Cilostazol, a phosphodiesterase inhibitor, prevents no-reflow and hemorrhage in mice with focal cerebral ischemia

Background and PurposeThe Cilostazol Stroke Prevention Study II has shown a similar efficacy in stroke prevention but markedly fewer hemorrhagic events with the phosphodiesterase inhibitor cilostazol versus aspirin. The purpose of this study is therefore to investigate how cilostazol affects cerebral hemodynamics and whether it prevents hemorrhagic transformation induced by recombinant tissue plasminogen activator (rtPA) in a mouse model of focal ischemia/reperfusion. Particular emphasis will be placed on the plasma-microvessel interface.MethodsAfter receiving food containing 0.3% cilostazol or standard food for 7 days, adult C57BL/6 J mice were subjected to middle cerebral artery occlusion/reperfusion with or without rtPA (10 mg/kg) intravenously administered prior to reperfusion. Cerebral blood flow was monitored at several time points by laser speckle imaging in the 24 hour period post reperfusion, before neurobehavioral and histological assessment. The long-term effect of cilostazol on cerebral ischemia was analyzed in the non-rtPA cohort.ResultsIn the non-rtPA cohort, pretreatment by cilostazol significantly decreased the endothelial expression of adhesion molecules (P-selectin and intercellular adhesion molecule-1) and prevented platelet aggregation and leukocyte plugging in the microvessels after cerebral ischemia/reperfusion in the acute phase. Cilostazol significantly reduced mortality rate and improved motor function at 7 days post-ischemia/reperfusion. In the rtPA cohort, cilostazol significantly suppressed edema formation and hemorrhagic transformation with reduced density of microglial cells positive for matrix metalloproteinase-9 in the cerebral cortex and the striatum. In both cohorts, cilostazol significantly suppressed focal no-reflow, mitigated cerebral infarct, and improved neurological outcome.ConclusionsCilostazol may possess protective properties against cerebral ischemic injury by preventing no-reflow and hemorrhagic transformation, via maintenance of microvascular integrity.     

Glutamine prevents intestinal mucosal injury induced by cyclophosphamide in rats

Purpose  

High doses of anticancer drugs often damage the intestinal mucosa. The purpose of the present study was to examine the effect of glutamine on mucosal damage induced by cyclophosphamide in a rat model, and to elucidate the mechanisms responsible for its protective effects.     

Difficulty of diagnosing Wegener's granulomatosis in the head and neck region

Objective

The objective of this study was to review the various clinical features associated with Wegener's granulomatosis (WG) in the head and neck region and to discuss the difficulty of diagnosing patients with early stage WG.

Methods

Between January 1998 and August 2007, WG was diagnosed and treated in 16 patients at the Department of Otolaryngology, Hyogo College of Medicine. Clinical and operating records of these patients were analyzed retrospectively. Diagnosis was based on the Japanese criteria proposed by the Japanese Ministry of Health and Welfare in 1998.

Results

Ten patients (62.5%) had a definite diagnosis of WG, and the other six patients (37.5%) had a probable diagnosis of WG. The period from the onset to diagnosis was between 1 month and 30 years. The generalized form of WG was observed in three patients (18.8%), and the limited form of WG was observed in the other 13 patients (81.2%). Nasal, aural, and ophthalmic symptoms were initially presented in 10, 3, and 3 patients, respectively. Cytoplasmic pattern antineutrophil cytoplasmic antibodies (cANCAs) and perinuclear pattern ANCA (pANCA) were positively detected in 68.8% (11/16) and 27.2% (3/11) of the patients, respectively. Five of 14 patients (35.7%) had pathologic features of WG in biopsy samples from the head and neck region. Three patients in whom a diagnosis of WG was difficult are presented, and immediate lessons of our experience were discussed.

Conclusions

This study emphasized the difficulty of diagnosing WG, particularly at an early stage and when limited to the head and neck region. The biggest challenge faced in diagnosing WG is that it requires a high index of suspicion. When WG was suspected, we should obtain an accurate medical history from patients and repeat serologic and histopathologic examinations.     

Efficacy of Combination Antifungal Therapy with Intraperitoneally Administered Micafungin and Aerosolized Liposomal Amphotericin B against Murine Invasive Pulmonary Aspergillosis

Targeted intrapulmonary delivery of drugs may reduce systemic toxicity and improve treatment efficacy. In the current study, we evaluated the effects of a combination treatment consisting of inhalation of aerosolized liposomal amphotericin B (L-AMB) with intraperitoneal administration of micafungin (MCFG) against murine invasive pulmonary aspergillosis. The combination of aerosolized L-AMB with intraperitoneal MCFG significantly improved the survival rate, and the fungal burdens and histopathology findings after this treatment were superior to those of the control and both monotherapy groups.Invasive pulmonary aspergillosis (IPA) results in significant morbidity and mortality in severely immunocompromised patients (6). Targeted intrapulmonary delivery of antifungals has the potential to reduce systemic toxicity and improve treatment efficacy as well as prophylaxis (1, 8) and may be used as an optional route in combination with other systemic antifungals. In the current study, we evaluated the efficacy of aerosolized liposomal amphotericin B (L-AMB) both singly and in combination with intraperitoneally administered micafungin (MCFG) in a murine model of IPA.Aspergillus fumigatus MF13 was clinically obtained from a patient admitted to the Nagasaki University Hospital. The minimum effective concentration of MCFG (Astellas Pharmaceuticals Inc., Tokyo, Japan) and the MIC of AMB (Sigma, St. Louis, MO) were determined using the microdilution method in accordance with Clinical Laboratory Standards Institute document M38-A2 (2). Drug interactions were assessed using the checkerboard titration broth microdilution-based method (3), and the fractional inhibitory concentration index was determined as previously described (5).Six-week-old female ICR mice (Charles River Breeding Laboratories, Shiga, Japan) were immunosuppressed and then challenged on day 0 with 5 × 10⁶ conidia of A. fumigatus MF13 intratracheally for monitoring of survival, as previously described (7, 11). Eight-week-old female ICR mice were used to determine fungal burdens and for histopathological examination. Mice were immunosuppressed by subcutaneous injection of cortisone acetate (Sigma, Tokyo, Japan) at 250 mg/kg of body weight and intraperitoneally administered cyclophosphamide (Sigma) at 200 mg/kg on days −2 and 0 for the survival study. Only cortisone acetate (200 mg/kg) was used on days −1, 0, and 1 for fungal-burden analysis and histopathological examination. Mice were assigned into the following groups: (i) control mice, (ii) mice receiving MCFG intraperitoneally, (iii) mice receiving aerosolized L-AMB, and (iv) mice receiving a combination treatment of intraperitoneally administered MCFG and aerosolized L-AMB. Each group consisted of 11 and 10 mice for survival and fungal-burden analyses, respectively. MCFG was administered intraperitoneally once daily at 1 mg/kg/day. L-AMB was administered once daily in an 8-ml suspension (at 1.2 mg/ml) per inhalation. Antifungals were initiated 16 h after inoculation and continued for 5 and 3 days for survival and fungal-burden analyses, respectively. The L-AMB solution was aerosolized using a nebulizer (Muromachi Kikai Co., Ltd., Tokyo, Japan), and mice were exposed to aerosol treatment for 60 min as previously described (9). Control mice were treated with sterile saline. Survival was observed until 11 days following the challenge. For fungal-burden and histopathological examinations, mice were sacrificed 4 h after the treatment on day 3. Numbers of CFU per lung tissue were calculated, and removed lungs were fixed and stained with Grocott''s methenamine silver nitrate and hematoxylin-eosin as previously described (11). Survival and fungal burden data are presented from a combination of two sets of experiments. The concentration in blood and the pharmacokinetics of aerosolized L-AMB were evaluated. Uninfected mice were also exposed to several concentrations of aerosolized L-AMB for 5 days, and blood samples and lungs were collected. AMB concentration was quantified as previously described (10). Survival curves were generated using the Kaplan and Meier method, and statistical differences were evaluated by the log rank test. To assess fungal burden in lung tissue, geometric means of numbers of CFU per organ were compared by Student''s t test. Statistical significance was defined as a P of <0.05.The MIC of AMB against A. fumigatus MF-13 was 1.0 μg/ml, and the minimum effective concentration of MCFG was 0.0315 μg/ml. The fractional inhibitory concentration index of AMB and MCFG was 1.5, and drug interaction was classified as indifferent (5).Survival periods of monotherapy groups, in which mice either were treated with intraperitoneally administered MCFG or inhaled aerosolized L-AMB were significantly longer than that of the control group (MCFG alone versus the control, P = 0.006; L-AMB versus the control, P < 0.001) (Fig. ​(Fig.1).1). The combination treatment group showed significantly longer survival than the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.037), and control (P < 0.001) groups. Numbers of CFU in the lungs of mice in the combination treatment group were significantly reduced compared to those in each of the intraperitoneal-MCFG (P < 0.001), aerosolized-L-AMB (P = 0.027), and control (P < 0.001) groups (Fig. ​(Fig.2).2). The lungs of aerosolized-L-AMB-administered and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than the intraperitoneal-MCFG and control groups (Fig. ​(Fig.3).3). The mean AMB concentrations in the lung tissue following L-AMB inhalation at 1.2, 2.6, and 4.0 mg/ml were 35.5, 73.2, and 94.2 μg/g, respectively. Recorded levels in sera were 0.02, 0.06, and 0.06 μg/ml when inhaled-L-AMB suspensions were administered at 1.2, 2.6, and 4.0 mg/ml, respectively.Open in a separate windowFIG. 1.Survival curves for mice with IPA (Kaplan-Meier plot). Groups of 11 mice were treated with a combination of intraperitoneal administration of MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [open squares]), inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml [filled triangles]), intraperitoneal administration of MCFG (1 mg/kg/day [open triangles]), and no therapy (control [filled circles]). *, P < 0.05 versus the control; **, P < 0.05 versus the control group, intraperitoneal-MCFG group, or aerosolized-L-AMB group (log rank test). The survival times for all treatment groups were longer than that for controls (P < 0.05). The survival time for the combination treatment group was significantly longer than those of the intraperitoneal-MCFG group and the aerosolized-L-AMB group (P < 0.05).Open in a separate windowFIG. 2.Numbers of CFU from homogenized lung tissues of mice with IPA. Groups of 10 mice were treated once per day with a combination of intraperitoneally administered MCFG (1 mg/kg/day) and inhalation of aerosolized L-AMB (8 ml at 1.2 mg/ml), aerosolized L-AMB (8 ml at 1.2 mg/ml), intraperitoneal MCFG (1 mg/kg/day), and saline (control). CFU counts, as a parameter of A. fumigatus burden in the lungs of IPA mice at 4 h after day 3 of treatment, are shown. *, P < 0.05 (Student''s t test).Open in a separate windowFIG. 3.Histopathology of lung tissues. Both lungs were obtained from IPA mice 4 h after 3 days of treatment with a combination of intraperitoneally administered MCFG and inhalation of aerosolized L-AMB, aerosolized L-AMB, intraperitoneal MCFG, and saline alone as a control. The lungs obtained from aerosolized-L-AMB-treated and combination treatment mice showed obviously smaller numbers of hyphae and fewer foci of inflammation than intraperitoneal-MCFG and control mice. HE, hematoxylin-eosin; GMS, Grocott''s methenamine silver nitrate stain.The current study demonstrated the efficacy of monotherapy of aerosolized L-AMB in a murine IPA model. The AMB concentrations in lung tissue in our study were relatively higher but extremely lower in serum than those from another report of a murine model of intravenously administered L-AMB, although experimental conditions were not the same (10). These results suggested that systemic toxicity generally caused by AMB treatment may be reduced by L-AMB inhalation therapy.The effect of combined intraperitoneal-MCFG and aerosolized-L-AMB treatment was an enhanced survival rate, even though this drug interaction was classified as indifferent in vitro. Since 78% of all control mice died in first 3 days in a survival analysis, we changed the experimental conditions for analysis of fungal burden and histopathological examination. In this model, no mice died before euthanasia, a prerequisite for the organ CFU assay. Both fungal-burden data and histopathological findings supported the survival data in our study.Unlike in our study, Graybill et al. previously reported that combination therapy demonstrated a lack of synergistic effects following intravenous-L-AMB and intraperitoneal-MCFG treatment in a model of murine IPA (4). These discrepancies are likely due to differences between our model and Graybill et al.''s model, including (i) the route of infection, (ii) the status of immunosuppression, and (iii) the administration route of antifungal drugs. These differences also suggest that targeted intrapulmonary delivery of drugs by inhalation raises the drug concentration at the active site of infection in the lungs, thus contributing to the efficacy of combination therapy. Further comparative efficacy studies in a clinical setting are warranted.     

Comparison of 3.0-and 1.5-tesla diffusion-weighted imaging in the visibility of breast cancer

Purpose The aim of this study was to compare diffusion-weighted imaging (DWI) at 3.0 T and 1.5 T by evaluating the apparent diffusion coefficient (ADC) value and visibility of breast cancer in the same patients. Materials and methods A total of 13 patients (16 lesions) with breast cancer underwent DWI at 3.0 T and 1.5 T. Tumors were classified into two groups based on the lesion size. The ADC values were measured, and visibility of the tumors was scored blindly. Results No significant difference was found for ADC values between 3.0 T and 1.5 T in either group (P > 0.05). All of the large lesions were visible clearly at both magnetic field strengths, and image scores were not different (P > 0.05). In contrast, small lesions were more clearly visible and had better image scores at 3.0 T than at 1.5 T (P < 0.001). Conclusion Small cancers were more clearly visible on DWI at 3.0 T than 1.5 T.     

Molecular Cloning and Heterologous Expression of a Biosynthetic Gene Cluster for the Antitubercular Agent d-Cycloserine Produced by Streptomyces lavendulae

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding d-alanyl-d-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, l-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-l-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-l-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the l-arginine derivative, N^G-hydroxy-l-arginine. The resulting O-ureido-l-serine is then racemized to O-ureido-d-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-d-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.The soil-dwelling genus Streptomyces undergoes a complex morphological differentiation and produces an enormous variety of bioactive secondary metabolites. Because they include clinically useful antibiotics and immunosuppressants, the genus Streptomyces occupies an important position as an industrial microorganism.d-Cycloserine (DCS), a cyclic structural analogue of d-alanine, is produced by “Streptomyces garyphalus” and Streptomyces lavendulae. This antibiotic is used as an antitubercular agent (21). Since DCS is similar to d-alanine, it prevents the action of both alanine racemase and d-alanyl-d-alanine ligase, which are necessary for the biosynthesis of a bacterial cell wall. Thus, DCS functions as an inhibitor of bacterial cell wall biosynthesis (16, 19). Although the structure of DCS is very simple, the biosynthetic genes for DCS have never been cloned until now.In general, antibiotic biosynthetic genes form a cluster and are adjacent to their self-resistance genes. We have previously cloned a gene (orfB) that confers resistance to DCS on Streptomyces lividans and Escherichia coli from DCS-producing S. garyphalus (CSH) 5-12 (17). The sequence analysis suggests that the gene may encode a membrane protein that is necessary for the excretion of the DCS outside the cell. We have found that the same gene is also present in S. lavendulae ATCC 25233 (20). We have also previously demonstrated that d-alanyl-d-alanine ligase, which is a target enzyme of DCS, functions as a self-resistance determinant in S. lavendulae ATCC 25233 (20). Interestingly, the gene encoding d-alanyl-d-alanine ligase, designated ddlS, was located just upstream of the putative membrane protein gene, orfB (20). Although the self-resistance genes in the DCS producers have been thoroughly analyzed, no attempt to clone the DCS biosynthetic genes has been carried out yet.To clone the biosynthetic genes for DCS from another DCS-producing S. lavendulae strain, ATCC 11924, we first investigated whether this strain harbors orfB and ddlS. Since both genes were found to be conserved, the flanking region was cloned from the chromosomal DNA. Here, we hypothesize about the biosynthetic pathway for DCS on the basis of our present data and previous studies by another research group (23, 24). The present study is the first report identifying the biosynthetic gene cluster for DCS.     

Bacterial contamination of stock solutions in storage cases for contact lens, and the disinfectant-resistance of isolates

To investigate the bacterial contamination of preservative solutions for contact lenses, contact lens cases were swabbed and the swabs were cultured. Various bacteria were isolated from 26 of 100 samples (26.0%). By preservative solution, the bacterial presence was low (16.7%) in the 30 samples from cases using ReNu, which was the most frequently employed solution, and 27.3% in 11 samples from cases using Complete, which was the second most frequently employed solution. Of 34 strains isolated and identified, gram-negative bacilli-glucose non-fermenting bacteria were the most frequently isolated, accounting for 61.8% (21 strains); particularly, 10 isolates were Stenotrophomonas maltophilia, accounting for 29.4%. The biofilm-forming ability of these isolates was investigated by staining. The mean absorbance of the gram-negative rods was 0.369, which was about 3 times higher than that (0.107) of the Staphylococcus group, confirming marked biofilm-forming ability. In addition, the bactericidal effects of the preservative solutions on the isolates were investigated. The effect varied among the preservative solutions in the suspension experiment, but the highest disinfection rate, which was achieved by Complete, was 99.9->99.99%, showing a favorable bactericidal effect. In contrast, none of the 3 test preservative solutions showed any bactericidal effect in the adhesion experiment.     

Identification of Legionella londiniensis isolated from hot spring water samples in Shizuoka, Japan, and cytotoxicity of isolates

As part of an epidemiological study on legionellosis, we attempted to isolate Legionella spp. from hot spring water and were able to isolate L. londiniensis HYKF-90505 (=JCM 16338), confirming that L. londiniensis inhabits hot spring water in Japan. To investigate the disease potential of L. londiniensis, we examined its ability to grow intracellularly within Acanthamoeba sp. JAC/E1 strain. The isolated HYKF-90505 was able to grow within Acanthamoeba sp. JAC/E1 strain, and we confirmed also that the HYKF-90505 strain showed cytotoxicity for cultured cells such as J774.1 (JCRB0018). However, in a culture of human U937 cells, the bacterial count was not increased by the intracellular growth of the HYKF-90505 strain. Cells infected for 24 h and stained using the Giménez method showed no intracellular growth of the HYKF-90505 strain. Thus, the isolate appears to be weakly pathogenic to humans.     

Expression of epithelial growth factor receptor and its two ligands, transforming growth factor-alpha and epithelial growth factor, in normal and neoplastic squamous cells in the vulva: an immunohistochemical study

Epithelial growth factor receptor (EGFR) sends signals to the proliferation signal transduction system, receiving two ligands: epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This immunohistochemical study examined the roles of EGFR and its ligands in the proliferation of normal and neoplastic vulvar squamous cells in 25 patients with vulvar squamous cell carcinoma (VSCC), 10 patients with vulvar condyloma acuminata (VCA), 15 patients with vulvar intra-epithelial neoplasm I-II or III (VIN I-II or III), and 5 subjects with vulvar normal squamous cells (VNSC). EGFR was detected in a few basal cells in 40% of the VNSC, in highly dysplastic cells in 40% of the VIN III, in many neoplastic cells in 80% of the VCA, and in some malignant cells in 64% of the VSCC. EGF was seen in the cytoplasm in 20% of the VIN I-II, 100% of the VIN III, 100% of the VCA, and 100% of the VSCC. Diffuse TGF-alpha was weakly expressed in the cytoplasm in 100% of the VNSC, more intensely in 100% of the VIN and 100% of the VCA, and intensely in 100% of the VSCC. These findings led to the suggestion that the TGF-alpha-EGFR system maintains the growth of normal squamous cells and, in part, maintains the growth of dysplastic and neoplastic squamous cells in the vulva. EGF expression was an early sign of neoplasia. The expression of EGFR with overexpression of its two ligands contributed to the proliferation of dysplastic and neoplastic squamous cells in VIN III and VCA. EGFR expression appeared to contribute to essential neoplastic abnormalities in 64% of the VSCC.