Indirect immunohistochemical evaluation of graft fibrosis and interface hepatitis after pediatric liver transplantation

Nagai S, Ito M, Kamei H, Nakamura T, Ando H, Kiuchi T. Indirect immunohistochemical evaluation of graft fibrosis and interface hepatitis after pediatric liver transplantation.
Pediatr Transplantation 2010:14:342–350. © 2009 John Wiley & Sons A/S. Abstract: Fibrosis or IH following pediatric liver transplantation is recognized as major causes of graft loss, but the etiology remains unclear. To determine this issue, we used an indirect immunohistochemistry technique with post‐transplant serum samples from recipients and normal human liver tissues from living liver donors, and the association between occult antibody reaction to the liver and the occurrence of fibrosis or IH was evaluated. Forty‐three recipients were evaluated, and both hepatocytes and biliary epithelial cells were evaluated for staining intensity. Fibrosis and IH occurred in 13 and six patients, respectively. According to staining results for the hepatocytes and biliary epithelial cells, 18 and 11 patients, respectively, were classified into the positive group. According to log‐rank analysis, positive reaction for hepatocytes was associated with increased rates of fibrosis and IH (p = 0.002 and 0.048, respectively), while positive reaction for biliary epithelial cells was associated with an increased rate of fibrosis (p = 0.014). Multivariate analysis revealed that positive reaction for hepatocytes and biliary epithelial cells was independently associated with fibrosis occurrence (p = 0.020 and 0.047, respectively). In conclusion, immune‐mediated reactions by occult antibodies may underlie the pathogeneses of fibrosis and IH.     

Topical hyaluronan alone promotes corneal epithelial cell migration whereas combination with benzalkonium chloride impairs epithelial wound healing

Abstract

Purpose: To evaluate the effects of topical hyaluronan (HA) on corneal epithelial wound healing when administered with or without benzalkonium chloride (BAC).

Methods: A cultured human corneal epithelial cell line (HCE-T) was subjected to in vitro scratch assays and in situ epithelial migration was evaluated in organ-cultured rabbit corneas. The corneal epithelium of C57BL/6J mice was also evaluated to determine in vivo wound healing. An in vivo imaging system was also used to evaluate the effects of HA on eye drop retention on the ocular surface.

Results: The findings revealed the promotion of HCE-T migration, in situ rabbit corneal epithelial migration, and in vivo wound healing in mouse corneal epithelium by HA. Pre-treatment with HA also protected against delayed epithelial wound healing in BAC in vitro. However, pre-treatment with 3?mg/mL HA did not show a protective effect against BAC in vivo, but instead delayed epithelial wound healing and increased detection of cleaved caspase-3. This suggested that HA promotes the retention of BAC on the ocular surface. The instilled HA was retained after 15?min, at a significantly higher rate than for phosphate-buffered saline.

Conclusions: The combination of HA and BAC impaired wound healing in the corneal epithelium.     

Female reproductive factors and risk of all-cause and cause-specific mortality among women: The Japan Public Health Center–based Prospective Study (JPHC study)

Purpose

We investigated the association between reproductive history and mortality from all and major causes among Japanese women.

刺人参甙Q和R的分离与结构鉴定

从刺人参叶中分离并鉴定了2种新甙,丝石竹皂甙元-3-O-β-D呲喃葡萄糖基-28-O-α-L-呲喃鼠李糖基(1→4)-β-D-呲喃葡萄糖基(1→6)-β-D-呲喃葡萄糖甙和3β-羟基羽扇豆-20(29)-烯-23-醛-28-酸-3-O-β-D-呲喃葡萄糖基-28-O-α-L-呲喃鼠李糖基(1→4)-β-D-呲喃葡萄糖基(1→6)-β-D-呲喃葡萄糖甙。     

CYP3A5 Contributes significantly to CYP3A-mediated drug oxidations in liver microsomes from Japanese subjects

The purpose of this study was to evaluate a contribution of polymorphic cytochrome P450 (CYP) 3A5 to the oxidation of diltiazem, midazolam and testosterone by liver microsomes from Japanese subjects. Twenty-seven liver samples were classified into three groups according to the CYP3A5 genotypes; CYP3A5(*)1/(*)1 (n=3), (*)1/(*)3 (n=12) and (*)3/(*)3 (n=12). The results of genotyping and immunochemical quantitation of CYP3A5 protein showed a good accordance between the CYP3A5 genotype and CYP3A5 content but not CYP3A4 content in liver microsomes. The expression levels of hepatic CYP3A5 protein ranged from 20 to 60% of the sum of CYP3A4 and CYP3A5 contents in subjects with at least one wild type allele ((*)1). The CYP3A5 contents correlated well with liver microsomal activities of diltiazem N-demethylation, midazolam 1'- and 4-hydroxylations and testosterone 6beta-hydroxylation among subjects carrying at least one (*)1 allele. In addition, the correlation coefficients of CYP3A5 contents with the rates of diltiazem N-demethylation, midazolam 1'-hydroxylation and testosterone 6beta- hydroxylation were higher than those of CYP3A4, although the value of CYP3A5 with the midazolam 4-hydroxylation rate was similar to that of CYP3A4. Kinetic analyses revealed a biphasic diltiazem N-demethylation in liver microsomes from subjects carrying the (*)1 allele. The apparent V(max)/K(m) values for recombinant CYP3A5 indicated the greater contributions to diltiazem N-demethylation and midazolam 1'-hydroxylation as compared with CYP3A4. These results suggest that polymorphic CYP3A5 contributes markedly to the drug oxidations, particularly diltiazem N-demethylation, midazolam 1'- hydroxylation and testosterone 6beta-hydroxylation by liver microsomes from Japanese subjects.     

Utility of a three-dimensional cultured human skin model as a tool to evaluate the simultaneous diffusion and metabolism of ethyl nicotinate in skin

The simultaneous diffusion and metabolism of ethyl nicotinate (EN) in a cultured human skin model, Living Skin Equivalent-high, was evaluated by the in vitro skin permeation and metabolism experiments, and esterase distribution was also determined. Theoretical calculations using Fick's 2nd Law of Diffusion with Michaelis-Menten kinetics were performed to obtain the permeation and metabolic parameters together with information on enzyme distribution. The obtained data was compared with the corresponding results in excised hairless rat skin. The partition coefficient of EN from the vehicle to the skin was of the same order of magnitude for the cultured human skin and hairless rat skin, but the diffusion and metabolic parameters were different. Esterase concentration in the epidermal membrane was greater than in the dermis of cultured skin, which was similar to hairless rat skin. Taking into account the similarities and differences between the membranes, the cultured human skin model can be utilized as a model membrane to rapidly predict simultaneous diffusion and metabolism of the prodrug through human skin.     

Studies on intestinal absorption of sulpiride (3): intestinal absorption of sulpiride in rats

The aim of this study was to investigate whether the concomitant administration of the substrates or inhibitors of PEPT1, OCTN1, OCTN2, and P-glycoprotein affects the intestinal absorption of sulpiride in rats. The absorption of sulpiride from rat intestine was decreased by the substrates or inhibitors of PEPT1, OCTN1, and OCTN2. On the other hand, the absorption was increased by the substrates of P-glycoprotein. The effects of these concomitantly administered drugs on the pharmacokinetic behavior of sulpiride after oral administration in rats were investigated. Peak concentration (C(max)) and area under the plasma concentration-time curve (AUC(0-8 h)) of sulpiride were decreased by the concomitant administration of the substrates or inhibitors of PEPT1, OCTN1, and OCTN2. However, the same parameters were significantly increased by the concomitant administration of the substrates of P-glycoprotein. The present results suggest the possibility of drug-drug interaction during the absorption process in the small intestine due to the coadministration of sulpiride and these agents. These findings provide important information for preventing adverse effects and for ensuring the effectiveness of sulpiride and concomitantly administered drugs.     

Effects of Decreased Vitamin D and Accumulated Uremic Toxin on Human CYP3A4 Activity in Patients with End-Stage Renal Disease

In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6β-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease.     

Effects of amiodarone on mutant Na+/Ca2+ exchangers expressed in CCL 39 cells

Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na+/Ca2+ exchange current (INCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na+/Ca2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. INCX was recorded by ramp pulses. Amiodarone at 30 microM inhibited INCX by 80% in cells expressing wild type NCX1. However, 30 microM amiodarone inhibited INCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (DeltaXIP) or 247-671 (Delta247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. INCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited INCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.