Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Prognostic significance of CD45RO+ memory T cells in renal cell carcinoma.

BACKGROUND: Memory T cells are well known to have a critical role for host defense in humans. However, their role in actual human cancer remains largely unknown. In this study, we tried to reveal the clinical importance of tumour-infiltrating CD45RO+ memory T cells in renal cell carcinoma (RCC). METHODS: We analysed 105 patients with RCC, who received radical or partial nephrectomy. Those were 65 in TNM stage I, 7 in stage II, 15 in stage III, and 18 in stage IV, respectively. CD45RO expression was evaluated by immunohistochemistry. CD4 and CD8 expressions were also systematically assessed in the same manner. RESULTS: Patients with higher TNM stage or high nuclear grade were found to have higher densities of CD45RO. Furthermore, CD45RO status was positively correlated with preoperative C-reactive protein level. In prognostic analysis, CD45RO+lo patients had a significantly better prognosis than CD45RO+hi patients. There was also a significant difference between CD4+lo and CD4+hi groups, whereas no significant difference was observed in CD8 T-cell status. Finally, multivariate analysis revealed that CD45RO+ status was the independent prognostic factor for patient overall survival. CONCLUSION: CD45RO+ memory T-cell status has a significant independent prognostic value, indicating that the adaptive immune response is functionally critical in human RCC.

Computerized method for estimation of the location of a lung tumor on EPID cine images without implanted markers in stereotactic body radiotherapy.

The purpose of this study was to develop a computerized method for estimation of the location of a lung tumor in cine images on an electronic portal imaging device (EPID) without implanted markers during stereotactic body radiotherapy (SBRT). Each tumor region was segmented in the first EPID cine image, i.e., reference portal image, based on a multiple-gray level thresholding technique and a region growing technique, and then the image including the tumor region was cropped as a 'tumor template' image. The tumor location was determined as the position in which the tumor template image took the maximum cross-correlation value within each consecutive portal image, which was acquired in cine mode on the EPID in treatment. EPID images with 512 x 384 pixels (pixel size: 0.56 mm) were acquired at a sampling rate of 0.5 frame s(-1) by using energies of 4, 6 or 10 MV on linear accelerators. We applied our proposed method to EPID cine images (226 frames) of 12 clinical cases (ages: 51-83, mean: 72) with a non-small cell lung cancer. As a result, the average location error between tumor points obtained by our method and the manual method was 1.47 +/- 0.60 mm. This preliminary study suggests that our method based on the tumor template matching technique might be feasible for tracking the location of a lung tumor without implanted markers in SBRT.

Exogenous mycoplasmal p37 protein alters gene expression, growth and morphology of prostate cancer cells.

We previously showed that the Mycoplasma hyorhinis-encoded protein p37 can promote invasion of cancer cells in a dose-dependent manner, an effect that was blocked by monoclonal antibodies specific for p37. In this study, we further elucidated changes in growth, morphology and gene expression in prostate cancer cell lines when treated with exogenous p37 protein. Incubation with recombinant p37 caused significant nuclear enlargement, denoting active, anaplastic cells and increased the migratory potential of both PC-3 and DU145 cells. Microarray analysis of p37-treated and untreated cells identified eight gene expression clusters that could be broadly classified into three basic patterns. These were an increase in both cell lines, a decrease in either cell line or a cell line-specific differential trend. The most represented functional gene categories included cell cycle, signal transduction and metabolic factors. Taken together, these observations suggest that p37 potentiates the aggressiveness of prostate cancer and thus molecular events triggered by p37 maybe target for therapy.

Effect of Gender Differences on Transplant Kidney Function.

BACKGROUND: Transplant kidney function is thought to be affected by sex differences, such as physical conditions including muscle volume, sex hormones, immune responses, and so forth. We examined the effect of sex differences on transplant kidney function. METHODS: The subjects were selected from kidney transplant recipients, who received kidney transplantation on our hospital between January 2000 and August 2015. Cadaveric donors and parent-child pairs with an age difference were excluded, then we included 47 recipients whose sex was different from the sex of the donor. We compared transplant kidney function between male donors and female recipients group (M→F, n = 20) and female donors and male recipients group (F→M, n = 27). RESULTS: Nadir creatinine value was higher in the F→M group than in the M→F group (1.09 mg/dL vs 0.76 mg/dL, P < .0001). The estimated glomerular filtration rate (eGFR) was significantly higher in the M→F group than in the F→M group (66.6 mL/min/1.73 m2 vs 50.1 mL/min/1.73 m2, P = .002), and eGFR ratio (recipient to donor) was significantly higher in the M→F group than in the F→M group (1.13 vs 0.57, P < .0001). Multiple linear regression analysis showed that the only the sex of the recipient was significant prognostic factor of eGFR after renal transplantation (P = .037). CONCLUSIONS: The short-term kidney function of the graft from male to female was better than that of the graft from female to male.

Hepatic alpha(2u)-globulin mRNA levels and diethylstilbestrol-associated testicular atrophy in rats.

Biosynthesis of alpha(2u)-Globulin (alpha(2u)-g) is under multihormonal regulation. In this study, we investigated histopathologic changes in the testis and hepatic alpha(2u)-g messenger ribonucleic acid (mRNA) levels in male rats after administration of the potent estrogen diethylstilbestrol (DES) at 0. 01, 0.1, or 1 mg/kg/day by gavage for 14 days. DES treatment decreased hepatic alpha(2u)-g mRNA levels in a dose-dependent manner accompanied by atrophic histopathologic changes in the testis. In addition, alpha(2u)-g mRNA levels were lowest in animals with the most marked testicular changes. Hepatic alpha(2u)-g mRNA may be a useful biomarker for the evaluation of endocrine disruption in male rats.

[Single-dose toxicity studies of tazobactam/piperacillin and tazobactam].

Tazobactam (TAZ) is a newly developed beta-lactamase inhibitor. Tazobactam/Piperacillin (TAZ/PIPC) is a formulation consisting of TAZ and PIPC in a ratio of 1:4. Singe-dose toxicity studies in TAZ/PIPC and TAZ were carried out using mice and rats of both sexes and male dogs. The results were as follows. 1. A common clinical sign in mice and rats administered TAZ/PIPC or TAZ by all routes was soft stool. Other signs in mice and rats included a decrease in spontaneous motor activity and/or a decreased respiratory rate for the intraperitoneal (i.p.), subcutaneous (s.c.) or intravenous (i.v.) route. The animals administered by the i.v. route showed tremor for mice and clonic convulsion for rats before death. Hyperemia, hemorrhage or edema of the lung, and hemorrhage of the digestive tract were observed in these animals at necropsy. An enlargement of the spleen was seen in some of the surviving animals treated with TAZ/PIPC. 2. In dogs, TAZ/PIPC caused vomiting, and TAZ caused vomiting, respiratory abnormality, soft stool and diarrhea by the intravenous (i.v.) administration. 3. TAZ/PIPC or TAZ caused clinical signs such as the loss of hair at the injection site for the s.c. route, and necrosis of the tail for the i.v. route in mice and rats, also caused limping of the injected anterior limb in dogs. Necrosis and hemorrhage at the injection site, and peritonitis by the i.p. injection were observed at necropsy. These findings were due to the irritation of TAZ/PIPC or TAZ.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation and determination of P-type-alpha amylase activity in human serum by monoclonal antibody].

We evaluated a kinetic procedure for determining pancreatic type of alpha-amylase isoenzyme (EC 3.2.1.1.) by using monoclonal salivary amylase antibody in human serum. The principle of this method is to measure residual pancreatic amylase activity after the reaction of monoclonal anti-salivary amylase antibody which is specifically inactivated human salivary amylase. The reaction of antibody with salivary alpha-amylase completed within 2 min of incubation and residual activity of pancreatic amylase was 100%, and the residual salivary amylase activity showed less than 3% of salivary amylase activity after incubation with antibody. The reproducibility and/or precision was 1.2% to 3.4% and the linearity obtained up to 1.500 U/l. Correlation coefficient between the proposed method (y) and electrophoresis (x1) and wheat germ inhibition method (x2) was well, respectively (r = 0.99 with both method, the regression curves was y = 1.02 x1-0.7 and y = 1.04x2-0.7). The reference value from healthy adults (male 98 and female 102) showed 13.6-48.2 U/l according to statistical parametric method and sex difference was not observed. The monoclonal anti-salivary amylase antibody was not cross-reacted with the other animal serum salivary type of amylase.

Can a belly board reduce respiratory-induced prostate motion in the prone position?--assessed by cine-magnetic resonance imaging.

The purpose of this study is to evaluate the real-time respiratory motion of the prostate and surrounding tissues/organs in the supine and prone positions and to investigate, using cine-MRI, whether a belly board can reduce respiratory-induced motion in the prone position. Cine-MRI scans were made of 13 volunteers in the supine and prone positions on a flat board and in two different prone positions using a belly board. Images in cine mode were recorded for 20 seconds. For each session, the points of interest (POIs) were located at the apex, base, mid-anterior surface and mid-posterior surface of the prostate; the tip of the seminal vesicle; the pubic symphysis; and the sacrum. The maximum range and standard deviation (SD) of the displacement from the mean value were calculated. The SDs for each of the four different positions were compared using a paired t-test. Respiratory-induced prostate motion was significantly larger in the prone position than in the supine position. However, when a belly board was used in the prone position, motion in the prostate and surrounding tissues/organs was significantly reduced. There were no significant differences between the two different positions using a belly board in any of the POIs.

SU-E-J-26: Automated Estimation Method of Patient Setup Errors Using Simulated Portal Images for Prostate Cancer Radiotherapy.

PURPOSE: We developed a novel automated estimation method for patient setup errors based on simulated and real portal images for prostate cancer radiotherapy. METHODS: The estimation of patient setup errors in this study was based on a template matching technique with a cross-correlation coefficient and Sobel filter between the real portal image and localized pelvic template of reference image, which were DRR (digitally reconstructed radiography) images and simulated portal images. The simulated portal image was derived by projecting a CT image according to an inverse exponential power law of x-ray attenuation for a water-equivalent path length of each voxel of the CT image on each ray from a source to each pixel on the EPID (electric portal imaging device). A localized pelvic template of each patient in AP (anterior-posterior) or lateral view was automatically extracted from the DRR or simulated portal images by cropping a rectangular region, which was determined by using the mean pelvic template and four anatomical feature points. We applied the proposed method to three prostate cancer cases, and evaluated it using the residual error between the patient setup error obtained by proposed method and the gold standard setup error determined by two radiation oncologists. RESULTS: The average residual errors of the patient setup error for the DRR and simulated portal images were 0.79 and 1.26 mm in the left-right (LR) direction, 3.17 and 2.05 mm in the superior-inferior (SI) direction, 1.69 and 5.82 mm in the anterior-posterior (AP) direction, 3.84 and 6.94 mm in Euclidean distance (ED), respectively. If we used the simulated portal image for LR and SI directions and the DRR image for AP direction, the Euclidean distance was 3.22 mm. CONCLUSIONS: The proposed method has a potential to correctly estimate patient setup errors for prostate cancer radiotherapy.

SU-E-J-117: Computer-Assisted Verification of Accumulated Dose Distribution during the Treatment Time Based on Estimation of Four-Dimensional Dose Distribution Using an Electronic Portal Imaging Device.

PURPOSE: The accumulated dose distributions during the course of radiation treatment are substantially important for verifying whether treatment dose distributions are produced according to planned dose distributions. The purpose of this study was to develop a computer-assisted verification method of accumulated dose distribution during the irradiation of a tumor based on estimation of four-dimensional (4D) dose distribution using an electronic portal imaging device (EPID). METHODS: The 4D 'treatment' computed tomography (CT) images during the irradiation were estimated based on affine transformations including respiratory motions, which were derived by registration between a planning portal dose image and treatment portal dose dynamic image. Planning portal dose images were calculated from planning CT images and an algorithm for calculation of dose spatial distribution. Treatment portal dose images were estimated from EPID dynamic images obtained during a treatment time. The planning portal dose images were registered to the treatment portal dose images to obtain the affine transformation, which could include respiratory motion in a patient body. The CT images at a treatment time were determined by deforming the planning CT images using the affine transformation matrix. 4D dose distributions during a treatment delivery were obtained by applying a dose calculation algorithm to the 4D treatment CT images. Finally, accumulated dose distributions during the course of radiation treatment were verified with planned dose distributions. RESULTS: We applied the proposed method to EPID dynamic images of 2 lung cancer patients, and evaluated the difference in accumulated dose distribution between the plan and treatment using a gamma evaluation (3mm/3%). The average pass rate for 2 cases was 78.2%. CONCLUSIONS: The proposed method can be used for adaptively modifying the plan based on the dose discrepancy between the plan and treatment. This work was partially supported by Grant-in-Aid for Scientific Research (C) (22611011) and Okawa Foundation for Information and Telecommunications.

Hypoplasia of the adenohypophysis in a Sprague-Dawley rat.

Pathological examination performed on a male 70-day-old Sprague-Dawley rat revealed formation of a large cyst in the hypophysis. The cyst, which completely occupied the adenohypophyseal area, was lined by the epithelial cells. Small masses of the adenohypophyseal cells were occasionally seen to sprout from the cyst wall epithelium.

Mammary adenocarcinoma in a young female hypercholesterolemic rat.

Pathological examination of a 12-week-old female rat with hypercholesterolemia revealed mammary adenocarcinoma. Grossly, one grayish-white nodule was found in the hypoderm near the right posterior mammary gland. Histologically, the nodule was composed of variable-sized epithelial cells which lined the tubules and ducts in one to several layers, accompanied by proliferation of connective tissue in the stroma. Mitotic figures, necrosis and hemorrhage appeared frequently throughout the lesion. The tumor cells had invaded the surrounding muscular tissue.

Changes in serum alpha2u-globulin levels in male rats given diethylstilbestrol and applicability to a screening test for endocrine-disrupting chemicals.

alpha2u-Globulin (AUG) is a major rat urinary protein, which has a molecular weight of 16 kDa (kidney type) or 19 kDa (native type). The biosynthesis of this protein is under multi-hormonal regulation. In this study, we investigated changes in serum AUG level and their association with changes in the reproductive organs of male rats after the administration of the estrogenic chemical, diethylstilbestrol (DES) at doses ranging from 0.01 mg/kg per day to 100 mg/kg per day by gavage for 14 days. Our aim was to establish basic data for the development of a new screening method for endocrine disrupting chemicals based on serum AUG levels. DES treatment decreased the weight of testes in a dose-dependent manner; and was accompanied by atrophic histopathological changes in testes. Testis weights were significantly decreased by the group given 1 mg/kg per day DES; however, histopathological abnormalities were found in the group given 0.1 mg/kg per day DES. In four of five animals in the group given 1 mg/kg per day there was no significant decrease in testis weight and only a slight or moderate degeneration of the pachytene spermatocytes. Despite these findings, serum AUG levels in this group decreased markedly, while the serum AUG level markedly decreased even in the animals with no histopathological change in the 1 mg/kg per day or 0.1 mg/kg per day groups with no histopathological change also showed decreased serum AUG level. These results suggest that the serum AUG level may be a sensitive parameter for detecting the activity of estrogenic chemicals in intact male rats. Although a uterotropic assay has been proposed for immature female or ovariectomized female rats and is currently undergoing validation studies internationally, there is no screening method for estrogenic chemicals in intact male animals. More data on AUG changes by treatment with other estrogenic chemicals are needed in order to determine the sensitivity and specificity of this response to estrogens. Nonetheless, an AUG-based screening test for estrogenic chemicals may be useful owing to its applicability to conventional toxicity studies and an apparently higher sensitivity of this parameter compared to organ weight change or histology of testis in intact male rats and applicability to conventional toxicity studies.