Characterization of hepatitis C virus (HCV) quasispecies dynamics upon short-term dual therapy with the HCV NS5B nucleoside polymerase inhibitor mericitabine and the NS3/4 protease inhibitor danoprevir.

In the INFORM-1 study, 73 patients with chronic hepatitis C virus infection received mericitabine plus danoprevir for up to 13 days. Seventy-two patients experienced a continuous decline in HCV RNA levels during treatment, and of these patients, 14 had viral loads that remained >1,000 IU/ml by day 13 and 1 met the definition for viral breakthrough. In-depth NS5B and NS3/4A population and clonal sequencing studies and mericitabine and danoprevir drug susceptibility testing were performed to assess the variability and quasispecies dynamics before and upon monotherapy or dual therapy. Sequence analysis of the viral quasispecies indicated that the mericitabine resistance mutation S282T was not present at baseline, nor was it selected (even at a low level) during treatment. Protease inhibitor resistance mutations, either as predominant or as minority species, were detected in 18 patients at baseline. No enrichment of minority protease inhibitor-resistant variants present at baseline was observed during treatment; viral population samples were fully susceptible to mericitabine and/or danoprevir, despite the presence within their quasispecies of minority variants confirmed to have reduced susceptibility to danoprevir or other protease inhibitors. It was also observed that certain NS3 amino acid substitutions affected protease inhibitor drug susceptibility in a compound-specific manner and varied with the genetic context. In summary, the slower kinetics of viral load decline observed in some patients was not due to the selection of danoprevir or mericitabine resistance during treatment. Over 2 weeks' therapy, mericitabine suppressed the selection of danoprevir resistance, results that could differ upon longer treatment periods.

Impact of inadequate initial antimicrobial therapy on mortality in patients with bacteraemic cholangitis: a retrospective cohort study.

OBJECTIVES: Acute cholangitis is a common cause of bacteraemia resulting in severe sepsis or septic shock. The impact of the appropriate initial antimicrobial therapy on short-term mortality in bacteraemic cholangitis has not been well investigated. METHODS: We conducted a retrospective cohort study of patients with bacteraemic cholangitis at two large tertiary care centres in Tokyo, Japan between 2009 and 2015. We determined the factors associated with 30-day all-cause mortality from the date of drawing the first positive blood culture, using a multivariate logistic regression analysis. RESULTS: We identified 573 patients with bacteraemic cholangitis (median age, 77 years; male, 58.3%). The 30-day all-cause mortality rate was 6.6% (38/573). Inadequate initial antimicrobial therapy occurred in 133 (23.2%) patients. Factors associated with 30-day all-cause mortality included the Charlson co-morbidity index score >3 (adjusted odds ratio (aOR) 4.12; 95% CI 1.18-14.38), jaundice (total bilirubin >2.5 mg/dL) (aOR 3.39; 95% CI 1.46-7.89), septic shock within 48 h of the first positive blood culture (aOR 3.34; 95% CI 1.42-7.89), biliary obstruction due to hepatobiliary malignancy (aOR 8.00; 95% CI 2.92-21.97), and inadequate initial antimicrobial therapy (aOR 2.78; 95% CI 1.27-6.11). CONCLUSIONS: Inadequate initial antimicrobial therapy was an important, modifiable determinant of survival.

Comparison of the computed tomography values of the bone fragment gap after sagittal split ramus osteotomy in mandibular prognathism with and without asymmetry.

The purpose of this study was to compare computed tomography (CT) Hounsfield unit values of bone fragment gaps after sagittal split ramus osteotomy (SSRO) in patients with and without asymmetry, and to evaluate differences between the deviated and non-deviated sides in asymmetric patients. Thirty-two patients who underwent a bilateral SSRO were included in this retrospective study. Patients were divided into symmetric and asymmetric groups by cephalometric analysis. CT values of the bone fragment gap were measured at 1 week and at 1 year after surgery. There were significant differences between CT values obtained at 1 week and at 1 year after surgery for all measurement points. However, there were no significant differences in CT values between symmetric and asymmetric patients at either 1 week or 1 year after surgery. Among asymmetric patients, there were no significant differences between the deviated and non-deviated sides at 1 week or 1 year after surgery. This study showed ossification of the bone fragments and adaptation to change the mandible form in patients with and without asymmetry following SSRO.

Changes in the computed tomography (pixel) value of mandibular ramus bone and fixation screws after sagittal split ramus osteotomy.

The purpose of this retrospective study was to evaluate the changes in computed tomography (CT) values of ramus bone and screws after sagittal split ramus osteotomy (SSRO) setback surgery. The subjects were 64 patients (128 sides) who underwent bilateral SSRO setback surgery. They were divided into six groups according to the fixation plate type used and the use or not of self-setting α-tricalcium phosphate (Biopex): group 1: titanium plate and screws; group 2: titanium plate and screws with Biopex; group 3: poly-l-lactic acid (PLLA) plate and screws; group 4: PLLA plate and screws with Biopex; group 5: uncalcined and unsintered hydroxyapatite particles and poly-l-lactic acid (uHA/PLLA) plate and screws; group 6: PLLA/uHA plate and screws with Biopex. CT values (pixel values) of the lateral cortex, medial cortex, osteotomy site, and screws were measured preoperatively, immediately after surgery, and 1 year postoperatively using horizontal CT images at the mandibular foramen taken parallel to the Frankfort horizontal plane. There were significant differences in the time-course change of pixel values for the lateral cortex (P<0.0001) and the osteotomy site (P<0.0001) among the six groups. This study suggests that the fixation plate type and use of bone alternative material may affect bone quality during the process of bone healing after SSRO.

Changes in occlusal function after orthognathic surgery in mandibular prognathism with and without asymmetry.

This study aimed to evaluate the postoperative changes in masticatory function in patients with jaw deformities with or without asymmetry treated by orthognathic surgery. Thirty female patients who underwent a Le Fort I osteotomy with sagittal split ramus osteotomy (SSRO) were enrolled. The patients were divided into symmetry and asymmetry groups. The bite force, occlusal contact area, and bite force balance were measured before and at 1, 3, and 6 months and 1 year after surgery; these measurements were compared statistically within and between the two groups. In the symmetry group, there was a significant difference in the preoperative bite force and the 1 month postoperative bite force (P=0.0033). In the asymmetry group, the bite force before surgery was significantly different from that at 1 month (P=0.0375) and at 1 year (P=0.0353) after surgery. Significant differences in the bite force were also observed between the following time points: 1 month and 1 year (P=0.0003), 3 months and 1 year (P=0.0034), and 1 month and 6 months (P=0.0486). The occlusal contact area, bite force, and occlusal balance tended to change after Le Fort I osteotomy with SSRO, with a significantly improved bite force in patients with asymmetry before surgery.

Molecular cloning, genomic characterization and expression of novel human alpha1A-adrenoceptor isoforms.

We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Acquisition of graft-versus-host reactivity by immature thymocytes in the coexistence of activated macrophages.

The collaborative role of macrophages in the acquisition of the rat graft-versus-host (GVH) reactivity by parental thymocytes was investigated by the popliteal lymph node enlargement assay. The addition of peritoneal macrophages activated by streptococcal bacteria injection resulted in significantly increased GVH reactivity of parental thymocytes, when assayed both in normal F1 hybrid hosts and in those depleted of effective macrophages and lymphocytes. The thymocytes that acquire that alloantigen reactivity in the presence of activated macrophages were shown to be cortisone sensitive and of high density. Hence, the cortisone-resistant and less dense population of thymocytes did not exhibit further reactivity even in the presence of macrophages; however, this subpopulation showed an enriched activity on its own. Moreover, the simultaneous injection of activated macrophages induced a further augmenting effect on the graft-versus-host reaction (GVHR) of freshly peripheralized thymocytes, but did not affect the reactivity of lymph lymphocytes from parental rats thymectomized 15 months previously. These data suggest that activated macrophages play an essential role in the acquisition of alloantigen reactivity by immature thymocytes, and also that the macrophage-dependent thymocytes in the reaction may reside among the subpopulation that is cortisone sensitive, highly dense, short-lived, and freshly peripheralized. The possible nature of immunological maturation of immature thymocytes in the periphery is discussed in relation to cellular interactions with activated macrophages.

Augmentation of the graft-versus-host reactivity of parental cells by the addition of small doses of alloantigen-activated T cells.

The direct action of alloantigen-activated T cells on the generation of graft-versus-host (GVH)-reactive T cells from their precursors was investigated in an in vivo system by the popliteal lymph node enlargement assay using normal and macrophage-depleted F1 hybrid rats. When very small numbers of alloantigen-activated parental T cells were inoculated in the foot-pads of F1 hosts 24 hr prior to the parental cell inoculum, a significant increase of the GVH reactivity of peripheral T cells was observed within the draining node in normal F1 rats and in F1 hosts in which an active participation of endogenous cells was greatly minimized. Moreover, a similar augmenting action of alloantigen-activated T cells was found in the thymocyte-GVH reaction, which was much higher than that of peripheral T cells when their stimulation indices were compared. These phenomena were produced by the addition of any of two different RT 1 species of alloantigen-activated T cells that were restimulated with F1 host-specific or nonspecific third-party alloantigen within the region of the assay, suggesting the importance of the restimulation of the alloantigen-activated T cell inoculum. A simultaneous injection of bacterial lipopolysaccharide had a cumulative effect on the augmenting action of alloantigen-activated T cells on the thymocyte GVH reaction. This study indicates that the preexistence of alloantigen-activated T cells at the assay site causes precocious maturation and proliferation of immature thymocytes, which allows them to function as GVH-reactive T cells in the periphery in vivo; it also suggests the presence of a serial stimulation network through T-T interactions in connection with macrophage-T-cell synergism and the interleukins.

Cloning, expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7b).

1. The rat 5-hydroxytryptamine (5-HT)7 receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human-5HT7 receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor. 2. A truncated splice variation in the human 5-HT7 receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be donated as the h5-HT7b receptor and the long form of the receptor as h5-HT7a. 3. The h5-HT7b receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [3H]-5-carboxyamidotryptamine (5-CT; Kd = 0.28 +/- 0.6 nM, Bmax = 7.3 +/- 17 pmol mg-1 protein). The rank order of affinities (pKi) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65) > 5-hydroxytryptamine (5-HT, 9.41) > methiothepin (8.87) > mesulergine (7.87) > 8-hydroxy-2 (di-n-propylamino)tetralin (8-OH-DPAT, 6.85) > ketanserin (6.44). 4. The h5-HT7b receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC50): 5-CT (8.7 +/- 0.11) > 5-MeOT (5-methoxytryptamine; 8.1 +/- 0.20) > 5-HT (7.5 +/- 0.13) tryptamine (5.6 +/- 0.36) > 8-OH-DPAT (5.3 +/- 0.28) > 5-methoxytryptamine (5.0 +/- 0.06). This rank order was comparable to that observed in the radioligand binding studies. 5. In a similar fashion to that described for the 5-HT7a receptor, PCR studies suggested that the 5-HT7b receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta. 6. It is concluded that the h5-HT7 receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT7 receptor gene.

Enzyme immunoassay for antibodies in serum using a covalent chromatographic method for separation of the bound label.

A sensitive enzyme immunoassay system for the measurement of antibodies in serum was developed by the use of a separation method based on the thiol-disulfide interchange reaction. Serum samples including antibodies (anti-insulin, anti-human chorionic gonadotropin, or anti-beta-D-galactosidase) were incubated with antigens labeled with beta-D-galactosidase from Escherichia coli. Each reaction mixture was then passed through a small column (0.1 ml) of (anti-IgG)IgG-Sepharose 4B, in which the (anti-IgG)IgG had been coupled by means of a disulfide bond. The column was washed to remove the unbound label, then the antibody-bound label was eluted from the column with a buffer containing 25 mM dithiothreitol, which cleaved the disulfide bonds between the Sepharose matrix and the anti-IgG antibody molecules. By measuring the enzyme activity in the eluate, the amount of antibodies could be determined even in a serum sample which contained antibodies corresponding to as little as 0.01% of the standard antisera. Preliminary experiments showed that the present method can be used to detect the anti-insulin antibody in the sera from insulin-treated diabetic patients.

Opposite regulation of the expression of cyclin-dependent kinase inhibitors during contact inhibition.

Contact inhibition is a well-known phenomenon, but the details of its mechanism are poorly understood. Recently, cyclin-dependent kinase inhibitors have been studied intensively with respect to their regulatory role in the cell cycle, and of them, p27(Kip1) is particularly involved in contact inhibition. p27(Kip1) is believed to be regulated primarily through posttranscriptional mechanisms. We now report that cyclin-dependent kinase inhibitors, including p27, are regulated differently at the mRNA level during contact inhibition in murine BALB/c-3T3 fibroblasts. The mRNA expression of p15, p16, and p27 was up-regulated as the cell density increased, but, on the contrary, the mRNA level of p21(Cip1/WAF1/Sdi1) markedly decreased when the cells became confluent. The protein levels of these genes were regulated in the same way as their mRNA levels, and cyclin-dependent kinase-2 activity was markedly inhibited on density-mediated growth arrest of the cells. These results indicate that the regulation of mRNA expression of cyclin-dependent kinase inhibitors appears to contribute to their protein levels and to the arrest of cell growth through contact inhibition.

Mechanism of pH-dependent photolysis of aliphatic amino acids and enantiomeric enrichment of racemic leucine by circularly polarized light.

It has been proposed that the origin of biological homochirality may be the result of irradiation of a racemic sample of amino acids by circularly polarized light (CPL). To determine the mechanism of enantiomeric enrichment, the irradiation of aliphatic amino acids by CPL was undertaken. An enantiomerically enriched sample (e.g., L isomer enrichment from r-CPL) was found to result from the preferential excitation/decomposition of one enantiomer over another via a Norrish Type II mechanism (leucine, valine, and isoleucine), with the enantiomeric excess dependent on the degree of protonation of the amino/carboxylic acid moiety.

Extension incision for renal carcinoma including invaded vena cava and right lobe of liver.

We report on a thirty-five-year-old woman with renal cell carcinoma who successfully underwent right radical nephrectomy and extended right hepatic lobectomy with resection and reconstruction of inferior vena cava (IVC). A temporary bypass was placed between the infrarenal IVC and right atrium using a heparin-coated synthetic tube. The tumor was resected en bloc including right kidney, adrenal gland, hepatic lobe, and IVC. The IVC was reconstructed using an expanded polytetrafluoroethylene (EPTFE) graft. Her postoperative course was uneventful with no signs of recurrence four years after surgery.

Enzymatic determination of bilirubin fractions in serum.

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (B delta). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and B delta are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH. The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively. The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. B delta is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slide method, and good results were obtained.

Triple carcinomas of the biliary tract associated with congenital choledochal dilatation and pancreaticobiliary maljunction.

We report a rare case of triple carcinomas of the biliary tract associated with congenital choledochal dilatation (CCD) and pancreaticobiliary maljunction (PBM). The patient was a 58-year-old Japanese man who complained of epigastralgia. Ultrasonography and computed tomography revealed an elevated lesion inside the markedly dilated extrahepatic bile duct, thickening of the gallbladder wall, and small polypoid lesions in the gallbladder. Magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography showed CCD and PBM. With a diagnosis of carcinoma of the bile duct and cholesterol polyps in the gallbladder, pylorus-preserving pancreaticoduodenectomy was performed. The resected specimen showed two elevated lesions in the dilated bile duct, cholesterol polyps, and an area of irregular mucosa in the gallbladder. Histopathological examination showed two carcinomas in the bile duct, an adenosquamous cell carcinoma, and a moderately differentiated tubular adenocarcinoma, and a well differentiated tubular adenocarcinoma of the gallbladder. Two years and 6 months after the operation, a solitary metastatic liver tumor was detected. Left hepatic lobectomy was performed. At present, 7 months after the second operation, the patient is doing well with no signs of recurrence. Multiple carcinomas in the biliary tract associated with CCD and PBM, including the details in the present patient, were reviewed.

Primary adenosquamous carcinoma of liver resected by right trisegmentectomy: report of a case and review of the literature.

A case of primary adenosquamous carcinoma of the liver in a patient with an elevated level of serum squamous cell carcinoma-related antigen is reported. A 68-year-old man was admitted to our hospital with a 10-day history of fever and jaundice. From the results of laboratory and imaging studies before surgery, a diagnosis of cholangiocellular carcinoma was made, and the patient underwent right trisegmentectomy with regional lymph node dissection. Histopathological examination of the resected specimen revealed adenocarcinoma, squamous cell carcinoma, and a transitional area containing both types of cancer cells. The number of argyrophilic nucleolar organizer regions and the labeling index of proliferating cell nuclear antigen were markedly elevated and the deoxyribonucleic acid ploidy pattern was aneuploid in the squamous component. The patient died due to liver metastases 3 months after the operation. We reviewed the 31 cases of adenosquamous carcinoma of the liver reported in the Japanese and English language literature, including the present case.

Hepatocellular carcinoma associated with anabolic steroid therapy: report of a case and review of the Japanese literature.

We report herein the case of a 35-year-old woman with aplastic anemia who developed hepatocellular carcinoma after long-term therapy with oxymetholone. She was treated with 60 mg/day of oxymetholone for 3 years (total dose 64.8 g). Alpha-fetoprotein, hepatitis B surface antigen, and hepatitis C antibody were all negative, but serum titers of carcinoembryonic antigen and carbohydrate antigen were elevated. Lateral segmentectomy of the liver was performed. The histopathological findings were compatible with those of multiple hepatocellular carcinoma without liver cirrhosis. Three years since the operation, the patient is doing well and no signs of tumor recurrence have been detected. According to our review of Japanese cases of hepatocellular carcinoma associated with anabolic steroid therapy, in all instances the tumors developed after long-term administration of anabolic steroids for hematologic diseases. In patients under long-term anabolic steroid therapy, routine screening of the liver by ultrasonography and computed tomography should be performed to detect liver tumors in the early stages.

Improved reaction buffers for solid-phase enzyme immunoassay without interference by serum factors.

A gelatin moderately digested by a protease, was as effective as original gelatin to remove the interference by serum factors with a sandwich immunoassay for insulin. Digestion of gelatin resulted in decrease in the viscosity, and the buffer containing the digested gelatin did not show gelation at 4 degrees C. Therefore the buffer involving the digested gelatin might be widely useful for the immunoassay without interference by serum factors.

Significance of cadmium concentrations in blood and hair as an indicator of dose 15 years after the reduction of environmental exposure to cadmium.

To evaluate the significance of cadmium (Cd) concentrations in blood (B-Cd) and hair (H-Cd) as an indicator of dose, a cross-sectional study was performed on 40 residents in a Cd-polluted area, Nagasaki Prefecture, Japan, in 1996. In the study area, soil replacement of Cd-polluted rice fields ended in 1981. B-Cd and H-Cd were significantly higher in the study population than in the control subjects. B-Cd was positively correlated with urinary Cd (U-Cd) (Spearman r=0.50, P=0.06 for males and r=0.72, P=0.0001 for females), while H-Cd was weakly or moderately correlated with U-Cd. After adjustment for gender using logistic regression analysis, log(B-Cd) and log(U-Cd), but not log(H-Cd), were significantly associated with the prevalence of increased urinary beta2-microglobulin (P for trend <0.05). These findings suggest that B-Cd is a good indicator of cumulative dose many years after the reduction of environmental exposure to Cd. H-Cd may be weakly or moderately correlated with body burden.

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability.

Tetrahymena thermophila could still swim after incubation of the cell body at 40 degrees C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (alpha, beta and gamma) subunits. Analysis by peptide mapping demonstrated that beta- and gamma-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.