Generation of Human iPSC-Derived Intestinal Epithelial Cell Monolayers by CDX2 Transduction.

BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.

Electron Spin Resonance Study of Molecular Orientation and Dynamics of Phenyl Imino and Nitronyl Nitroxide Radicals in Organic 1D Nanochannels of Tris( o-phenylenedioxy)cyclotriphosphazene.

Imino and nitronyl nitroxide (IN and NN, respectively) radicals such as phenyliminonitroxide (PhIN) and phenylnitronylnitroxide (PhNN), respectively, were dispersed in the organic 1D nanochannels of tris( o-phenylenedioxy)cyclotriphosphazene (TPP). Electron spin resonance (ESR) measurements were conducted on these inclusion compounds (ICs) in the temperature range 4.2-300 K. The modulated-septet ESR spectra of TPP ICs using PhIN observed in the range 165-258 K were reproduced with the EasySpin program package using a model in which some of the PhIN molecules underwent uniaxial rotational diffusion in the TPP nanochannels around the molecular long axis corresponding to the principal y-axis of the g tensor. However, for the TPP IC using PhNN, complicated ESR spectra were observed, which were not consistent with the modulated quintet observed in solution or in the fast-motion limit in solids. These spectra were reproduced by the superposition of a quintet originating from rotational diffusion of PhNN molecules and a septet based on rotational diffusion of PhIN molecules generated in the synthetic process. The rotational diffusion activation energies of PhIN and PhNN in the TPP nanochannels were estimated to be 19 and 45 kJ mol-1 using an Arrhenius plot, respectively. These were consistent with that for NN radicals in the 1D nanochannels of 2,4,6-tris(4-chlorophenoxy)-1,3,5-triazine (CLPOT) (37-54 kJ mol-1) with a larger pore diameter than TPP reported in our previous study, or with that for 4-substituted-2,2,6,6-tetramethyl-1-piperidinyloxyl (4-X-TEMPO) in TPP nanochannels (5-26 kJ mol-1) with regard to molecular size or host-guest or guest-guest interactions. These results indicate that not only the NN group but also IN may be used for the clarification of chemical or biological structures of nanomaterials such as nanosized cavities.

Analysis of Nonlinear Pharmacokinetics of a Highly Albumin-Bound Compound: Contribution of Albumin-Mediated Hepatic Uptake Mechanism.

The cause of nonlinear pharmacokinetics (PK) (more than dose-proportional increase in exposure) of a urea derivative under development (compound A: anionic compound [pKa: 4.4]; LogP: 6.5; and plasma protein binding: 99.95%) observed in a clinical trial was investigated. Compound A was metabolized by CYP3A4, UGT1A1, and UGT1A3 with unbound Km of 3.3-17.8 µmol/L. OATP1B3-mediated uptake of compound A determined in the presence of human serum albumin (HSA) showed that unbound Km and Vmax decreased with increased HSA concentration. A greater decrease in unbound Km than in Vmax resulted in increased uptake clearance (Vmax/unbound Km) with increased HSA concentration, the so-called albumin-mediated uptake. At 2% HSA concentration, unbound Km was 0.00657 µmol/L. A physiologically based PK model assuming saturable hepatic uptake nearly replicated clinical PK of compound A. Unbound Km for hepatic uptake estimated from the model was 0.000767 µmol/L, lower than the in vitro unbound Km at 2% HSA concentration, whereas decreased Km with increased concentration of HSA in vitro indicated lower Km at physiological HSA concentration (4%-5%). In addition, unbound Km values for metabolizing enzymes were much higher than unbound Km for OATP1B3, indicating that the nonlinear PK of compound A is primarily attributed to saturated OATP1B3-mediated hepatic uptake of compound A.

Newly established ELISA for N-ERC/mesothelin improves diagnostic accuracy in patients with suspected pleural mesothelioma.

Pleural mesothelioma is an aggressive tumor, commonly caused by exposure to asbestos. The prognosis of mesothelioma remains disappointing despite multimodal treatment. We reported previously that N-ERC/mesothelin could be a useful biomarker for the early diagnosis of pleural mesothelioma and developed an enzyme-linked immunosorbent assay (ELISA) system for its detection. However, the reproducibility of our previous 7-16 ELISA system has been revealed to be unsatisfactory. To measure N-ERC/mesothelin more precisely, we developed a new 7-20 ELISA system. The subjects of this study were patients who were referred to our department with suspected pleural mesothelioma. The current study demonstrated that the newly established 7-20 ELISA system improved the sensitivity and specificity for diagnosing pleural mesothelioma compared with the previous system. Moreover, the 7-20 ELISA system showed better reproducibility and displayed the tendency of both higher sensitivity and higher specificity in plasma than in serum. Particularly for the epithelioid type, the area under the curve (AUC) and the diagnostic accuracy of N-ERC/mesothelin were excellent; the AUC was 0.91, the sensitivity was 0.95, and the specificity was 0.76 in plasma. In conclusion, assessment of N-ERC/mesothelin with our newly established 7-20 ELISA system is clinically useful for the precise diagnosis of pleural mesothelioma.

Cystic, nodular and cavitary metastases to the lungs in a patient with endometrial stromal sarcoma of the uterus.

A 57-year-old woman, who had undergone hysterectomy for uterine myoma 11 years earlier presented with cystic, nodular and cavitary lesions simultaneously visible on computed tomography images of the chest. Histological examinations of both the resected lung and past "myoma" specimens demonstrated that the original uterine tumor was a low-grade endometrial stromal sarcoma (ESS) that had metastasized to the lungs. No previous reports have described the coexistence of cystic, nodular and cavitary lesions with pulmonary metastasis of ESS; however, we successfully correlated the radiologic appearance with the corresponding pathologic findings. Medroxyprogesterone acetate therapy has effectively kept the patient asymptomatic for approximately five years.

Substrate- and dose-dependent drug interactions with grapefruit juice caused by multiple binding sites on OATP2B1.

PURPOSE: OATP2B1-mediated grapefruit juice (GFJ)-drug interactions are substrate-dependent; for example, GFJ ingestion significantly reduces bioavailability of fexofenadine, but not pravastatin. In the present study, we aimed to establish whether this observation can be explained by the presence of multiple binding sites (MBS) on OATP2B1. METHODS: OATP2B1-mediated drug uptake was evaluated using a Xenopus oocyte expression system. Drug concentration was quantified by LC/MS/MS analysis. RESULTS: OATP2B1-mediated uptake of pravastatin and fexofenadine exhibited biphasic saturation kinetics, indicating the presence of MBS on OATP2B1. GFJ strongly inhibited pravastatin uptake mediated by the high-affinity site on OATP2B1, while no significant inhibition of the low-affinity site was observed. In contrast, high-affinity transport of fexofenadine was only modestly inhibited by GFJ, while significant inhibition of the low-affinity site was observed. Contribution analysis indicated that both drugs are transported via the low-affinity site on OATP2B1 at therapeutically relevant concentrations. These findings indicate that only fexofenadine is expected to interact with GFJ on OATP2B1 at therapeutic concentrations, in accordance with the clinical observations. CONCLUSION: Substrate- and dose-dependent GFJ-drug interactions mediated by OATP2B1 might be explained in terms of the presence of MBS: interaction occurs only when drug and GFJ components share the same binding site on OATP2B1.

Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC50 ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC50 values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC50 ratios ([C]/IC50 ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC50 ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.

The N-ERC index is a novel monitoring and prognostic marker for advanced malignant pleural mesothelioma.

BACKGROUND: Although N-ERC/mesothelin (N-ERC) is an attractive diagnostic and treatment monitoring biomarker for malignant pleural mesothelioma (MPM), its clinical utility for predicting the prognosis has not yet been clarified. The aim of this study is to investigate whether the serum N-ERC level can accurately predict the outcome in patients with MPM. METHODS: Twenty-six patients with MPM were enrolled. Serum N-ERC level was measured before and after chemotherapy. The N-ERC index was determined by the logarithm of the division of the N-ERC level after two courses of chemotherapy by the prior level. RESULTS: The median N-ERC index in the partial response (PR) group was significantly lower than that in patients with the stable disease (SD) plus the progressive disease (PD) group. The overall survival in the group whose median N-ERC index was lower than its median value was significantly longer than the group whose median N-ERC index was higher than its median value. CONCLUSIONS: The N-ERC index is therefore considered to be a useful biomarker for predicting not only the chemotherapeutic response, but also the prognosis in patients with advanced MPM.

Major active components in grapefruit, orange, and apple juices responsible for OATP2B1-mediated drug interactions.

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1-mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone-3-sulfate. All three juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with half-maximum inhibition (IC(50) ) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1-mediated estrone-3-sulfate uptake with IC(50) values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration-IC(50) ratios ([C]/IC(50) ) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC(50) ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.

Long-lasting inhibitory effect of apple and orange juices, but not grapefruit juice, on OATP2B1-mediated drug absorption.

Enzyme-based grapefruit juice (GFJ)-drug interactions are mainly due to mechanism-based irreversible inhibition of metabolizing enzyme CYP3A4 by GFJ components, but the transporter organic anion transporting polypeptide (OATP)2B1 is also a putative site of interaction between drugs and fruit juices (FJ) in the absorption process. Here we aimed to investigate the effect of preincubation with FJ on OATP2B1-mediated transport of drugs in vitro. When OATP2B1-expressing Xenopus oocytes were preincubated with GFJ, orange juice (OJ), or apple juice (AJ), AJ induced a remarkable decrease in OATP2B1-mediated estrone-3-sulfate uptake in a concentration-dependent manner (IC(50) = 1.5%). A similar but less potent effect was observed with OJ (IC(50) = 21%), whereas GFJ had no effect. Similar results were obtained in preincubation studies using fexofenadine. Preincubation with OJ and AJ resulted in time-dependent inhibition of OATP2B1. Again, AJ had the more potent effect; its action lasted for at least 240 minutes, suggesting that AJ irreversibly inhibits OATP2B1-mediated drug uptake. Kinetic analysis revealed that coincubation and preincubation with AJ reduced OATP2B1-mediated estrone-3-sulfate uptake via competitive and noncompetitive mechanisms, respectively. Thus, OATP2B1 is functionally impaired through both competitive and long-lasting inhibition mechanisms by AJ and OJ, but not GFJ. Interestingly, although GFJ but not AJ is able to irreversibly inhibit CYP3A4, in the case of OATP2B1, AJ but not GFJ has a long-lasting inhibitory effect. Accordingly, complex FJ-drug interactions may occur in vivo, and their clinical significance should be examined.

Synergism of Staphylococcus aureus colonization and allergic reaction in the nasal cavity in mice.

BACKGROUND: The aim of this study is to investigate the reciprocal effect of Staphylococcus aureus colonization and allergic rhinitis in an allergy model of mice. METHODS: BALB/c mice with intraperitoneal ovalbumin (OVA) sensitization and/or intranasal S. aureus inoculation were prepared. The following 4 groups were designed: an OVA-sensitized S. aureus-inoculated (AR-SA) group, an OVA-sensitized uninoculated (AR) group, a nonsensitized S. aureus-inoculated (SA) group, and a nonsensitized uninoculated (control) group. After intranasal OVA challenge, nasal lavage fluid, peripheral blood, and nasal mucosa were collected. Polymorphonuclear cells in the nasal lavage fluid were counted, serum OVA-specific IgE and IgG1 were measured by enzyme immunoassays, and IL-4, IL-5, and IFN-γ mRNAs in the nasal mucosa were assessed by quantitative real-time reverse transcription-PCR. The number of S. aureus in the nasal mucosa and lavage fluid was counted. RESULTS: Both eosinophil and neutrophil counts were larger in the AR-SA group than in the other groups. Both IgE and IgG1 levels were higher in the AR and AR-SA groups than in the SA and control groups, and the IgG1 level was higher in the AR-SA group than in the AR group. The expression of IL-4 mRNA was higher in the AR-SA group than in the other groups, and the expression of IL-5 mRNA was higher in the AR-SA group than in the SA group. The AR-SA group showed higher counts of S. aureus in the nasal mucosa than the SA group. CONCLUSION: These results indicate the mutually potentiating effect of S. aureus colonization and allergic rhinitis.

Coexistence of pyriform sinus fistula, ectopic lingual thyroid, and ectopic cervical thymus.

OBJECTIVE: We report an extremely rare case of coexistence of pyriform sinus fistula, ectopic lingual thyroid, and ectopic cervical thymus. CASE REPORT: A 16-year-old girl with ectopic lingual thyroid had suffered repetitive painful swelling in the left anterior neck. Barium swallow radiography and computed tomography revealed a fistula arising from the pyriform sinus running down to the anterior neck. She underwent transcervical fistulectomy with preservation of the recurrent laryngeal nerve. Histopathological examination of the surgical specimen showed coexisting thymic tissue. CONCLUSION: This illustrative case exemplifies the different embryological origin of the thyroid from that of the third and fourth pharyngeal pouch derivatives.

Functional pleiotropy of organic anion transporting polypeptide OATP2B1 due to multiple binding sites.

The purpose of this study was to examine whether the presence of multiple binding sites can explain the pleiotropy of substrate recognition by OATP2B1, using Xenopus oocytes expressing OATP2B1. OATP2B1-mediated uptake of estrone-3-sulfate apparently exhibited biphasic saturation kinetics, with Km values of 0.10 ± 0.05 and 29.9 ± 12.1 µM and Vmax values of 14.1 ± 6.4 and 995 ± 273 fmol/min/oocyte for high- and low-affinity sites, respectively. Contribution analysis revealed that transport of estrone-3-sulfate mediated by high- and low-affinity sites on OATP2B1 could be evaluated at the concentrations of 0.005 and 50 µM, respectively. pH-dependence study of OATP2B1-mediated estrone-3-sulfate uptake suggested that high- and low-affinity sites show different pH sensitivity. When the inhibitory effect of 12 compounds on estrone-3-sulfate uptake by high- and low-affinity sites on OATP2B1 was examined, 4 compounds appeared to be inhibitors of the high-affinity site on OATP2B1. Two other compounds appeared to be inhibitors for the low-affinity site and four others were inhibitory at both sites. These results indicated the presence of multiple binding sites on OATP2B1 with different affinity for drugs. Accordingly, it is likely that drug-drug and drug-beverage interactions occur only when two drugs share the same binding site on OATP2B1.

Nasopharyngeal Streptococcus pneumoniae carriage in Japanese children attending day-care centers.

OBJECTIVES: We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Streptococcus pneumoniae carriage in healthy children in day-care centers (DCCs). METHODS: A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n=181) and the following winter (n=182). We obtained 157 S. pneumoniae isolates and analyzed them by antibiotic susceptibility testing, PCR assay for the penicillin-binding protein (PBP) genes and macrolide-resistance gene, and pulsed-field gel electrophoresis (PFGE). RESULTS: The overall carriage rate was 43.3% (157/363). The percentages of penicillin-intermediately resistant S. pneumoniae (PISP) strains, penicillin-resistant S. pneumoniae (PRSP) strains, erythromycin-intermediately resistant S. pneumoniae strains and erythromycin-resistant S. pneumoniae strains were 35.7% (56/157), 0.6% (1/157), 1.9% (3/157), and 69.4% (109/157), respectively. The percentages of S. pneumoniae strains with the pbp mutation(s) and mefA and/or ermB gene(s) were 92.4% (145/157) and 71.3% (112/157), respectively. Fifty strains with different PFGE patterns were obtained from among the 157 isolates. Thirteen strains were observed in both seasons, but only one of these strains was isolated from the same carrier. Twenty-one strains (42.0%) were isolated from two or more children, and 17 of these were each isolated from children attending the same DCC. CONCLUSIONS: These results indicate the spread of S. pneumoniae, particularly those with antibiotic-resistance genes, and the vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan.

Prediction of hearing outcomes by distortion product otoacoustic emissions in patients with idiopathic sudden sensorineural hearing loss.

OBJECTIVE: To investigate whether distortion product otoacoustic emissions (DPOAEs) can be a prognostic indicator of hearing outcomes in patients with idiopathic sudden sensorineural hearing loss (ISSNHL). METHODS: Seventy-eight consecutive patients with ISSNHL were enrolled. DPOAEs were measured at the first hospital visit. Two primary pure tones with a frequency ratio (f2/f1) of 1.2 were used at non-equal sound pressure levels (L1/L2=80/70dB SPL). The DPOAE amplitude was measured at the 11 frequencies of 2f1-f2 with f2 varying from 593 to 6031Hz. All the patients received steroid administration in combination with hyperbaric oxygen (HBO) therapy. Hearing recovery was evaluated by the improvement in hearing compared to the unaffected contralateral ear. Correlations between the hearing improvement rate and five potential prognostic factors (the DPOAE amplitude, patient's age, days from onset to the start of treatment, initial hearing level, and presence of vertigo) were examined by simple and multiple regression analyses. RESULTS: The net DPOAE amplitude in patients with hearing improvement rate ≥50% was significantly larger than that with hearing improvement rate <50% at f2 frequencies of 3031 and 4812Hz (unpaired Student's t-test, p<0.05). A simple regression analysis showed that the hearing improvement rate significantly correlated with the net DPOAE amplitude at f2 frequencies of 3031 and 4812Hz, but not with that at the other f2 frequencies tested. The correlation coefficients were 0.528 and 0.522 for 3031 and 4812Hz, respectively, with p values <1×10(-6). In a multiple regression analysis, the partial correlation coefficients of the net DPOAE amplitude were 0.308 and 0.246 with p values of 0.008 and 0.036 for 3031 and 4812Hz, respectively. CONCLUSION: The significant correlation between hearing recovery and DPOAEs measured before treatment indicates that DPOAEs are a potentially useful means of predicting hearing prognosis in ISSNHL.

Prediction model for hearing outcome in patients with idiopathic sudden sensorineural hearing loss.

The aim of this study is to develop a regression model for predicting hearing outcome in patients with idiopathic sudden sensorineural hearing loss (ISSNHL). A total of 174 consecutive patients with ISSNHL (average of the hearing levels at 250, 500, 1,000, 2,000, and 4,000 Hz was ≥40 dB; time from onset to treatment was ≤30 days) were retrospectively analyzed. They received steroid administration (400 mg/day of hydrocortisone sodium succinate followed by tapered doses) in combination with hyperbaric oxygen therapy. The hearing improvement rate compared to the unaffected contralateral ear was calculated. Correlations between the hearing improvement rate and four prognostic factors (patient's age, days from onset to treatment, initial hearing level, and the presence of vertigo) were examined by simple and multiple regression analyses. In the simple regression analysis, significant correlations were observed between the hearing improvement rate and all four prognostic factors. In the multiple regression analysis, the correlation was significant for patient's age, days from onset to treatment, and the presence of vertigo with partial correlation coefficients of -0.221, -0.324, and -0.329, respectively, but was not significant for the initial hearing level. We subsequently formulated a multiple regression equation for predicting the hearing improvement rate. The multiple correlation coefficient was 0.495 with a p value of 1.42 × 10(-9). Using this regression model, the hearing improvement rate is still difficult to predict with 95% probability, but is predictable with 70% probability.

[Two cases of familial summer-type hypersensitivity pneumonitis requiring differentiation from bird breeder's lung].

A 74-year-old-man (case 1) was admitted to our hospital because of dry cough, fever, and dyspnea on effort. His daughter-in-law, a 53-year-old-woman (case 2), was also admitted to our hospital on suspicion of hypersensitivity pneumonitis (HP). Their diagnoses of HP were established by radiological, serological, and histological examinations. It was necessary to differentiate between summer-type hypersensitivity pneumonitis (SHP) and bird breeder's lung due to their special environment. Several examinations, including immunological findings of BALF, returning-home provocation test, and antigen inhalation challenge test, enabled us to establish their diagnoses of SHP.

[Evaluation of clinical effectiveness of Pazufloxacin Mesilate in acute exacerbation of chronic respiratory diseases].

Patients with chronic respiratory disease have increased susceptibility to infection, because of impairment of the local immunologic defense mechanism in the airway system, which often results in acute exacerbation. Acute exacerbation of chronic respiratory disease is one of the most important predictors of increased morbidity and mortality, and thus the management in the acute phase is essential for better prognosis. Although the clinical guidelines for the management of respiratory tract infections published by the Japanese Respiratory Society recommend administering fluoroquinolones intravenously in case of hospitalized patients, the clinical evidence is still limited. In this study, we evaluated the efficacy of Pazufloxacin Mesilate (PZFX). an intravenous fluoroquinolone. in patients with chronic respiratory diseases complicated with acute exacerbation caused by acute respiratory infections. As a result, 16 out of 18 cases were successfully treated with PZFX. No adverse event was observed during this study. These results may support the validity of administering intravenous fluoroquinolone in hospitalized patients with acute exacerbation caused by infections, as recommended by the Japanese Respiratory Society.

Immunoreactivities for glutathione S-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea.

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.

Nasopharyngeal Haemophilus influenzae carriage in Japanese children attending day-care centers.

We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Haemophilus influenzae carriage in healthy children in day-care centers (DCCs). A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 184) and the following winter (n = 179). We obtained 172 H. influenzae isolates and analyzed them by antimicrobial susceptibility testing, PCR for bla(TEM-1) and the penicillin-binding protein (PBP) gene, and pulsed-field gel electrophoresis (PFGE). The overall carriage rate was 47.4% (172/363), and 37.2% of the isolates (64/172) were ampicillin (AMP) resistant. All the resistant isolates had a PBP mutation(s), while only three isolates had TEM-1. The carriage rate was significantly higher in the winter than in the summer (56.4% and 38.6%, respectively), owing to the increase in the numbers of AMP-susceptible H. influenzae isolates in the winter. Children aged < or = 3 years showed a higher rate of carriage of H. influenzae isolates with an AMP resistance gene(s) than those aged > or = 4 years (21.9% and 12.6%, respectively). Forty-two strains with different PFGE patterns were obtained from among the 172 isolates. Only five strains were observed in both seasons. None of the strains isolated in the summer was isolated from the same carrier in the winter. Twenty-seven strains (64.3%) were isolated from two or more children, and 25 of these were each isolated from children belonging to the same DCC. These results indicate the spread of H. influenzae, particularly those with a PBP mutation(s), and the highly vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan.