Yoshihiko Wakabayashi

Diagnostic potential in bladder cancer of a panel of tumor markers (calreticulin, γ‐synuclein, and catechol‐o‐methyltransferase) identified by proteomic analysis

Using proteomic analysis, we previously identified calreticulin (CRT) as a potentially useful urinary marker for bladder cancer. Now, we have also identified γ‐synuclein (SNCG) and a soluble isoform of catechol‐o‐methyltransferase (s‐COMT) as novel candidates for tumor markers in bladder cancer, by means of proteomic analysis. In the process of establishing a superior tumor marker system, we investigated the diagnostic value of a combination assay of these three proteins. Voided urine samples were obtained from 112 bladder cancer and 230 control patients. Urinary CRT, SNCG, and s‐COMT were measured as a combined marker by quantitative western blot analysis. Relative concentration of each protein was calculated and the diagnostic value of a concomitant examination of these markers was evaluated by receiver operator characteristic analysis. With the best diagnostic cutoff, the overall sensitivity of the combined markers was 76.8% (95% confidence interval, 69–81%) with a specificity of 77.4% (72–80%), while those of a single use of CRT were 71.4% and 77.8%, respectively. When evaluated in relation to tumor characteristics, such as grade, stage, size, and outcome of urinary cytology, the diagnostic capacity of the combined markers was equal to or better than that of CRT in all categories. Concomitant use of CRT, SNCG, and s‐COMT had higher sensitivity for detection of bladder cancer than did single use of CRT. Our study suggests that use of this panel of markers will improve the diagnosis of bladder cancer and may allow the development of a protein microarray assay or multi‐channel enzyme‐linked immunosorbent assay.

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy.

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.

Increase of p75 immunoreactivity in rat urinary bladder following inflammation

PREVIOUS studies suggest that NGF may function as a mediator of inflammatory pain. Here, we examined the effect of inflammation on expression of the low affinity neurotrophin receptor p75, using the model of cyclophosphamide-induced cystitis in rats. In control rats, p75-positive thick fibre bundles were scattered in the muscle layer. At 2 and 3 days after injection of cyclophosphamide, numbers of p75-positive fine fibres in the muscle layer were dramatically increased. Electron microscopy revealed that p75 immunoreactivity was localized on the surface of Schwann cells and at the sites where they were apposed to axons. Results show that p75 is up-regulated in inflamed tissues, suggesting that p75 may bind to and take up nerve growth factor (NGF), thus participating in NGF-induced hyperalgesia.

Low-affinity nerve growth factor receptor immunoreactivity in the human urinary bladder

The localization of low-affinity nerve growth factor receptor (LNGFR) in the human urinary bladder was examined immunohistochemically using the mouse monoclonal antibody (ME20-4) against human LNGFR. LNGFR immunoreactivity was present in the human urinary bladder. The distribution of LNGFR-positive fibers was more abundant in the mucosa than in the muscle layer. Results also showed that some LNGFR-positive fiber bundles contained tyrosine hydroxylase immunoreactivity. Electron microscopic examination revealed that LNGFR immunoreactivity was located on the surface of Schwann cells, and frequently on the interface of axons and Schwann cells.

INCREASE OF LOW-AFFINITY NEUROTROPHIN RECEPTOR p75 AND GROWTH-ASSOCIATED PROTEIN-43 IMMUNOREACTIVITIES IN THE RAT URINARY BLADDER DURING EXPERIMENTALLY INDUCED NERVE REGENERATION

PURPOSE Nerve regeneration in the urinary bladder after pelvic nerve plexus injury remains uncertain. The objectives were to establish a rat model of nerve regeneration in the bladder and to examine possible changes of low-affinity neurotrophin receptor p75 and growth-associated protein-43 (GAP-43) immunoreactivities during denervation and nerve regeneration. MATERIALS AND METHODS Adult male rats were divided into 3 groups: controls, crush of the nerve bundles from the right major pelvic ganglion (MPG) to the bladder (nerve crush group) and removal of the right MPG (MPG removal group). Bladders were collected at 3, 7, 14, 30 and 60 days, and immunohistochemically stained for protein gene product 9.5 (PGP9.5: an axonal marker), p75 and GAP-43. RESULTS In the nerve crush group, PGP9.5 positive nerves were decreased in number at 3 and 7 days, and then increased after 14 days in the muscle layer of the operated side. By 60 days, the density returned to control levels. However, MPG removal resulted in a decrease of the density of PGP9.5 positive nerves throughout the experimental periods. These findings indicate that nerve regeneration occurred in the nerve crush group. The density of p75 labeled fibers was significantly increased at 3 to 30 days postoperatively in the nerve crush group. p75 immunoreactivity showed smooth surface and cytoplasmic staining, indicating that Schwann cells were p75 positive. GAP-43 labeled fibers showed significantly greater density at 3 to 14 days postoperatively. Schwann cells were GAP-43 immunoreactive and, in particular, regenerating nerve fibers appeared to be GAP-43 positive at 14 days. CONCLUSION The present study suggests that p75 and GAP-43 are involved in the mechanism(s) of nerve regeneration in the urinary bladder.

Expression of neurotrophin messenger RNAs during rat urinary bladder development

The family of neurotrophins, encompassing nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), is important in the regulation of neuronal development and function. We examined the expression of neurotrophin messenger RNAs (mRNAs) in the rat urinary bladder during pre- and postnatal development using competitive reverse transcription-polymerase chain reaction. The mRNA levels showed a biphasic pattern of expression; one peak was at prenatal ages (embryonic day (E)15-E18) and the other peak was at postnatal ages (postnatal day (P)14-P28). NT-4/5, BDNF and NGF mRNA levels were greatest at E15, E16 and E18, respectively. In contrast, NT-3 mRNA levels were significantly highest at P14. These data suggest that neurotrophins are involved in the mechanisms of bladder nerve growth for the prenatal period and reorganization of bladder reflex pathways during the second to the fourth postnatal week.

Horseshoe kidney in russell-silver syndrome

We describe a case of Russell-Silver syndrome associated with horseshoe kidney in a three-year-eleven-month-old boy. Several urologic anomalies are reported in this syndrome, but to our knowledge, horseshoe kidney has not been reported previously in the English literature.

Simple Techniques For Atraumatic Peritoneal Dissection From The Abdominal Wall And For Preventing Peritoneal Injury During Trocar Placement Under Retroperitoneoscopy

PURPOSE Inadvertent peritoneal tearing causes pneumoperitoneum and makes retroperitoneal laparoscopic procedures technically more difficult. We describe some simple techniques of atraumatic peritoneal dissection and the prevention of peritoneal injury during trocar placement under retroperitoneoscopic guidance. MATERIALS AND METHODS After balloon dilation and the establishment of pneumoretroperitoneum a laparoscopic swab stick was used for peritoneal dissection from the abdominal wall under retroperitoneoscopic guidance. Exploratory puncture using a Cathelin (Terumo, Tokyo, Japan) needle was performed before trocar placement in close proximity to the lateral peritoneal reflection. RESULTS We applied this technique in our last 10 consecutive retroperitoneal laparoscopic procedures. No peritoneal rents occurred during dissection of the lateral peritoneal reflection or trocar insertion. CONCLUSIONS The laparoscopic swab stick technique described facilitates atraumatic peritoneal dissection as well as creation of an adequate working space. Exploratory puncture using a Cathelin needle is useful for preventing inadvertent peritoneal injury during trocar placement.

Increase of growth-associated protein-43 immunoreactivity following cyclophosphamide-induced cystitis in rats

We examined the effect of inflammation on immunoreactivity of growth-associated protein (GAP-43) in the rat urinary bladder in which acute cystitis was induced with cyclophosphamide (CPA). Following CPA injection, the number of GAP-43 labeled nerves was significantly increased in the muscle layer. Immunoreactivity of PGP9.5, which was used as an axonal marker, was not augmented following CPA injection. Double fluorescence immunohistochemistry revealed that substance P immunoreactivity was present in most GAP-43 immunoreactive fibers (90.2%) in the inflamed bladder. Electron microscopic examination showed that GAP-43 immunoreactivity was localized on axons. Some GAP-43 positive axons showed degeneration. Possible significance of the increase of GAP-43 immunoreactive afferent nerve fibers in the muscle layer of acutely inflamed bladder was discussed.

Immuno-electron microscopic study of tyrosine hydroxylase in the cat urinary bladder and proximal urethra

The distribution and density of tyrosine hydroxylase (TH)-immunoreactive nerves in the cat urinary bladder and proximal urethra were similar to those of glyoxylic acid fluorescent nerves. Both TH-immunoreactive and fluorescent nerve fibres markedly decreased after 6-hydroxydopamine treatment. Hence, immuno-electron histochemistry of TH was employed to investigate adrenergic termination to the muscle layers of the cat urinary bladder and proximal urethra. A total number of 3728 TH-immunoreactive axon terminals in the lower bladder body, lateral bladder base, trigone, bladder neck and proximal urethra were examined. In the bladder base (the lateral bladder base, trigone and bladder neck) and proximal urethra, most of the TH-immunoreactive axon terminals (57.1-89.2%) lay outside smooth muscle cell fascicles, while most (69.7%) in the bladder body were inside muscle cell fascicles. The proportions of TH-immunoreactive axon terminals accompanying non-TH-immunoreactive axon terminals in the five regions were almost the same (about 70%). The present study demonstrated that the mode of adrenergic termination in the bladder base and proximal urethra notably differed from that in the bladder body, and that approximately 70% of adrenergic axon terminals were associated with non-adrenergic or cholinergic axon terminals in each region.