Solid dispersion of spironolactone with porous silica prepared by the solvent method.

Solid dispersions of spironolactone (SPI) with porous silica (Sylysia 730 and Sylysia 350) were prepared by the solvent method. The physicochemical properties of the prepared solid dispersions were evaluated by powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and atomic force microscopy (AFM). In the SEM study, no differences in the surface condition between Sylysia 350 and the solid dispersion of a Sylysia 350:SPI system in a weight ratio of 1:1 were observed. However, AFM phase images showed that the surface of the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) was rather smooth due to the adsorption of SPI as compared with that of a Sylysia 350 intact. The results of PXRD and DSC data in the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) indicated that the molecular state of the adsorbed SPI changed from crystalline to amorphous. Although the decrease in the SPI concentration increased with the amorphous fraction in the solid dispersion, the diffraction peaks due to SPI crystals still remained in the solid dispersion of a Sylysia 730:SPI system (weight ratio of 1:1), indicating that the mean pore diameter and specific surface area of an additive are some of the important factors for the amorphization of SPI crystals. The dissolution property of the SPI from the solid dispersions was remarkably improved in comparison with that of SPI crystals. The dissolution rate of the SPI from the solid dispersions with Sylysia 350 was faster than that of the SPI from the solid dispersions with Sylysia 730. The difference in the dissolution properties of SPI from both the solid dispersions was attributed to the difference in the molecular state of the SPI in both the solid dispersions. In the stability test, the amorphous state of the SPI in the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) was maintained for 2 weeks at 25 degrees C and 0% RH, while the amorphous SPI without Sylysia 350 crystallized under the same conditions.

EXpression of ErbB proteins in human prostate.

ErbB proteins are widely expressed in human and animal tissues, notably in cells of epithelial or neuroendocrine origin. Protein expression and interactions of ErbBs were examined in prostate cancer specimens. Expression of ErbB1-4 proteins was determined with immunohistochemical methods using each monoclonal antibody in 20 prostatic adenocarcinomas. The 4 ErbB proteins were widely expressed in normal, hyperplastic and cancerous tissues of the prostate. ErbBs may contribute to normal development or tumor growth and progression in human prostate.

A Study of observation system of time-sequenced images of multi-sliced CT based on a PC.

We developed a method for an observation system of time-sequenced images of heart, vessels, and surrounding organs. The observation images are constructed using multi-sliced CT tomogram images by applying texture mapping technology, one of which is namely 3D-pseudo solid image. The generated images could be observed like as a solid modeled image and be generated faster than by using solid model. The method can be realized on a contemporary PC (which means movable) and also applicable to be a part of the intuitive observation system developed already.

A method of extraction and graphical presentation of diaphragms for an observation system with an intuitive control.

The observation of movement of diaphragm is one of important methods for diagnosis of lung diseases. So, we have been developing an observation system which generates a movie of pseudo solid images using numerous sagittal and frontal MRI images and the movie on a flat panel monitor. And also the direction of the solid images can be changeable dynamically by operator's tilting its monitor, which means the intuitive control. We refined our observation system and developed a method to extract a diaphragm boundary and a new presentation to display the boundary of a diaphragm and body outlines to observe and discriminate their movement.

PKCdelta and zeta mediate IL-4/IL-13-induced germline epsilon transcription in human B cells: a putative regulation via PU.1 phosphorylation.

We have investigated the role of PKC isozymes in the function of IL-4 and IL-13 in human B cells. In a Burkitt's B lymphoma cell line, DND39, IL-4 induced the translocation of PKCdelta and zeta from the cytosol to the membrane fraction. The activation of germline epsilon promoter by IL-4 was abrogated not only by the expression of dominant negative mutants of PKCdelta and zeta but also by isozyme-selective PKC inhibitors, rottlerin and PKCzeta pseudosubstrate peptide. These inhibitors also suppressed IL-4/IL-13-induced germline epsilon transcription in the IL-13Ralpha1-transfected DND39 cells as well as in normal human B cells, but had no influence on the induction of CD23b in the latter cells. As a downstream event of PKC, we found threonine phosphorylation of PU.1 in IL-4-stimulated DND39 cells. This phosphorylation was suppressed by the PKC inhibitors, although STAT6 activation was unaffected. These results suggest that, in human B cells, IL-4/IL-13 utilize PKCdelta and zeta for the STAT6-independent signaling pathway and thereby modulate the transcriptional activity of PU.1.

[Inflammatory cytokines (IL-4, IL-5 and IL-13)].

The polarized Th2 cells play an important role in the pathogenesis of atopic asthma as well as in the induction of airway inflammation. Th2 cytokines, such as IL-4, IL-5 and IL-13, are pivotal in regulating the allergic phenotype, the IgE response or the inflammatory cell-mediated function. Selective inhibition of Th2 cytokines by pharmacologic agents, including anti-cytokine blocking antibody, cytokine mutant and soluble cytokine receptor, will contribute to asthma therapy. Strategies based on blocking key signaling cytokines are also discussed.

flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp.

For establishment of a rapid-identification method of Leptospira species, a flaB gene of Leptospira was investigated and the following results were obtained. 1) HaeIII- or HindIII-restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products (793 bp) of flaB gene was effectual for the classification of species of Leptospira. 2) Twenty cells of Leptospira in 1 ml of coagulated blood and 100 cells of Leptospira in 1 ml of anti-coagulated blood could be detected by flaB-PCR. These results suggested that PCR-RFLP based on the flaB gene was an efficient tool for rapid detection and identification of species of infected Leptospira from clinical specimens.

Negative incidence of Lyme disease-related Borrelia spp. in Alishan, Taiwan.

To investigate the prevalence of Lyme disease-related Borrelia species, wild rodents were captured around Yushan National Park and Alishan Forest Recreation Area Park in Taiwan 2,000 to 3,000 meters above sea level. Borrelia was not isolated from 67 small mammals of 7 species. Sera from rodents showed no positive reactivity against whole cell antigens of B. garinii, B. afzelii or B. valaisiana by ELISA. These results suggested that Lyme disease is not endemic to the Alishan area.

Involvement of CYP2B6 in n-demethylation of ketamine in human liver microsomes.

Ketamine is metabolized by cytochrome P450 (CYP) leading to production of pharmacologically active products and contributing to drug excretion. We identified the CYP enzymes involved in the N-demethylation of ketamine enantiomers using pooled human liver microsomes and microsomes from human B-lymphoblastoid cells that expressed CYP enzymes. The kinetic data in human liver microsomes for the (R)- and (S)-ketamine N-demethylase activities could be analyzed as two-enzyme systems. The K(m) values were 31 and 496 microM for (R)-ketamine, and 24 and 444 microM for (S)-ketamine. Among the 12 cDNA-expressed CYP enzymes examined, CYP2B6, CYP2C9, and CYP3A4 showed high activities for the N-demethylation of both enantiomers at the substrate concentration of 1 mM. CYP2B6 had the lowest K(m) value for the N-demethylation of (R)- and (S)-ketamine (74 and 44 microM, respectively). Also, the intrinsic clearance (CL(int): V(max)/K(m)) of CYP2B6 for the N-demethylation of both enantiomers were 7 to 13 times higher than those of CYP2C9 and CYP3A4. Orphenadrine (CYP2B6 inhibitor, 500 microM) and sulfaphenazole (CYP2C9 inhibitor, 100 microM) inhibited the N-demethylase activities for both enantiomers (5 microM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 microM) failed to inhibit these activities. In addition, the anti-CYP2B6 antibody inhibited these activities in human liver microsomes by 80%, whereas anti-CYP2C antibody and anti-CYP3A4 antibody failed to inhibit these activities. These results suggest that the high affinity/low capacity enzyme in human liver microsomes is mediated by CYP2B6, and the low affinity/high capacity enzyme is mediated by CYP2C9 and CYP3A4. CYP2B6 mainly mediates the N-demethylation of (R)- and (S)-ketamine in human liver microsomes at therapeutic concentrations (5 microM).

Serological analysis of human leptospirosis in the Philippines.

Leptospirosis in the Philippines is an underrepresented disease. To achieve an accurate means of serodiagnosis, we demonstrated antibodies to the prevalent Leptospira serovars in sera of 71 patients from three major hospitals in Manila by the microscopic agglutination test and Western blot analysis. Sera of 53 patients contained antibody against 8 serovars poi, tarassovi, manilae, pyrogenes, australis, grippotyphosa, javanica, and autumnalis.

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma.

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid-liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane-2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.

Atopy and the human IL-4 receptor alpha chain.

BACKGROUND: Atopy is a common inherited disorder characterized by increased IgE responsiveness, but no functional analysis of the candidate genes related to atopy has been performed. IL-4 is important for B-cell production of IgE, and the human IL-4 receptor alpha chain (hIL-4Ralpha) is crucial for the binding and signal transduction of IL-4, so hIL-4Ralpha may be a candidate gene related to atopy. OBJECTIVE: We examined the relationship between the variation at amino acid 50 of hIL-4Ralpha and atopic asthma. METHODS: We performed a genetic study to investigate the relationship between the variation of amino acid 50 (isoleucine [Ile(50)] or valine [Val(50)]) and atopic asthma in a Japanese population and a functional study with the use of transfectants that expressed hIL4Ralpha bearing either Ile(50) or Val(50). Furthermore, we analyzed CD23 expression and IgE synthesis after IL-4 stimulation of peripheral blood mononuclear cells bearing either Ile(50) or Val(50). RESULTS: The prevalence of Ile(50) was higher than that of Val(50) in individuals with atopic asthma, especially during childhood. In transfectants, germline epsilon transcription activity and Stat6 activity were upregulated by the Ile(50) variant, compared with Val(50), but receptor affinity for IL-4 was similar between the two. CD23 expression and IgE synthesis in response to IL-4 were augmented in Ile(50)-expressing peripheral mononuclear blood cells compared to cells expressing Val(50). CONCLUSION: The Ile(50) variant of hIL-4Ralpha may be related to atopic asthma, particularly in children.

The initial fixation of the press-fit acetabular shell--clinical observation and experimental study.

The initial solid fixation of an uncemented acetabular component affects the amount of bone ingrowth. We had several problems with broken screws in cases of acetabular revision. In recent years, the development of uncemented components without screws has attempted to improve these problems. We started to use "press-fit"-type acetabular shells in November 1996. Our thirty cases undergoing 2 mm under-reaming show good initial stability. The aim of our biomechanical study was to assess the most suitable degree of under-reaming of the bony acetabulum for the implantation of an uncemented hemispherical porous coated component.

Possible involvement of Shc in IL-4-induced germline epsilon transcription in a human B cell line.

The IL-4Ralpha contains the I4R motif which binds to the phosphotyrosine binding domain of several adaptor proteins, including IRS-1/2 and Shc. Although the involvement of IRS-1/2 in IL-4-induced PI3-kinase activation is known, there is little information on the role of Shc in IL-4 signaling. In this study, we found the preferential utilization of Shc by the IL-4Ralpha in a human Burkitt's B lymphoma cell line, DND39. IL-4 induced the association of tyrosine-phosphorylated Shc with the IL-4Ralpha, whereas no detectable tyrosine phosphorylation of IRS-1 or IRS-2 was induced. IL-4-induced germline epsilon promoter activation was enhanced by overexpression of Shc and was inhibited by truncated Shc lacking the collagen-homologous domain. We further found the association of Shc with PLCgamma1. Although direct tyrosine phosphorylation of PLCgamma1 was not detectable, the amount of PLCgamma1 coprecipitable with anti-phosphotyrosine was increased after IL-4 stimulation. These results suggest that Shc can function as an adaptor protein of the IL-4Ralpha and mediate the germline epsilon transcription.

Bilateral transcapholunate dislocation.

An uncommon case has been reported of bilateral transscaphoid-perilunate dislocation fracture following trauma. A 35-year-old male with bilateral transscapholunate dislocation was treated by closed reduction and internal fixation. The case was examined in detail, and compared to the findings in the literature; observations regarding fracture prognosis were also made. Two years post-surgery, the patient remained asymptomatic.

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands.

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.

In-vitro and in-vivo antibiotic susceptibilities of Lyme disease Borrelia isolated in China.

The antibiotic susceptibilities of seven Borrelia burgdorferi sensu lato isolates from Ixodes persulcatus in China were examined by in-vitro microdilution minimum inhibition concentration (MIC) and macrodilution minimum bactericidal concentration (MBC) methods. All isolates tested were susceptible to amoxicillin, erythromycin, and minocycline. The MICs of these drugs for the Chinese isolates were 0.025-0.1 microg/ml, <0.012-0.05 microg/ml, and <0.012-0.05 microg/ml, respectively. The MBCs were 0.1-0.39 microg/ml, <0.012-0.2 microg/ml, and 0.025-0.39 microg/ml, respectively. The in-vivo antimicrobial susceptibilities of the Chinese Borrelia isolates to two test drugs, amoxicillin and minocycline, were evaluated using ddY mice. Mice were infected by subcutaneous inoculation into the right hind footpad. When infection was confirmed, the mice were treated by subcutaneous injection of the test drugs into the back. Amoxicillin and minocycline, which possessed high in-vitro activities against Lyme disease Borrelia, provided good protection against borreliosis in this animal model. Higher doses of these drugs resulted in elimination of the Lyme disease spirochete from all animals receiving this course of treatment. The 50% curative doses (CD50) of amoxicillin and minocycline were 8.7 mg/kg and 3.1 mg/kg, respectively. This suggested that amoxicillin and minocycline could be useful for the treatment of Chinese Borrelia infection.

The relationship between postoperative results and bone dynamics in RA patients who underwent cementless Ortholoc II TKA: histomorphometric study.

Abstract We previously reported a histomorphometric study of iliac bone obtained at the time of cementless total knee arthroplasty (TKA) in patients with rheumatoid arthritis (RA). In this paper, we further extended our analysis to investigate the postoperative results and radiographs after TKA in association with bone metabolism. All the patients were female, ranging in age from 38 to 68 years (mean, 56.8 years). Histomorphometric results demonstrated that bone volume and trabecular thickness were decreased, whereas bone absorption and bone formation rate (BFR) were increased. The bone in patients with mutilating disease (MUD) showed remarkably increased bone turnover and osteoporosis compared with those of the more erosive subset (MES) patients. Sixteen out of the 20 patients were alive at the time of follow-up (follow-up period between 5 years 10 months and 8 years 11 months). Among them, 10 patients could be further examined. Radiolucent lines (RLL) were assessed by the modified Knee Society evaluation (scoring) system. The number of RLL (N-RLL) were correlated with BFR in morphometric examination. In addition, the weight-bearing zones in the N-RLL were more significantly correlated with BFR. The Japanese Orthopedic Association (JOA) score and pain score negatively correlated with eroded bone surface. The present study indicated that bone dynamics, rather than the bone volume at the time of TKA, were involved in the presence of RLL and in pain after surgery for TKA.

Determination of members of a Borrelia afzelii-related group isolated from Ixodes nipponensis in Korea as Borrelia valaisiana.

The 16S rRNA sequences of the Korean Borrelia strains 5MT and 9MT, isolated from Ixodes nipponensis, showed identities of 99.0-99.1% to that of B. afzelii. The strains were tentatively classified as belonging to the B. afzelii-related group. In this study, Korean isolates, including these strains, were characterized further and compared with recently described new species. These strains generated a RFLP pattern that has not been found previously in RFLP analysis of the 5S-23S rRNA intergenic spacer and the flagellin gene. When phylogenetic trees were constructed, based on the 5S-23S rRNA intergenic spacer, flagellin gene and 16S rRNA sequences, these Korean isolates formed a cluster with the Borrelia strain Am501 isolated from Ixodes columnae in Japan and Borrelia valaisiana strains VS116T and UK isolated from Ixodes ricinus in Europe and were distinguishable from the other species. However, these three groups of strains were divergent from each other in the molecular masses of the putative outer surface protein A (OspA) and in the sequences of the ospA gene. These findings suggest that these Korean isolates and one Japanese isolate are members of B. valaisiana and that OspA of this species is divergent, as is that of Borrelia garinii. This led to the speculation that B. valaisiana strains are adapted to the vector ticks found in each locality.

Natural killer (NK) T cells are significantly decreased in the peripheral blood of patients with rheumatoid arthritis (RA).

The number of NK T cells was measured in relation to the Th1/Th2 imbalance observed in RA. Peripheral blood samples of patients with RA (n = 60) and healthy controls (n = 36) were stained with anti-NK receptor 1A (anti-NKR-P1A), anti-CD56, and anti-CD3 MoAbs, and examined by three-colour flow cytometry. NK T (NKR-P1A+CD3+) cells in the peripheral blood were decreased in RA compared with the controls: 25 +/- 20/microl versus 143 +/- 53/microl (P < 0.0001). CD56+CD3+ cells were also decreased in RA: 60 +/- 46/microl versus 116 +/- 54/microl (P < 0.0001). The decrease was significant when adjusted to the number of total lymphocytes (P < 0.0001) or NK (CD56+CD3-) cells (P < 0.0001), and showed no correlation with age, sex, disease duration, disease activity, functional class, x-ray stage, drug treatment, joint score, grip strength, C-reactive protein, rheumatoid factor or erythrocyte sedimentation rate of the patients. The results show that the levels of NK T cells are depressed in the peripheral blood of patients with RA, suggesting that the measurement of NK T cells in peripheral blood may have clinical importance for a Th1-type autoimmune disease like RA.