EDTA-induced urothelial cell shedding for the treatment of superficial bladder cancer in the mouse

AIM: The aim of this study was to determine the effect of intravesical EDTA instillation on the development of intravesically implanted tumor cells in normal mice. METHODS: The mouse bladder tumor (MBT-2) model was used in female C3H/eb mice to evaluate the amount of normal urothelial cell shedding, and the degree of tumor growth inhibition following intravesical EDTA instillation in comparison with phosphate-buffered saline (PBS) instillation. RESULTS: At 1 h after instillation, the number of urothelial cells aspirated was 500-1000 per PBS-treated mouse and 10,000-20,000 per EDTA-treated mouse (P < 0.00001). The bladder weight, which reflected the effect of the agent on the tumor, was similar in the untreated and PBS-treated mice (105.46 +/- 46 mg and 106.2 +/- 50 mg, respectively). It was significantly lower in the EDTA-treated mice (80.4 +/- 42 mg) (P = 0.0045). CONCLUSIONS: Intravesical administration of EDTA results in significant normal and neoplastic urothelial cell shedding. Intravesical irrigation with EDTA may prevent adherence of the malignant cells to the bladder wall following tumor resection.     

AN APPROACH FOR HIGH SENSITIVITY DETECTION OF PROSTATE CANCER BY ANALYSIS OF CHANGES IN STRUCTUREDNESS OF THE CYTOPLASMIC MATRIX OF LYMPHOCYTES SPECIFICALLY INDUCED BY PSA-ACT

PURPOSE: An alternative procedure for detection of prostate cancer was examined based on the observation that cells reexposed in vitro to antigenic or mitogenic stimulation will change their intracellular structuredness as measured by polarization of fluorescent light emitted by labeled cells (SCM test). MATERIALS AND METHODS: Lymphocytes derived from patients bearing a nonmalignant prostate tumor and healthy individuals were exposed to PSA-ACT, PHA, and MUC-1. RESULTS: Of sixty-five patients with prostate carcinoma (CaP), sixty-two were correctly diagnosed by the test. Of the eighty males in the control group, five were incorrectly diagnosed as having the disease and seventy-five were correctly diagnosed as healthy subjects. The sensitivity of the test was 96.8%. The specificity was 91.1%. The BPH (Benign Prostatic Hyperplasia) control group exhibited a sensitivity of 9.38%, but the specificity was 91.1%. Similar percentages for specificity and sensitivity were observed in the NRT (Non-Relevant Tumor) control group. CONCLUSIONS: The results shown here indicate the possibility of a different use of PSA-ACT for detection of prostate cancer with high specificity and sensitivity.     

Semen Quality and Hormonal Status of Patients Following Testicular Torsion

16 patients, 4 months to five years following unilateral torsion of the testis were evaluated as to semen quality and hormonal status. In patients operated within 12 hours of the onset of pain 44% had normal semen quality while in those operated following more than 12 hours only 20% had normal semen analysis. FSH, LH and testosterone levels were normal in 14 of the 16 patients. One patient had a low testosterone level and slightly elevated FSH, another patient had slightly elevated levels of both FSH and LH.     

Permanent Pacemaker Implantation Following Cardiac Surgery: Indications and Long-Term Follow-Up

Background: Conduction disturbances requiring permanent pacemaker implantation after heart surgery occur in about 1.5% of patients. Early pacemaker implantation may reduce morbidity and postoperative hospital stay. We reviewed our experience with patients undergoing surgery to try and identify predictors for pacemaker requirements and patients who will remain pacemaker dependent.
Methods: We performed a retrospective review of 4,999 patients undergoing surgery between the years 1993 and 2005. Patient age was 64 ± 12 years, and 71% were males. Coronary bypass was performed in 4,071 (81%), aortic valve replacement in 675 (14%), and mitral valve replacement in 968 (18%) patients.
Results: Seventy-two patients (1.4%) required implantation of a permanent pacemaker after surgery. Indications for pacemaker implantation included complete atrioventricular block in 59, symptomatic bradycardia/slow atrial fibrillation in nine, second-degree atrioventricular block in two, and other conduction disturbances in two patients. Predictors for pacemaker requirement by multivariate analysis were left bundle branch block and aortic valve replacement (P < 0.001). Late follow-up was available in 58 patients, at 72 ± 32 months. Thirty-seven (63%) were pacemaker dependent. Predictors for late pacemaker dependency were third-degree atrioventricular block after surgery and preoperative left bundle branch block (P < 0.001).
Conclusions: Patients at high risk for pacemaker implantation after heart surgery include those with preexisting conduction disturbances, and those undergoing aortic valve replacement. Of those receiving a pacemaker, about one-third will recover at late follow-up. For patients in the high-risk group who are pacemaker dependent after surgery, we recommend implanting a permanent pacemaker at 5 days after surgery, thus enabling early mobilization and early discharge.     

DIFFERENTIAL EFFECTS OF SEX HORMONES AND PHYTOESTROGENS ON PEAK AND STEADY STATE CONTRACTIONS IN ISOLATED RABBIT DETRUSOR

PURPOSE: Recent evidence suggests that sex steroids may produce rapid inhibition of voltage operated Ca2+ channels (VOCCs). Detrusor smooth muscle is highly dependent upon Ca2+ influx for receptor-activated contractions. Thus, we examined the relative effectiveness of a select group of sex steroids and dietary phytoestrogens to relax detrusor contracted with the muscarinic receptor agonist, bethanechol (BE) and the purinergic P2X receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP). MATERIALS AND METHODS: Isolated strips of rabbit detrusor were secured to isometric force transducers in a tissue bath and length-adjusted until maximum contractions were achieved. Peak (P) contractile responses were recorded for alpha,beta-MeATP (P(ATP)) and BE (P(BE)) and steady-state (SS) responses were recorded for BE (SS(BE)) in the presence and absence of selected sex steroids and phytoestrogens (10 microM, unless indicated). RESULTS: The L-type VOCC inhibitor, nifedipine (1 to 10 microM), completely inhibited P(ATP) but reduced SS(BE) by approximately 50%, whereas the VOCC and non-VOCC inhibitor, SKF 96365, inhibited SS(BE) by approximately 95%, suggesting that P(ATP) was entirely dependent on L-type VOCCs, but (BE)-induced contractions depended also on activation of non-VOCCs. 17Beta-estradiol (estradiol) and progesterone inhibited P(ATP) by approximately 60% and 20%, respectively, and 32 microM estradiol and ethinyl estradiol inhibited SS(BE) by approximately 80 and 95%, respectively. Inhibition by estradiol was potentiated, rather than blocked, by the nuclear estrogen receptor antagonist, tamoxifen. Moreover, tamoxifen alone nearly completely relaxed SS(BE). The inactive metabolite of estradiol, 17alpha-estradiol, inhibited both P(ATP) and P(BE) by approximately 40%. Testosterone had no effect on P(ATP) and P(BE). The phytoestrogen and tyrosine kinase inhibitor, genistein, inhibited SS(BE) by 44%, whereas daidzein, a phytoestrogen without tyrosine kinase inhibitory activity, produced only a 7% inhibition. None of the phytoestrogens examined inhibited P(BE), whereas all inhibited P(ATP) by approximately 20 to 35%. A comparison of inhibition of (BE) and alpha,beta-MeATP-induced contractions by selected estrogen isomers showed some distinct differences. For example, estrone did not inhibit P(BE) or SS(BE), but inhibited P(ATP) by approximately 20%, whereas DES inhibited SS(BE) by nearly 90%, but P(ATP) by a lesser degree (approximately 70%). CONCLUSIONS: Our data support the hypothesis that 17beta-estradiol, ethinyl estradiol, DES, tamoxifen and genistein may relax detrusor contractions by inhibition of both VOCCs and non-VOCCs. Moreover, our data show that genistein, a dietary phytoestrogen with tyrosine kinase inhibitory activity, selectively reduced alpha,beta-MeATP-induced peak and BE-induced steady-state contractions, sparing the maximum response to BE. Lastly, the inactive isomer, 17alpha-estradiol, inhibited both BE- and alpha,beta-MeATP-induced contractions. These data suggest that certain dietary phytoestrogens (for example, genistein) or sex steroids, especially those with weak activity at the nuclear steroid site (for example, 17alpha-estradiol), or tamoxifen may prove therapeutically useful in treating overactive bladder caused by elevated muscarinic and purinergic receptor activation.     

Evolution of Action Potential Propagation and Repolarization in Cultured Neonatal Rat Ventricular Myocytes

INTRODUCTION: Cultured neonatal rat ventricular myocytes (NRVM) reestablish gap junctions as they form synchronously and spontaneously beating monolayers, thus providing a useful model for studying activation and repolarization. METHODS AND RESULTS: We used the multielectrode array data acquisition system with 60 unipolar electrodes to investigate the functional organization of cultured NRVM, by determining propagation and repolarization patterns. Activation maps were constructed from the local activation times at each electrode. During days 3 to 8 in culture, QRS amplitude and dV/dt(max) increased with age. Concomitantly, with the culture maturation, QT interval (representing action potential duration) decreased, and T wave amplitude and slopes of the T wave ascending and descending limbs progressively increased. The changes in conduction velocity were different than those of the electrogram properties, slightly increasing during the first 3 to 5 days and gradually declining toward day 8 in culture. CONCLUSION: Establishment of uniform activation patterns in spontaneously firing or driven myocytes in monolayer cultures is accompanied by organization of activation and repolarization whose evolution appears in concert with that of a mature connexin43 staining pattern. The experimental techniques developed in this study provide useful tools to investigate the complex relations among gap junctions, conduction velocity, and propagation patterns, as well as a means to learn how gap junctional remodeling under pathophysiologic conditions predisposes the myocardium to arrhythmias.     

Novel ψ-S-CH2 peptide-bond replacement and its utilization in the synthesis of nonpeptidic surrogates of the Leu-Asp-Val sequence that exhibit specific inhibitory activities on CD4+ T cell binding to fibronectin

The Leu-Asp-Val-(LDV)-containing amino acid sequence, derived from the alternatively spliced first connecting segment region of fibronectin (FN), was shown to be recognized primarily by the α₄β₁-integrin receptor expressed on the surface of various cell types. This adhesion epitope may therefore inhibit integrin-mediated cell interactions with the extracellular matrix glycoprotein, including adhesion, migration, activation and differentiation. To probe the structural requirements for LDV recognition by integrins and examine the feasibility of inhibition of LDV-dependent cell-FN interactions, we have designed and constructed a novel ψ-S—CH₂ peptide bond surrogate that was employed in the formation of LDV surrogates. The synthesis of the ψ-S–CH₂ surrogates reported herein is based on Michael addition of 4-methylpentane thiol to an itaconic acid diester to form an S–CH₂ bond. We have found that the LDV surrogates comprises of 4-methylpentanoate-Asp-i-butyl amide and 8-methyl-3-(2-methylpropylaminocarbonyl)-5-thianonanoic acid interfered with CD4⁺ human T-cell adhesion to FN in vitro, with an ED₅₀ of 280 μg/mL. A control structural mimetic of the Leu-Glu-Val (LEV) peptide did not interfere with the T-cell-FN interaction. The specificity of the reaction was substantiated by the finding that the LDV mimetics did not interfere with T-cell adhesion to laminin, another major cell-adhesive glycoprotein of the extracellular matrix. That the nonpeptidic mimetics of LDV interfered markedly with T-cell-FN adhesive interactions indicate that the peptide bond and the amine and carboxyl end groups of the tripeptide makes only a minor contribution to the integrin binding affinity. Thus, consistent with our recent report on the production of Arg-Gly-Asp surrogates, we suggest that these constructs could provide novel insights into the fundamental mechanisms of integrin-ligand interactions, and serve as competitive antagonists of conceivable therapeutic value.