosis. The aim of this study was to describe time trends and distribution of pancreaticoduodenectomies (PDs) performed for treating PC in Brazil in recent years.

Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma

Patients in complete clinical remission after myeloablative allogeneic stem cell transplantation (allo-SCT) were enrolled in a longitudinal study to assess the predictive value of molecular monitoring. Using polymerase chain reaction (PCR) for immunoglobulin gene rearrangements it was possible to generate a clone-specific molecular marker in 48 of 70 patients. Of these 48 patients, 16 (33%) attained durable PCR-negativity after transplantation, whereas 13 (27%) remained persistently PCR-positive and 19 (40%) showed a mixed pattern. The cumulative risk of relapse at 5 years was 0% for PCR-negative patients, 33% for PCR-mixed patients, and 100% for PCR-positive patients. Within the group studied it was not possible to identify any clinical feature predictive of durable PCR-negativity. We believe that these findings could prompt the design of prospective studies to evaluate if the treatment of molecular disease can extend remission duration and survival.     

Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system

Transcobalamin II (TCII) is a cobalamin (Cbl, vitamin B12)-binding protein in mammalian plasma that facilitates the cellular uptake of the vitamin. To obtain human TCII in sufficient quantity for analytical studies, the complementary DNA (cDNA) encoding TCII was inserted into the plasmid PVL 1393, and the baculovirus expressing TCII was obtained by homologous recombination in Spodoptera frugiperda (SF9) insect cells by cotransfection with the wildtype virus. Under optimized conditions, SF9 cells infected with the recombinant virus secreted 2 to 4 micrograms of TCII per milliliter of culture medium. TCII did not accumulate in the SF9 cells and seemed to be constitutively secreted as observed previously in cultured human endothelial cells. The purified recombinant TCII has the same molecular weight by SDS-PAGE as purified human TCII. The recombinant TCII cross-reacts with an antiserum to native human TCII, binds Cbl and facilitates the uptake of Cbl in eukaryotic cells by binding to the receptor for TCII-Cbl on the plasma membrane of K562 cells. Amino acid sequence analysis of the purified recombinant TCII identified two polypeptides, one identical to the amino acid sequence deduced from the cDNA and a second lacking the first and second N-terminal residues. These sequences are identical to two TCII polypeptides purified from Cohn fraction III of pooled human plasma. The two forms of recombinant TCII have the same isoelectric points as the two predominant isoprotein forms of TCII in human serum. Since the baculovirus construct contains a single cDNA that can encode only one amino acid sequence, the two isoproteins in recombinant TCII must be generated by a mechanism other than allele specific expression. A plausible mechanism for generating isoproteins of nonglycosylated peptides, such as TCII, may be by splicing of the leader peptide at alternative sites.     

Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies

An abnormal increase in numbers of CCGG sites methylated in the 5' region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.