Micronucleus scoring in liver fine needle aspiration cytology.

OBJECTIVE: This study evaluated the role of the micronucleus (MN) in liver fine needle aspiration (FNA) cytology. METHODS: Histological features of 75 cases of hepatocellular carcinoma (HCC), of which 25 were well differentiated, 37 moderately differentiated and 13 poorly differentiated, and 58 benign hepatic lesions (total, 133 cases) were correlated with MN expression observed in FNA smears reported as benign (n =40), atypical (n = 14), suspicious (n = 30) and malignant (n =49). RESULTS: Stepwise increases in the MN score (0.4 ± 0.6, 1.2 ± 1.3, 6.3 ± 4.2 and 14.8 ± 8.8) correlated with the degree of cytological abnormality: benign, atypia, suspicious and malignant, respectively. The mean MN scores for well-, moderately and poorly differentiated HCC were 5.4 ± 2.2, 11.5 ± 4.5 and 24.9 ± 9.1, respectively, which was significantly different between malignant and suspicious (P < 0.0001), between suspicious and atypical (P= 0.008) but not between atypical and benign. The MN scores differed significantly between all degrees of differentiation of HCC and between the HCC and benign hepatic lesions (P < 0.0001). High sensitivity, specificity and accuracy of liver FNA for diagnosing HCC (96%, 98%, and 96%, respectively) were obtained at a cutoff of three for the MN score. CONCLUSIONS: The MN score is an effective HCC biomarker and has a good potential use as an ancillary tool for diagnosing HCC using FNA cytology.

Accelerating Advanced MRI Reconstructions on GPUs.

Computational acceleration on graphics processing units (GPUs) can make advanced magnetic resonance imaging (MRI) reconstruction algorithms attractive in clinical settings, thereby improving the quality of MR images across a broad spectrum of applications. This paper describes the acceleration of such an algorithm on NVIDIA's Quadro FX 5600. The reconstruction of a 3D image with 128(3) voxels achieves up to 180 GFLOPS and requires just over one minute on the Quadro, while reconstruction on a quad-core CPU is twenty-one times slower. Furthermore, relative to the true image, the error exhibited by the advanced reconstruction is only 12%, while conventional reconstruction techniques incur error of 42%.

NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3.

Antibody response to hepatitis C virus infection after liver transplantation.

OBJECTIVE: To determine whether liver transplantation and the subsequent immunosuppression affect the antibody response to hepatitis C virus (HCV) infection. METHODS: Sera from 46 patients were compared before and after liver transplantation for markers of HCV infection. Serum HCV RNA was determined by polymerase chain reaction (PCR). Anti-HCV antibody was determined by first- and second-generation immunoassays as well as a quantitative assay of the titer of anti-HCV core antibody. RESULTS: Among individuals who acquired hepatitis C infection in association with liver transplantation, only 15% (3/12) developed antibody to the core antigen and only 25% (3/12) reacted to any antigen present on the second-generation recombinant immunoblot assay after a mean follow-up period of 18 months. Thirty-eight percent (5/13) were positive, by the second-generation enzyme immunoassay (EIA-2) Whereas 94% (16/17) of the individuals who had detectable anti-HCV core antibodies pretransplant continued to have such antibodies after transplant, the titer of these antibodies declined an average of 4-fold. No significant change was seen in the antibody titer toward rotavirus, a common viral pathogen. Patients who acquired HCV infection or in whom the allograft became reinfected had a significantly increased incidence of posttransplant hepatitis (61% vs. 33%, respectively). CONCLUSIONS: Liver transplantation and posttransplant immunosuppression lead to an attenuated antibody response to hepatitis C viral infection. Currently available assays for anti-HCV antibodies may be unreliable in the posttransplant setting.

'Albumin-mediated transport phenomenon' observed for ligands with high membrane permeability. Effect of the unstirred water layer in the Disse's space of rat liver.

In this paper, we offer experimental evidence of the rate-limiting diffusion of ligands through the unstirred water layer (UWL) as an explanation for the so-called albumin-mediated transport phenomenon. The relative membrane permeability of various ligands was first evaluated using isolated rat hepatocytes. Then, the effect of albumin on the uptake of ligands of a wide range of membrane permeabilities was examined using the perfused rat liver. The results were similar to those expected from the UWL model: ligands with high membrane permeability (warfarin, diazepam and taurocholate) clearly exhibited albumin-mediated transport, those with medium membrane permeability (tolbutamide and salicylate) showed less albumin-mediated transport, and ligand with low membrane permeability (cefodizime) did not show albumin-mediated transport. These results were explained by simulation studies of two separate cases based on the UWL model; one assuming the rapid equilibrium of ligand binding with albumin, and the other considering the slow dissociation of ligands from albumin. In light of these findings, we suggest that the rate-limiting diffusion through the UWL plays an important role in the so-called albumin-mediated transport phenomenon.

Metabolite inhibition of parent drug biotransformation. Studies of diltiazem.

Previously, we have demonstrated in vivo, in humans, nonlinear diltiazem disposition with an elimination half-life 50-100% greater after chronic diltiazem as compared to single-dose diltiazem administration. At least two metabolites, desmethyldiltiazem (MA) and desacetyldiltiazem (M1), accumulate significantly in human plasma during chronic diltiazem administration. To test the hypothesis that nonlinear diltiazem accumulation is associated with inhibition of biotransformation, we studied diltiazem disappearance during incubation with a number of its identified metabolites in an isolated rat hepatocyte system. Apparent kis for disappearance of diltiazem were: MA, 88.3 microM; M1, 608 microM; M2 (desacetyl N-desmethyldiltiazem), 495 microM; M4 (desacetyl O-desmethyldiltiazem), 152 microM; and M6 (desacetyl N,O-desmethyldiltiazem), 448 microM. These apparent ki values are similar to those derived for diltiazem-mediated inhibition of other drug substrates such as antipyrine, the clearance of which is inhibited by diltiazem in vivo in humans. Nonlinear diltiazem accumulation in vivo may be explained in part by progressive metabolite accumulation, particularly MA, which results in the inhibition of parent drug diltiazem biotransformation.

Protein-mediated hepatic uptake of rose bengal in analbuminemic mutant rats (NAR). Albumin is not indispensable to the protein-mediated transport of rose bengal.

Recent kinetic studies using in situ perfused rat liver suggested that the hepatic uptake of extensively albumin-bound ligands is mediated primarily by direct interaction of the albumin-ligand complex with the hepatocyte surface rather than by the small unbound fraction of ligand, as has been generally believed [Ockner et al., Am. J. Physiol. 245, G13 (1983)]. In order to investigate this mechanism in vivo, rose bengal (RB) was injected iv to the normal and Nagase analbuminemic mutant rats (NAR) and both the pharmacokinetic parameters and the serum protein binding parameters for the two groups were compared. The serum disappearance curves of RB in normal rats and NAR were almost superimposed, and no significant difference in various pharmacokinetic parameters including the hepatic uptake clearance (k12V1) was observed between the two groups. Nevertheless, the unbound fractions of RB in serum were approximately 4-fold (equilibrium dialysis method) and 10-fold (spectrophotometric method) higher than those in normal rats. However, in both groups of rats RB is extensively bound to plasma proteins and more than 99.8% of RB in the plasma exists as the protein-bound form. The intrinsic ability of the two groups of rats to take up unbound RB was compared using isolated liver cells. No significant difference between the two groups was observed in the initial velocity of uptake. From these findings, we concluded that the hepatic uptake of RB is primarily driven by the serum protein-bound form and not by the unbound form and that the serum protein-mediated uptake mechanism of RB was not specific only for serum albumin but also for other serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic analysis of albumin-mediated uptake of warfarin by perfused rat liver.

We previously found that the uptake of warfarin in the presence of albumin by perfused rat liver could not be explained simply by the unbound warfarin concentration. The aim of the present study is to develop a kinetic model to account for this albumin-mediated uptake of warfarin. Single circulation indicator dilution studies on warfarin uptake were carried out in the isolated perfused rat liver in the absence and presence of various concentrations of bovine serum albumin (BSA) in the perfusate. A distributed model was fitted to the dilution data and the estimates of the influx, efflux, and sequestration rate constants were obtained. The results showed that the predicted concentration of the unbound warfarin is not high enough to explain the observed uptake rate; the liver cell surface appears to reduce the binding affinity of warfarin for BSA to 1/20 of that observed in vitro. A kinetic model which considers the interaction between albumin and the liver cell surface was fitted to the uptake rates of warfarin over a wide range of BSA concentration. The model gave a dissociation constant of the cell surface for albumin of 160 microM, which is comparable with those reported by others for the hepatic extractions of free fatty acids and rose bengal. Based on this kinetic model, the contributions of the unbound and bound warfarin to its hepatic uptake were estimated, and the bound warfarin was found to contribute most in the physiological albumin concentration range.

Effect of albumin on hepatic uptake of warfarin in normal and analbuminemic mutant rats: analysis by multiple indicator dilution method.

Multiple indicator dilution studies of warfarin uptake were carried out on isolated perfused rat livers in the presence and absence of bovine serum albumin (BSA) in the perfusate using normal rats and Nagase analbuminemic rats (NAR). A distributed model was fitted to the dilution data and estimates of influx, efflux, and sequestration rate constants were obtained. In both groups of rats, the intrinsic clearance for unidirectional hepatic uptake (CLint,influx) of warfarin in the presence of 1.6 g/dl BSA was approximately 37-45% of that in the absence of BSA, while the unbound fraction of warfarin with 1.6 g/dl BSA in the perfusate was only 4.2% of that in the absence of BSA. Thus the degree of BSA-induced reduction of the value of CLint,influx and that of the unbound fraction are different. From these observations, it was found that the hepatic uptake of warfarin is not driven solely by the unbound concentration of warfarin, supporting the recent concepts of albumin-mediated transport for tightly albumin bound ligands as reported by Ockner et al. In addition, the fact that the same hepatic uptake mechanism of warfarin was also observed in NAR suggested that the hepatic uptake of warfarin may not necessarily require a special albumin receptor on the hepatocyte surface.

Effect of chlorpromazine on hepatic transport of indocyanine green in rats.

The effect of chlorpromazine hydrochloride (CPZ) on the hepatic transport of indocyanine green (ICG) was studied in the rat, in an attempt to elucidate the mechanisms of hepatotoxicity of CPZ in vivo, by comparing the pharmacokinetic parameters of ICG after bolus and chronic administration of CPZ. Delays were shown in both plasma disappearance and biliary excretion of ICG in the CPZ-treated rats (10 and 15 mg/kg intraportal bolus administration). Significant decreases were observed in the pharmacokinetic parameters, V2 and total body clearance (CLtot) in CPZ 10 mg/kg treated rats and k34, V2 and CLtot in CPZ 15 mg/kg treated rats, while a significant increase was observed in k21 in both CPZ-treated groups; V1 was not altered. The apparent liver-to-plasma concentration ratio (Kp,app) of ICG at 50 min after i.v. administration was decreased significantly in CPZ 15 mg/kg treated rats when compared to control rats, suggesting an alteration in the distribution of ICG to the liver by CPZ. Bile flow rates decreased immediately after bolus intraportal administration of CPZ in both CPZ-treated groups, and they then returned progressively to the basal levels. The output of bile acids was also inhibited by CPZ in a time-dependent and reversible manner and the bile acid independent fraction of bile flow was decreased significantly in both CPZ-treated groups. Chronic treatment with CPZ (10 or 20 mg/kg, i.p., per day for 3 weeks) did not alter either the pharmacokinetic parameters or the bile secretion profile of ICG, although there were significant decreases in body and liver weights in CPZ-treated groups. This may have been due to the rapid metabolism and excretion of CPZ in the rat when compared to humans. It is proposed that the acute toxic effect of CPZ on hepatic transport of ICG in the rat may be due mainly to the time-dependent and reversible cholestasis induced by CPZ, and that chronic treatment with CPZ may not alter the hepatic transport of ICG in the rat.

Effect of chlorpromazine on isolated rat hepatocytes.

The effect of chlorpromazine hydrochloride (CPZ) (1-500 microM) on plasma membrane permeability and mitochondrial respiratory function of isolated rat hepatocytes was studied. The endogenous oxygen consumption stimulated by 1 mM succinate was increased significantly by 5 microM CPZ, whereas the ability to exclude trypan blue (TB) was decreased significantly by 100 microM CPZ. The release of a cytosomal enzyme, lactate dehydrogenase (LDH), was increased significantly by 50 microM CPZ, whereas the release of glutamic-opalacetic transaminase (GOT) was increased significantly by 100 microM. The endogenous oxygen consumption was decreased significantly by 150 microM CPZ. The respiration control ratio by 2 microM carbonylcyanide-m-chlorphenyl hydrazon (CCP) showed significant decreases at all concentrations of CPZ studied; and this might be attributable to the suppression by CPZ of the respiratory stimulation induced by CCP. The results indicated that CPZ at a low concentration (5 microM) first produced a significant change in plasma membrane permeability to low molecular substances such as succinate and then at higher concentrations (50-100 microM) produced significant release of the cytosomal and mitochondrial enzymes, LDH and GOT. They also indicated that the concentrations of CPZ which produced significant effects on respiratory function were higher (above 150 microM) than those which produced significant changes in plasma membrane permeability of hepatocytes.