Time course of the antibody response in humans compared with rats experimentally infected with hepatic alveolar echinococcosis.

Human alveolar echinococcosis (AE) is caused by the accidental ingestion of Echinococcus multilocularis eggs. Early detection is essential as surgical resection is the only treatment for a complete cure. However, details are unclear about changes in the antibody response during the initial stages of infection, yet such information is useful for early serodiagnosis. Therefore, a long-term investigation was performed into the time course of the antibody response before 'positive' detection. Patient sera were used for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) analysis using crude antigens extracted from E. multilocularis protoscoleces. Rats were experimentally infected with AE and similarly analysed by ELISA and WB. Among the markers for diagnoses, the 18 kDa band (main marker) appeared last in the preoperative stages and disappeared first after operation in a WB test. Although the 18 kDa antigen can be useful as a marker for AE diagnosis, it cannot contribute to the detection of some patients before the 18 kDa band appearance. To avoid misdiagnosis, different diagnostic antigens such as the 26-28 and 7-8 kDa bands should also be considered. These bands tend to appear earlier than the 18 kDa band and thus offer the potential for early detection of AE. We first observed changes in the antibody response in a relatively early stage after infection in human AE cases. Notably, changes in the antibody response of two intermediate species were similar. These findings provide valuable information for the early detection of human AE cases in the future.

Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury.

Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery.

Galbeta1-6Gal, antigenic epitope which accounts for serological cross-reaction in diagnosis of Echinococcus multilocularis infection.

Parasitic helminths express various antigenic carbohydrates, which often account for serological cross-reactions. In serodiagnosis, it is essential to inspect cross-reactivity between the target parasite and other parasites in order to assess diagnostic performance. Our previous study showed that the Galbeta1-6Gal sequence was a common epitope between Echinococcus multilocularis (Em) and E. granulosus (Eg). Furthermore, compounds with this sequence from Fasciola hepatica (Fh) reportedly were recognized by sera with Eg infection. Our aim is to investigate whether this sequence is one of the widely common epitopes in many kinds of parasites. For various parasites, sera with Fh infection cross-reacted at the highest frequency (71.4%) against Em antigen. In patients with other parasitic infections, sera showed cross-reactions against Fh antigen bound to Em antigen with a high frequency (23.7%). Binding inhibition tests with commercial Galbeta1-6Gal disaccharide showed that Galbeta1-6Gal was the common epitope between not only Em, Eg and Fh, but also between various other parasites. Furthermore, the presence of the Galbeta1-6Gal epitope in Em antigen was confirmed by immunoblot testing with the specific antibody for this sequence. This study showed that the Galbeta1-6Gal sequence is one of the antigenic epitopes that accounts for serological cross-reactivity between Em and various other parasites.

Diagnosis of alveolar echinococcosis using immunoblotting with plural low molecular weight antigens.

Alveolar echinococcosis (AE) is endemic to Hokkaido, Japan. For the past 20 years, detection of AE among inhabitants has involved serological screening using an enzyme-linked immunosorbent assay (ELISA) followed by Western blotting (WB). Between the years 1987 and 2000, antigens targeted on 66, 55 and 30-35 kDa bands were routinely used in the WB step of AE diagnosis. However, since 2001 diagnosis has been dependent on three smaller molecular weight antigens (26-28, 18 and 7-8 kDa). Due to its higher sensitivity, this improved WB approach has been used as a confirmation step in the screening process and also for the testing of suspected AE cases in hospital outpatients. Using the improved WB technique, a total of 1745 serum samples were examined in 2001-2006 with 81 patients detected and registered with AE. Interestingly, sera from 76 of the 81 diagnosed AE patients (93.8%) demonstrated reactivity with all three antigens. However, sera from the remaining five patients (6.2%) demonstrated no reactivity with the 18 kDa antigen, even though they exhibited clearly detectable levels of reactivity with the 26-28 and 7-8 kDa bands. These results suggest that medical practitioners need to pay particular attention to the specific reactions to some different diagnostic antigens to minimize the risk of misdiagnosing AE patients. In turn, these results may also provide important diagnostic information for cystic echinococcosis (CE).

Serodiagnostic potential of chemically synthesized glycosphingolipid antigens in an enzyme-linked immunosorbent assay for alveolar echinococcosis.

In the serodiagnosis of alveolar echinococcosis, the detection of specific reactions against not only protein but also carbohydrate antigen is useful and both antigens supplement each other. Though recombinant protein antigens have recently advanced, the preparation of carbohydrate antigen still depends on extraction from crude antigens. In the latter case, it is not conventional to obtain carbohydrate antigen as a single component for examination and research. Therefore, chemically synthesized carbohydrate antigens were prepared for serodiagnosis by the enzyme-linked immunosorbent assay (ELISA). Four antigens with the structure of glycosphingolipids from Echinococcus multilocularis were examined and one antigen, Galbeta1-6(Fucalpha1-3)Galbeta1-6Galbeta1-ceramide, was found to show significant serodiagnostic potential in differentiating alveolar from cystic echinococcosis.

[Surgical fixation for flail chest].

Flail chest occurs by blunt chest trauma and is associated with pulmonary contusion, atelectasis, pneumothorax, hemothorax, and respiratory failure. Because of its severity, it may need internal pneumatic stabilization or surgical fixation. Some patients do not need the internal stabilization and are observed conservatively. Some of these patients, however, increase the flail after palliating the pain and getting up. These patients show inefficient ventilation and surgical fixation is needed. The operation should be performed after the improvement of pulmonary contusion. In this paper, we presented 2 patients who showed such course and clarified the surgical methodology.

The HLA-DOB gene displays limited polymorphism with only one amino acid substitution.

The HLA-DO molecule is a non-classical class II heterodimer composed of alpha and beta chains. We have previously recognized that all eight of the allelic variations of the HLA-DOA gene represent non-synonymous amino acid substitution. In the present study, to analyze genetic polymorphism and allelic variation of the HLA-DOB gene which may affect the efficiency of class II restricted antigen presentation thereby being involved in the susceptibility of HLA associated diseases, we conducted direct DNA sequencing of HLA-DOB in 36 HLA class II homozygous typing cells and identified six new allelic variations (DOB*0101101, *0101102, *01012, *01022, *0104101 and *0104102) including five single nucleotide polymorphisms with only one amino acid substitution. Furthermore, strong linkage disequilibrium was detected between DOB*01022 and DRB1*1502 only, with no linkage disequilibrium between the DOA and the DOB genes. The HLA-DOB gene has been identified in other mammals, and their nucleotide sequences are well conserved. These facts suggest that limited polymorphism in the DOB gene is profitable to execute their unique function as a co chaperone and so strong selective pressure is operating to prevent generic variation against the DOB molecule interacting with the DM molecule and thus maintaining the specified immunological function of regulating antigen presentation.

In-vivo kinetics of the interaction between midazolam and erythromycin in rats, taking account of metabolic intermediate complex formation.

To predict, quantitatively, the extent of drug interaction during repeated administration of a metabolic inhibitor, we analysed the effects of erythromycin treatment under several regimens on the area under the concentration curve (AUC) of midazolam in rats. Midazolam was administered into the portal vein 12 h after erythromycin treatment for 1, 2 or 3 days, or 12, 24, 36, 48, 72 and 96 h after erythromycin treatment for 4 days, and the plasma-concentration profiles of midazolam were analysed to assess the AUC. Moreover, the contents of total cytochrome P450 and inactive metabolic intermediate (MI) complex were simultaneously quantitated. While the AUC value of midazolam was not affected by the administration of erythromycin for 1 day, repeated administration of erythromycin evoked an increase in AUC ratio (AUC in erythromycin-treated rats/AUC in vehicle-treated rats), which reached a maximum value of 1.99 at 12 h after 4 days' treatment with erythromycin. The total content of cytochrome P450 in liver microsomes was unaffected by erythromycin treatment. Although the MI complex was undetectable after 1 day's treatment with erythromycin, its content increased with duration of erythromycin treatment, and the complex disappeared after the end of erythromycin treatment with a half-life of 12.3 h. In conclusion, the interaction between erythromycin and midazolam could be well predicted when the formation of MI complex in the liver was taken into account.

Prediction of midazolam-CYP3A inhibitors interaction in the human liver from in vivo/in vitro absorption, distribution, and metabolism data.

The extent of decreases in apparent hepatic clearance and intrinsic hepatic clearance of midazolam (MDZ) after intravenous administration of MDZ with concomitant oral administration of cimetidine (CIM), itraconazole (ITZ), or erythromycin (EM) was predicted using plasma unbound concentrations and liver unbound concentrations of inhibitors. When MDZ was concomitantly administered with CIM, the observed increase in MDZ concentration was successfully predicted using inhibition constants assessed by human liver microsome and liver-to-plasma unbound concentration ratios in rats. However, the extent of interaction with ITZ or EM was still underestimated even taking into account the concentrative uptake of inhibitors into liver. We could predict the degree of "mechanism-based" inhibition by EM on the hepatic metabolism of MDZ, after repeated administration of EM, by a physiological model incorporating the amount of active enzyme as well as the concentration of inhibitor. The maximum inactivation rate constant and the apparent inactivation constant of EM on MDZ metabolism were 0.0665 min(-1) and 81.8 microM, respectively. These kinetic parameters for the inactivation of the enzyme were applied to the physiological model with pharmacokinetic parameters of EM and MDZ obtained from published results. Consequently, we estimated that cytochrome P450 3A4 in the liver after repeated oral administration of EM was inactivated, resulting in 2.6-fold increase in the plasma concentration of MDZ. The estimated extent of increase in MDZ concentration in our study correlated well with the observed value based on metabolic inhibition by EM from published results.

The blood supply of the lateral epiphyseal arteries in Perthes' disease.

We performed superselective angiography in 28 hips in 25 patients with Perthes' disease in order to study the blood supply of the lateral epiphyseal arteries (LEAs). Interruption of the LEAs at their origin was observed in 19 hips (68%). Revascularisation in the form of numerous small arteries was seen in ten out of 11 hips in the initial stage of Perthes' disease, in seven of eight in the fragmentation stage and in five of nine in the healing stage. Penetration of mature arteries into the depths of the epiphysis was seen in four of nine hips in the healing stage. Vascular penetration was absent in the weight-bearing portion of the femoral head below the acetabular roof. Interruption of the posterior column artery was seen where it passed through the capsule in seven hips when they lay either in internal rotation or in abduction with internal rotation. We suggest that in Perthes' disease the blood supply of the LEAs is impaired at their origin and that revascularisation occurs from this site by ingrowth of small vessels into the femoral epiphysis. This process may be the result of recurrent ischaemic episodes.

Quantitative prediction of metabolic inhibition of midazolam by erythromycin, diltiazem, and verapamil in rats: implication of concentrative uptake of inhibitors into liver.

To evaluate the degree of drug-drug interaction concerning metabolic inhibition in the liver quantitatively, we tried to predict the plasma concentration increasing ratio (R) of midazolam (MDZ) by erythromycin (EM), diltiazem (DLZ), or verapamil (VER) in rats. MDZ was administered through the portal vein at the steady state of plasma concentration of these inhibitors. The R values in the area under the plasma concentration curve of MDZ in the presence of EM, DLZ, and VER were 2.02, 1.64, and 1.30, respectively. The liver to plasma unbound concentration ratios of EM, DLZ, and VER at the steady state after infusion were 20.8, 1.02, and 3.01, respectively, suggesting concentrative uptake of EM and VER into the liver. The predicted R value in the presence of EM calculated by use of plasma unbound concentration was 1.03, whereas the value calculated with liver unbound concentration was 1.61, which was very close to the observed value. These findings indicated the need to consider the concentrative uptake of inhibitors into the liver for the quantitative prediction of metabolic inhibition. However, the predicted values in the presence of DLZ or VER calculated by use of liver unbound concentration were still underestimated. This result may be due to the metabolic inhibition by the metabolites of both inhibitors. Therefore, when predicting the degree of metabolic inhibition quantitatively, the inhibitory effect by coadministered drugs and the disposition of these metabolites in the liver must also be considered.

Establishment of the anti-Klotho monoclonal antibodies and detection of Klotho protein in kidneys.

A novel gene, klotho (kl), which is involved in the development of a syndrome resembling human aging in mice, was recently identified. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. There also exists a splice variant of kl mRNA which encodes a putative secreted protein in both human and mouse. In this study, to characterize the physiological roles of Klotho protein, we established three monoclonal antibodies (mAbs) against the recombinant human Klotho protein. The mAbs are named KM2076 (rat IgG(2)a), KM2119 (rat IgG(2)b), and KM2365 (mouse IgG(1)). In Western blots, KM2076 and KM2119 specifically recognized a 130 kDa Klotho protein in the mouse and human kidney membrane fractions. To detect the human Klotho protein, the sandwich-type ELISA system with KM2076 and KM2365 was established. Using the ELISA system, we detected the human Klotho protein as low as 20 ng/ml in the supernatant of Chinese hamster ovary cells (CHO cells), introduced the human klotho gene. KM2076 and KM2119 specifically gave a positive staining by immunohistochemical staining in paraffin or frozen sections of the kidneys from wild-type mice but not in those from kl mice. Strong staining was observed especially in cortical renal tubules of the mouse kidney, where expression of klotho transcripts overlaps. KM2076 also showed a similar reaction pattern in the paraffin sections of rat and human kidneys. The mAbs established in this paper will serve as useful analytical, pathological, and diagnostic tools to disclose the role of Klotho protein in the suppression of a syndrome resembling human aging.

Correlation between in vivo and in vitro hepatic uptake of metabolic inhibitors of cytochrome P-450 in rats.

To predict the degree of accumulation of hepatic metabolic inhibitors in the liver from the in vitro data, we investigated the relationship between cell/medium concentration ratios (C/M ratios) in isolated rat hepatocytes and liver/blood unbound concentration (K(Bf)) after i.v. administration of various metabolic inhibitors such as itraconazole, ketoconazole, verapamil, diltiazem, enoxacin, ciprofloxacin, clarithromycin, cimetidine, and nizatidine. The C/M ratios of itraconazole were approximately 6,000 and 200 at the concentrations of 0.1 and 10 microg/ml, respectively, and the uptake of ketoconazole and verapamil into the hepatocytes also showed a concentration dependence, although the degree was smaller than that of itraconazole. The uptake of diltiazem, enoxacin, ciprofloxacin, and clarithromycin into the hepatocytes showed linear profiles on concentration dependence. There was an excellent correlation between C/M ratios and K(Bf) values of all nine drugs with a slope of 1. This finding suggested the possibility of predicting drug concentrations in the liver (C(H)) from C/M ratios, the blood concentrations of drugs (C(B)) and unbound fraction in blood (f(B)), which was expressed by C(H) = (C/M). C(B). f(B). It may be possible to predict the drug concentrations in human liver from K(Bf) values estimated with isolated human hepatocytes and concentrations in the blood in a similar manner as in rats.

Recent epidemiologic trends in alveolar echinococcosis prevalence in humans and animals in Hokkaido.

We investigated chronological and geographical changes of alveolar echinococcosis (AE) prevalence in 14 administrative districts of Hokkaido based on the data of our epizootiologic and seroepidemiologic surveys. The results suggest that the chronological transitions of the enzootic state of AE in Hokkaido markedly reflect those of human AE prevalence, and that new prevalence of human AE has been emerging from central and western Hokkaido.

Influence of spironolactone on neonatal screening for congenital adrenal hyperplasia.

AIM: To determine if the diuretic spironolactone cross reacts with 17alpha-hydroxyprogesterone (17OHP) in an enzyme linked immunosorbent assay (ELISA) kit used for the mass screening of congenital adrenal hyperplasia. METHODS: Concentrations of 17OHP on a blood filter paper disc were measured using an ELISA kit (kit C-7: ENZAPLATE N-17alpha -OHP-7; Chiron, Tokyo, Japan). The cross reactivity of spironolactone and its metabolites with 17OHP was determined. The concentrations of spironolactone and its metabolites in blood were measured using HPLC (high performance liquid chromatography). RESULTS: Spironolactone cross reacted with 17OHP using kit C-7 (0.01%), by increasing 17OHP concentration in a dose dependent manner. The blood concentration of spironolactone and its metabolites was nearly 900 ng/ml, high enough to show an additive effect on the 17OHP concentration. About 12% of the false positive cases screened using the kit were due to the administration of spironolactone. CONCLUSIONS: Spironolactone interferes with 17OHP concentrations, leading to false positive test results for CAH.

Retinoic acid stimulates development of adult-type chromatophores in the flounder.

Premetamorphic flounder larvae were administered different doses of 9-cis retinoic acid (9cRA). 9cRA at 25 nM (the highest dose) not only stimulated adult-type (ad-) chromatophore development on the ocular (eyed) side, but also induced the development of ad-chromatophores on the blind (non-eyed) side of the metamorphosed fish. The ad-chromatophore development was stimulated by 9cRA only when administered to the larvae that were at late premetamorphosis and at early prometamorphosis. Ad-chromatophores actually appear much later, at the end of metamorphosis. 9cRA was not effective at later stages of metamorphosis. These results suggest that 9cRA stimulated the development or determination of the developmental fate of neural crest cells for chromatophores. The present results also suggest the presence of immature chromatophores or neural crest cells on both sides of the larval body of the flounder and that the ad-chromatophore development is somehow inhibited on the blind side in spontaneous metamorphosis. All-trans retinoic acid (atRA) had a similar effect on the ad-chromatophore development. In addition, both types of RAs affected the development of fin rays of the fish, resulting in deformity of fins when administered at high doses early in metamorphosis. The teratogenic effect of atRA was greater than that of 9cRA.

Quantitative prediction of metabolic inhibition of midazolam by itraconazole and ketoconazole in rats: implication of concentrative uptake of inhibitors into liver.

To evaluate the extent of drug-drug interaction concerning metabolic inhibition in the liver quantitatively, we tried to predict the plasma concentration increasing ratio of midazolam (MDZ) by itraconazole (ITZ) or ketoconazole (KTZ) in rats. MDZ was administered at a dose of 10 mg/kg through the portal vein at 60 min after bolus administration of 20 mg/kg ITZ or during 0.33 mg/h/body of KTZ infusion. The ratio values in the area under the plasma concentration curve of MDZ in the presence of ITZ and KTZ was 2.14 and 1.67, respectively. The liver-unbound concentration to plasma-unbound concentration ratios of ITZ and KTZ were 11 approximately 14 and 1.3, respectively, suggesting a concentrative uptake of both drugs into the liver. ITZ and KTZ competitively inhibited the oxidative metabolism of MDZ in rat liver microsomes, and Ki values of ITZ and KTZ were 0.23 microM and 0.16 microM, respectively. We predicted the ratio values of MDZ in the presence of ITZ and KTZ, using Ki values and unbound concentrations of both drugs in the plasma or liver. The predicted ratio values in the presence of ITZ or KTZ calculated by using unbound concentration in the plasma were 1.03 approximately 1.05 and 1.39, whereas those calculated using unbound concentration in the liver were 1.73 approximately 1.97 and 1.51, respectively, which were very close to the observed ratio values. These findings indicated the necessity to consider the concentrative uptake of inhibitors into the liver for the quantitative prediction of the drug-drug interactions concerning metabolic inhibition in the liver.

P-glycoprotein-mediated transport of itraconazole across the blood-brain barrier.

The mechanism for the accumulation of itraconazole (ITZ) in its elimination from the brain was studied in rats and mice. The concentration of ITZ in liver tissue declined in parallel with the plasma ITZ concentration until 24 h after intravenous injection of the drug (half-life, 5 h); however, the ITZ in brain tissue rapidly disappeared (half-life, 0.4 h). The time profiles of the brain/plasma ITZ concentration ratio (Kp value) showed a marked overshooting, and the Kp value increased with increasing dose; these phenomena were not observed in the liver tissue. This finding indicates the occurrence of a nonlinear efflux of ITZ from the brain to the blood. Moreover, based on a pharmacokinetic model which hypothesized processes for both nonlinear and linear effluxes of ITZ from the brain to the blood, we found that the efflux rate constant in the saturable process was approximately sevenfold larger than that in the nonsaturable process. The Kp value for the brain tissue was significantly increased in the presence of ketoconazole or verapamil. The brain Kp value for mdr1a knockout mice was also significantly increased compared with that of control mice. Moreover, the uptake of vincristine or vinblastine, both of which are substrates of the P glycoprotein (P-gp), into mouse brain capillary endothelial cells was also significantly increased by ITZ or verapamil. In conclusion, P-gp in the brain capillary endothelial cells participates in a process of active efflux of ITZ from the brain to the blood at the blood-brain barrier, and ITZ can be an inhibitor of various substrates of P-gp.

Quantitative prediction of the interaction of midazolam and histamine H2 receptor antagonists in rats.

To quantitatively evaluate drug-drug interactions involving metabolic processes in the liver, we attempted to predict the increasing ratio of the plasma concentration of midazolam (MDZ) in the presence of cimetidine (CIM) or nizatidine (NZD) in rats. Under steady-state conditions for the plasma concentration of CIM or NZD, MDZ was administered through the portal vein. The AUC of MDZ in the presence of CIM was 2.5-fold higher than that in the absence of CIM. There was no effect of NZD on the AUC of MDZ. The liver/plasma concentration ratios for CIM and NZD were 4.0 and 2.7, respectively. The estimated liver unbound concentration (CH,f)/plasma unbound concentration (Cp,f) ratios for CIM and NZD were 1.9 and 2.4, respectively, suggesting concentrative hepatic accumulation of both drugs. The oxidative metabolism of MDZ in rat liver microsomes was competitively inhibited by CIM or NZD, and the Ki values of CIM and NZD were 110 and 2600 microM, respectively. Based on these data obtained in vivo and in vitro, the increasing ratios for MDZ in the presence of CIM or NZD were predicted using the equations Rp = 1 + Cp,f/Ki and RH = 1 + CH,f/Ki. The observed increasing ratios in the presence of CIM were very close to RH, compared with Rp. However, Cp, f and CH,f were much less than Ki and there was no difference between Rp and RH in the presence of NZD. Consequently, Cp,f and CH, f were greater than or equal to Ki and Cp,f was not equal to CH,f, as in the presence of CIM, and it was indicated that CH,f was more suitable for quantitatively predicting the drug-drug interactions than was Cp,f.