Excellent results of immunocomplex capture fluorescence analysis-I for cross-match test in renal transplantation.

A flow cytometry cross-match (FCXM) test is the gold standard for detection of human leukocyte antigen (HLA) antibodies in renal transplantation because of its high sensitivity. However, this technique can produce false-positive results when non-HLA antibodies or low-titer donor-specific antibodies (DSA) are detected. To determine the clinical relevance of the recently introduced novel cross-match test termed immunocomplex capture fluorescence analysis (ICFA), we retrospectively compared the results of ICFA and FCXM, including a single-antigen bead test for detection of DSA in renal transplant recipients. We found a correlation of 71.4% (235/329) between the results of ICFA-I and FCXM-T, whereas that between ICFA-II and FCXM-B was 41.1% (134/326). Ninety-four patients were ICFA-I negative and FCXM-T positive, and 188 were ICFA-II negative and FCXM-B positive, whereas 46.8% (44/94) and 61.7% (116/188) were found to be DSA-I and DSA-II negative, respectively, which classified them into the non-HLA antibody and low-titer DSA groups, respectively. The mean value of molecules of equivalent soluble fluorochrome for DSA-I was 22,994 in the ICFA-I-positive group, which was significantly higher than 2117 in the negative group (P < .0001), whereas there was no significant difference for DSA-II between the ICFA-II-positive and ICFA-II-negative groups. Graft survival in the ICFA-I-negative group was significantly higher than that in the ICFA-I-positive group (P = .0058). Our results indicate that ICFA-I does not respond to non-HLA antibodies or low-titer DSA, which have influence on graft survival. Therefore, this novel hybrid test, which combines cross-match testing and HLA antibody detection functions, may be useful for clinical pretransplantation evaluation of renal transplantation patients.

Intraoperative fluorescence vascular imaging using indocyanine green for assessment of transplanted kidney perfusion.

INTRODUCTION AND OBJECTIVE: Indocyanine green (ICG) emits infrared light with exposure to laser light. When intravenously injected, it binds to plasma proteins and predominantly persists in the vasculature, which is very useful for definition of the vascular network. The HyperEye Medical System (HEMS; Mizuho Ikakogyo Co., LTD, Tokyo, Japan) is a new device able to identify both near-infrared and visible rays "in situ" without needing to dim the operation room lighting. We speculated that intraoperative ICG imaging would be applicable for kidney transplantation, by providing "in situ" determination of successful vascular anastomosis. MATERIALS AND METHODS: Four patients underwent intraoperative ICG imaging following intravenous administration of 1 mL of a solution containing 0.25% ICG. After performing vascular anastomosis, the allograft was examined using the HEMS light source device. Fluorescent signals were transmitted to a digital video processor connected to a television monitor and evaluated in real time. RESULTS: In all 4 patients, intraoperative ICG imaging provided excellent resolution of blood flow at each step in real time, namely, coming from the recipient's artery to the allograft renal artery, circulating throughout the whole grafted kidney, and draining through the allograft renal vein to the recipient's vein. HEMS provides ICG fluorescence image in color, allowing surgeons to clearly discriminate the positional relationship between the target tissue and the surrounding tissue. No complications associated with ICG injection were noted. CONCLUSION: Our preliminary results indicate that HEMS is a feasible and safe ICG imaging system that helps prevent technical failure during vascular anastomosis, and also demonstrates blood supply to the grafted kidney.

Superoxide dismutase analog (Tempol: 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) treatment restores erectile function in diabetes-induced impotence.

We hypothesized that the administration of the superoxide dismutase (SOD) mimetic Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) may reverse diabetes-induced erectile dysfunction. To test this hypothesis, reactive oxygen species-related genes (SOD1, SOD2, GP x 1, CAT, NOS2, NOS3) were tested, erectile functional studies and immunohistochemical analysis were carried out in diabetic rats treated with or without Tempol. Thirty Sprague-Dawley (3-4 months old) rats were divided into three groups (n=10 each), 20 with diabetes (diabetic control and Tempol treatment) and 10 healthy controls. At 12 weeks after the induction of diabetes by streptozotocin and Tempol treatment, all groups underwent in vivo cavernous nerve stimulation. Rat crura were harvested and the expression of antioxidative defense enzymes were examined by semi-quantitative reverse transcriptase PCR (RT-PCR). To confirm the RT-PCR results, we carried out immunohistochemistry (IHC) for catalase (CAT) and iNOS (NOS2). Nitration of tyrosine groups in proteins was also examined by IHC. Mean intracavernous pressure in the diabetic group was significantly lower than in the healthy controls (P <0.001) and was reversed by Tempol treatment (P <0.0108). NOS2 protein expression was significantly increased in diabetic animals compared with healthy controls and Tempol restored NOS2 protein level. Nitrotyrosine was also higher in diabetic animals and although Tempol treatment decreased its formation, it remained higher than that found in healthy controls. This study suggests that Tempol treatment increased erectile function through modulating oxidative stress-related genes in diabetic rats. This is the first report about the relationship between diabetes-induced erectile dysfunction and oxidative stress, and antioxidative therapy using the superoxide dismutase mimetic, Tempol, to restore erectile function.

Knockdown of astrocyte-elevated gene-1 inhibits prostate cancer progression through upregulation of FOXO3a activity.

Astrocyte-elevated gene-1 (AEG-1) has been reported to be upregulated in several malignancies and play a critical role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase/AKT signaling pathway. However, the role of AEG-1 in prostate cancer (PC) has never been reported. We now show that AEG-1 is overexpressed in clinical PC tissue samples and cultured PC cells compared to benign prostatic hyperplasia tissue samples and normal prostate epithelial cells. Interestingly, AEG-1 knockdown induced cell apoptosis through upregulation of forkhead box (FOXO) 3a activity. This alteration of FOXO3a activity was dependent on reduction of AKT activity in LNCaP and PC-3 cells with high constitutive AKT activity, but not in DU145 cells with low constitutive AKT activity, although AEG-1 knockdown had no impact on phosphatase and tensin homolog expression in these cells. AEG-1 knockdown also attenuated the constitutive activity of the nuclear factor kappaB (NF-kappaB) and the activator protein 1 (AP-1) with a corresponding depletion in the expression of NF-kappaB and AP-1-regulated genes (interleukin (IL)-6, IL-8 and matrix metalloproteinase-9) and significantly decreased cell invasion properties of PC-3 and DU145 cells. Overall, our findings suggest that aberrant AEG-1 expression plays a dominant role as a positive auto-feedback activator of AKT and as a suppressor of FOXO3a in PC cells. AEG-1 may therefore represent a novel genetic biomarker to serve as an attractive molecular target for new anticancer agents to prevent PC cell progression and metastasis.

Downregulation of androgen, estrogen and progesterone receptor genes and protein is involved in aging-related erectile dysfunction.

We hypothesize that downregulation of sex hormone receptors (androgen, estrogen and progesterone receptors) is involved in aging-related erectile dysfunction. To test this hypothesis, we investigated the expression of sex hormone receptors in penile crura of aging rats. A total of 40 rats were divided into four groups based on age (6, 12, 18 and 24 months), and the erectile function was analyzed by the measurement of intracavernous pressure. Gene and protein expressions of sex hormone receptors were analyzed by RT-PCR and immunostaining, respectively. The mean intracavernous pressures of 6-, 12-, 18- and 24-month-old rats were 110.1, 89.6, 73.5 and 42.7 cm H(2)O, respectively. Gene and protein expressions for androgen receptor, estrogen receptor-beta and progesterone receptor were present in similar levels in 6-, 12- and 18-month-old rat crura, but significantly lower or absent in 24-month-old crura. This is the first study to demonstrate that downregulation of sex hormone receptors in aging rat crura is associated with erectile dysfunction.

Transrectal colour Doppler ultrasonography for quantifying angiogenesis in prostate cancer.

OBJECTIVE: To assess the correlation between angiogenesis and Doppler signal intensity using transrectal colour Doppler ultrasonography (CDUS) in patients with prostate cancer. PATIENTS AND METHODS: The study comprised 56 patients who underwent radical prostatectomy and had untreated tumours with a volume of> 0.1 mL in the peripheral zone. CDUS images were recorded on videotape before surgery. The Doppler signal intensity in tumours was evaluated using the colour pixel intensity (PI). Microvessel density (MVD) and vascular endothelial growth factor (VEGF) immunoreactivity were determined in the prostatectomy specimens. Microvessels were identified by immunohistochemical staining of endothelial cells for CD31. RESULTS: The PI in the tumour correlated with MVD (P < 0.001) and increased with higher levels of VEGF immunoreactivity (P = 0.004). There was no correlation between Gleason score and MVD or PI in the tumour. CONCLUSION: Blood flow assessed by CDUS may reflect the state of angiogenesis in prostate cancer. CDUS may be a useful technique for predicting tumour progression or prognosis, and may be useful for monitoring the effects of anti-angiogenic agents in the future.

Modulation of heat-induced cell death in PC-3 prostate cancer cells by the antioxidant inhibitor diethyldithiocarbamate.

OBJECTIVE: To examine the relationships between the form of cell death (apoptosis or necrosis), reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and the level of heat-shock protein 70 (hsp 70) expression after thermotherapy of PC-3 prostate cancer cells; also assessed were the tumoricidal effects of combined treatment with both heat and the antioxidant inhibitor diethyldithiocarbamate (DDC). MATERIALS AND METHODS: PC-3 cells were treated with thermotherapy at 42, 43 or 44 degrees C for 30, 60, 90 or 120 min. Cell proliferation, ROS generation, SOD activity and cellular hsp 70 level were determined using tetrazolium-based cytotoxicity, fluorescent dichlorofluorescein (DCF) and nitroblue tetrazolium assays, Western blot analysis and flow cytometry, respectively. The apoptotic and necrotic cells were determined by staining with propidium iodide and fluorescein isothiocyanate-labelled annexin V. These variable were also measured after combined treatment of PC-3 cells with 1 mmol/L DDC and thermotherapy at 43 or 44 degrees C for 60 min. RESULTS: Cell survival was significantly lower after heating cells at 43 degrees C for 60, 90 and 120 min and at 44 degrees C for all periods tested (P<0.05). At 43 degrees C apoptosis increased with the duration of heating and was similarly enhanced after heating at 44 degrees C for 30 min. Necrosis was not increased by heating at 42 or 43 degrees C, but was markedly enhanced after heating at 44 degrees C with both the duration of heating and with time after heating. Significant increases in DCF production were induced by heating at 43 degrees C for 60, 90 and 120 min (P<0.05) and at 44 degrees C at all times (P<0.010-0.005). There was a significant correlation between the level of ROS generation and necrosis (P<0.001) but no correlation between the ROS level and apoptosis. SOD activity increased in cells after heating at 43 degrees C, with significant differences among cells heated for 60, 90 and 120 min (P<0.05). After heating at 44 degrees C, SOD activity was maximal in cells heated for 30 min (P<0.005), by 30 min and then decreased with time after heating. There were significant increases in hsp 70 level in cells heated at 43 degrees C for 90 and 120 min (P<0.05) and at 44 degrees C for 30 and 60 min (P<0.05 and <0.025, respectively). Hsp 70 levels decreased after heating at 44 degrees C for 90 and 120 min. The combination of DDC and heating significantly increased ROS generation and the percentage of cell death, and decreased SOD activity (P<0.05). CONCLUSION: These findings show a qualitative change in the form of cell death induced by thermotherapy of PC-3 cells, which changed from apoptosis to necrosis according to the degree and duration of heating. Mild thermotherapy induced marginally low occurrence of apoptosis of PC-3 cells and DDC may represent a useful future strategy for the treatment of prostate carcinoma.

Cardiac electrophysiologic and hemodynamic effects of sildenafil, a PDE5 inhibitor, in anesthetized dogs.

A recent in vitro study demonstrated that supratherapeutic concentrations of sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, blocked I(Kr) and prolonged cardiac repolarization. This study assessed the in vivo cardiohemodynamic and electrophysiologic effects of sildenafil using a halothane-anesthetized, closed-chest canine model (n = 5) to bridge the gap between basic observation and clinical experience. Intravenous administration of sildenafil citrate in doses of 0.03, 0.3, and 3.0 mg/kg for 10 min, which provided sub-to supratherapeutic plasma drug concentrations, did not affect the monophasic action potential duration or effective refractory period of the right ventricle during the sinus rhythm as well as the ventricular pacing at the cycle length of 400 and 300 ms. However, sildenafil decreased the total peripheral resistance, simultaneously inducing positive chronotropic and inotropic effects at the top dose, which gave plasma concentrations at least 10 times higher than the therapeutic range. This cardiohemodynamic profile of sildenafil can be largely explained by reflex sympathetic activation associated with its vasodilator effect. Meanwhile, the lack of prolongation of the ventricular repolarization phase at the therapeutically relevant to moderately supratherapeutic sildenafil concentrations supports the earlier clinical studies that indicate that sildenafil has no effect on electrocardiogram.

Alterations of beta- and gamma-catenin in N-butyl-N-(-4-hydroxybutyl)nitrosamine-induced murine bladder cancer.

Abnormal degradation of beta-catenin caused by alteration of the glycogen synthase kinase-3beta (GSK-3beta) consensus motif is an important step for carcinogenesis. We hypothesize that beta- and gamma-catenin may play an important role in the pathogenesis of bladder cancer. We tested this hypothesis through analysis of beta- and gamma-catenin in both murine and human bladder cancers. A murine bladder cancer model was prepared by use of N-butyl-N-(-4-hydroxybutyl)nitrosamine (BBN) in 6-week-old male B6D2F1 mice. After 4, 8, 12, 16, 20, 24, and 28 weeks of BBN treatment, bladder specimens were harvested and analyzed for both protein and gene expression for beta- and gamma-catenin. Mutational analysis of the NH(2)-terminal regulatory domains of beta- and gamma-catenin was performed in each specimen by PCR-single-strand conformational polymorphism (SSCP) analysis. Mutations were further confirmed by direct DNA sequencing with a dye terminator method. Human bladder cancer specimens with normal tissues, dysplasia, carcinoma in situ, and carcinoma of grades, 1, 2, and 3 were also analyzed for beta- and gamma-catenin expression. beta- and gamma-catenin were analyzed for mutations by SSCP and direct DNA sequencing. Intracellular accumulation of beta- and gamma-catenin was observed in 6 of 20 invasive carcinoma specimens. There was no intracellular accumulation of beta- and gamma-catenin in mucosal dysplasia, papillary or nodular dysplasia, and carcinoma in situ specimens. On an SSCP analysis for beta-catenin, abnormal bandshifts were detected in two invasive carcinomas with intracellular beta-catenin accumulation. Further sequencing revealed two mutations [AGT(S) to ATT(I) and TCT(S) to CCT(P)] within the consensus motif for GSK-3beta phosphorylation. On the other hand, SSCP analysis for gamma-catenin followed by sequencing revealed three mutations in two invasive carcinomas with intracellular accumulation of gamma-catenin. These three alterations affected the 3' downstream region outside the GSK-3beta phosphorylation site [ACC(T) to GCC(A), CTC(L) to ATC(I), and CTC(L) to ATG(M)]. In human bladder cancer, beta- and gamma-catenin expression was significantly weaker than in normal bladder. On SSCP analysis one abnormal bandshift was observed in high-grade human bladder cancer with intracellular beta-catenin accumulation. DNA sequencing revealed mutation TCT(S) to TGT(C). In summary, alterations in beta- and gamma-catenin are late events favoring tumor progression in mouse BBN-induced bladder cancer. Changes affecting the GSK-3beta phosphorylation site appear to be associated with activation of beta-catenin, but not with activation of gamma-catenin. In human blabber cancer, beta- and gamma-catenin expression is similar to the expression in the mouse model. The present study demonstrates that beta- and gamma-catenin may play an important role in bladder cancer progression.

Multilocular spermatocele: a case report.

We describe a case of a multilocular spermatocele. Ultrasound examination revealed several cystic spaces at the head of the left epididymis. Epididymal tumor could not be excluded, and therefore surgical exploration was performed. Histopathological examination of the specimen revealed a multilocular spermatocele arising from the rete testis. Most spermatoceles remain small and rarely present marked clinical problems. but they are occasionally large, and may simulate a solid tumor.

Proarrhythmic effects of fluoroquinolone antibacterial agents: in vivo effects as physiologic substrate for torsades.

Drug-induced prolongation of the QT interval is often associated with the onset of Torsades de Pointes (TdP) resulting in a life-threatening ventricular arrhythmia. The potential of the proarrhythmic effects of the new fluoroquinolone antibacterial agents, levofloxacin and sparfloxacin, was examined in the chronic complete atrioventricular block dogs with stable idioventricular automaticity using Holter ECG monitoring in conscious state (Experiment 1). Next, to better analyze the mechanisms of the proarrhythmic property, the cardiovascular effects of these two drugs were compared in the halothane-anesthetized dogs under the monitoring of ECG, His bundle electrogram, systemic and left ventricular pressure, monophasic action potential, cardiac output, and effective refractory period (Experiment 2). In Experiment 1, oral administration of 6 mg/kg (n = 4) as well as 60 mg/kg (n = 4) of levofloxacin did not induce any ventricular premature depolarization. On the other hand, oral administration of 60 mg/kg of sparfloxacin (n = 4) induced TdP leading to ventricular fibrillation in all animals within 24 h, while 6 mg/kg of sparfloxacin (n = 4) did not induce any ventricular premature depolarization. In Experiment 2, intravenous administration of 0.3 mg/kg as well as 3.0 mg/kg of levofloxacin slightly increased cardiac output, but no significant changes were detected in the other parameters (n = 6). On the other hand, intravenous administration of 0.3 mg/kg of sparfloxacin prolonged the effective refractory period. Additional administration of 3.0 mg/kg of sparfloxacin decreased the heart rate and prolonged the effective refractory period and ventricular repolarization phase in a similar extent, but no significant changes were detected in the other parameters (n = 6). These results suggest that backward shift of the relative repolarization period in a cardiac cycle may be the mechanism responsible for the torsadegenic effect of sparfloxacin.

Comparison of the electropharmacological effects of verapamil and propranolol in the halothane-anesthetized in vivo canine model under monophasic action potential monitoring.

The cardiovascular profile of verapamil was assessed in the halothane-anesthetized canine model and compared with that of propranolol. Verapamil was infused at the rates of 1, 3 and 10 microg x kg(-1) x min(-1) (n=6), whereas propranolol was administered at a fixed rate of 10 microg x kg(-1) x min(-1) (n=6). Each infusion was performed over 30 min, and the parameters were assessed for 20-30 min after the start of each infusion. Verapamil in a dose of 10 microg x kg(-1) x min(-1) significantly suppressed atrio-ventricular (AV) node conduction and slightly decreased the mean blood pressure, but no significant change was detected in the left ventricular end-diastolic pressure, maximum upstroke velocity of the left ventricular pressure, sinus automaticity, double product, cardiac output, intraventricular conduction, and ventricular repolarization phase and refractoriness. Propranolol suppressed AV node conduction to an extent similar to that of verapamil, but it also inhibited intraventricular conduction, sinus automaticity and ventricular contraction, increased the ventricular refractoriness, and decreased the double product and cardiac output, without any significant change in the other variables measured. These results suggest that verapamil can selectively affect the AV node, and that the greater part of the suppressive action of propranolol on the multiple cardiovascular performance is through a beta-blocking action and direct membrane effect, although the halothane inhalation itself might have modified each of the drug's effects. The abbreviation of the relative refractory period of the ventricle by propranolol may show its potential utility for re-entry type ventricular tachycardia.

Concomitant presence of bladder cancer and neurogenic bladder in a patient with HTLV-1 carrier: a case report.

We describe a case of an HTLV-1 carrier who developed bladder cancer and neurogenic bladder. HTLV-1 is thought to alter host immune function and to contribute to the development of other malignancies. It is also sometimes reported that urinary symptoms precede pyramidal symptoms in patients with HAM. To our knowledge, concomitant presence of bladder cancer and neurogenic bladder in an HTLV-1 carrier has not been previously reported.

The role of colour Doppler ultrasonography in detecting prostate cancer.

OBJECTIVE: To determine the usefulness of colour Doppler ultrasonography (CDUS) in detecting prostate cancer, by comparing CDUS with grey-scale transrectal ultrasonography (TRUS) and magnetic resonance imaging (MRI). PATIENTS AND METHODS: In all, 278 patients who underwent prostate biopsies because of an abnormal digital rectal examination, elevated prostate specific antigen levels, and/or abnormal TRUS between May 1998 and November 1999 were evaluated. The diagnostic accuracies of TRUS, CDUS, MRI and combinations of these imaging techniques in detecting prostate cancer were compared, based on the biopsy results. RESULTS: Carcinoma was detected in 233 of 1696 specimens, and 87 patients were diagnosed with prostate cancer. For each detected cancer site, the sensitivity of CDUS was lower than those of other imaging techniques, but CDUS had high a specificity and positive predictive value. The combination of grey-scale TRUS and CDUS or MRI improved the sensitivity and negative predictive value. The specificity and positive predictive value of the combination of grey-scale TRUS and MRI were less than those for grey-scale TRUS alone, while those for the combination of grey-scale TRUS and CDUS were higher than those for grey-scale TRUS alone. Five tumours were isoechoic but seen as hypervascular lesions with CDUS. CONCLUSION: CDUS provides information useful for detecting prostate cancer when used in combination with grey-scale TRUS, and should be included in the routine examination for prostate cancer.

Heat-induced cellular damage and tolerance in combination with adriamycin for the PC-3 prostate cancer cell line: relationships with cytotoxicity, reactive oxygen species and heat shock protein 70 expression.

OBJECTIVE: To analyze the relationships among the antitumor effect of chemothermotherapy, generation of reactive oxygen species (ROS), and expression of heat shock protein 70 (HSP-70) in the PC-3 prostate cancer cell line. MATERIALS AND METHODS: Changes in cell proliferation, cell cycle fractions, intracellular ROS accumulation and HSP-70 expression were examined after thermotherapy of PC-3 cells at 41, 42, 43 and 44 degrees C and/or simultaneous treatment with Adriamycin for 1 h, using the trypan blue dye exclusion method, flow cytometry, fluorescent 2', 7'-dichlorofluorescein (DCF) assay, and Western blot analysis. RESULTS: A significant decrease in the number of viable cells was observed with chemothermotherapy compared with thermotherapy at 42, 43, or 44 degrees C (p<0.05). DNA distribution histograms revealed cell accumulation in the S-G(2)/M phase after thermotherapy at 43 degrees C and after chemothermotherapy at 37, 41, 42 and 43 degrees C. After thermotherapy and chemothermotherapy at 44 degrees C, DNA histograms revealed no accumulation of cells with S-G(2)/M DNA content and cells exhibited a marked loss of viability. A significant increase in DCF production was observed with chemothermotherapy compared with thermotherapy at 42, 43 or 44 degrees C (p<0.05, p<0.01 and p<0.01, respectively). HSP-70 levels increased linearly with increasing temperature. HSP-70 levels after thermotherapy and chemothermotherapy increased with time and reached plateaus at 30 min, whereas the level after thermotherapy at 44 degrees C decreased at 60 min. CONCLUSIONS: In conclusion, one possible synergism in cytotoxic effects of chemothermotherapy and Adriamycin could be evaluated by the relationship between ROS accumulation and HSP-70 expression in the PC-3 prostate cancer cell line.