Algorithm-based design of synthetic growth media stimulating virulence properties of Metarhizium anisopliae conidia.

AIMS: Synthetic media should be designed for the production of Metarhizium anisopliae conidia with improved virulence properties. METHODS AND RESULTS: A genetic algorithm (GA), demonstrated to be suitable for the design of media for spore mass production (Hutwimmer et al. 2008), was utilized for a multi-objective medium design to improve conidia yield and three proposed virulence properties of conidia: C : N ratio, germination speed and amount of spore-bound Pr1 protease. After five iterative optimizations, 52 media were improved over Sabouraud dextrose agar (SDA). Four media exhibited medium performances (a factor derived from the four single optimization variables) of around 0.7; cf. SDA = 0.532; media with enhanced properties were reached for each single optimization variable; Bioassays against Tenebrio larvae indicated also a slight improvement in virulence of conidia from designed media. A degenerated phenotype of the same strain did not exhibit differences in colony appearance, spore characteristics and virulence if grown on designed media. CONCLUSIONS: The application of a problem-oriented GA is a practical and rapid method to design media for multi-objective purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The applicability of a GA for multi-objective medium design was demonstrated for the cultivation of anamorphic fungi on solid media.

Algorithm-based design of novel synthetic media for Metarhizium anisopliae simulating its nutritional conditions in the environment.

AIMS: The objective of this study was to develop a novel synthetic growth medium for increased conidium production of the entompathogenic fungus Metarhizium anisopliae. The medium should simulate natural nutritional conditions. METHODS AND RESULTS: Macro- and micronutrients were selected in concentrations found in insects or from sources which are believed to be advantageous for virulence. Medium development was guided by a problem-oriented genetic algorithm (GA) implemented with 'mutation' and 'recombination' operators specific for this optimization problem. Following five optimization steps and a total of 181 medium variations, 19 synthetic media led to increased conidium production as compared with the standard Sabouraud dextrose agar medium: increases in conidia yields of up to 120% and a 17-fold higher conidium production per square centimetre of mycelium were achieved. Rapid germination of conidia, conidial C : N ratio, as well as spore-bound Pr1 protease production were monitored as proposed quality control markers for a virulent inoculum. CONCLUSIONS: It was demonstrated that our problem-oriented GA is a powerful tool for the design of chemically defined cultivation media with special/enhanced properties for anamorphic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study, which demonstrates the advantage of the use of a GA for the rapid design of solid synthetic media for maximum production of virulent conidia.

Sonographic measurement of renal pelvis wall thickness as diagnostic criterion for acute pyelonephritis in adults.

PURPOSE: Inflammatory processes may increase the urothelial thickness of the renal pyelon. Purpose of the study was to assess sonographic measurement of pyelon wall thickness (PWT) in adult patients with acute pyelonephritis, chronic urinary tract infection (UTI) and indwelling ureteral stents. MATERIALS AND METHODS: Four study groups (acute pyelonephritis n=50, chronic UTI n=10, indwelling ureteral stents n=10, controls n=25) underwent renal ultrasonography (Acuson Seqouia, Mountain View, CA; 6 MHz Transducer). The renal pyelon was imaged in transverse and longitudinal planes. PWT measurements of patients with acute pyelonephritis were repeated after successful antibiotic treatment. RESULTS: Mean PWT in healthy controls was 1.0 mm+/-0.19. In patients with acute pyelonephritis, PWT was significantly increased to 2.9 mm+/-0.89 (p<0.001). PWT decreased significantly after antibiotic treatment to 1.4 mm+/-0.47 (p<0.001). Kidneys with indwelling stents presented with a PWT of 2.7 mm+/-0.68, kidneys with chronic UTI demonstrated a PWT of 2.8 mm+/-0.62. PWT in these patient groups was significantly greater than PWT in healthy volunteers (p<0.001). The interobserver agreement was excellent (p<0.001). CONCLUSION: PWT is a reproducible diagnostic criterion for acute pyelonephritis. Based upon our experience, we suggest a cut-off value of 2.0 mm to distinguish healthy kidneys from those with urothelium thickened by inflammation. PWT cannot be used to distinguish acute pyelonephritis from chronic inflammation of the urothelium.

Transurethral ultrasonography-guided injection of adult autologous stem cells versus transurethral endoscopic injection of collagen in treatment of urinary incontinence.

In the last years preclinical studies have paved the way for the use of adult muscle derived stem cells for reconstruction of the lower urinary tract. Between September 2002 and October 2004, 42 women and 21 men suffering from urinary stress incontinence (age 36-84 years) were recruited and subsequently treated with transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. In parallel, 7 men and 21 women (age 39-83 years) also diagnosed with urinary stress incontinence were treated with standard transurethral endoscopic injections of collagen. Patients were randomly assigned to both groups. After a follow-up of 12 months incontinence was cured in 39 women and 11 men after injection of autologous myoblasts and fibroblasts. Mean quality of life score (51.38 preoperatively, 104.06 postoperatively), thickness of urethra and rhabdosphincter (2.103 mm preoperatively, 3.303 mm postoperatively) as well as contractility of the rhabdosphincter (0.56 mm preoperatively, 1.462 mm postoperatively) were improved postoperatively. Only in two patients treated with injections of collagen incontinence was cured. The present clinical results demonstrate that, in contrast to injections of collagen, urinary incontinence can be treated effectively with ultrasonography-guided injections of autologous myo- and fibroblasts.

Comparison of contrast enhanced color Doppler targeted biopsy to conventional systematic biopsy: impact on Gleason score.

PURPOSE: Prostate cancer grading with Gleason score is an important prognostic factor. This prospective randomized study compares ultrasound systematic biopsy vs contrast enhanced color Doppler targeted biopsy for the impact on Gleason score findings. MATERIALS AND METHODS: We examined 690 men (mean age 56 years, range 41 to 77) with a serum total prostate specific antigen of 1.25 ng/ml or greater, a free-to-total prostate specific antigen ratio less than 18% and/or a suspicious digital rectal examination. Contrast enhanced color Doppler targeted biopsies with a limited number of cores (5 or less) were performed in hypervascular areas of the peripheral zone during administration of the ultrasound contrast agent Sonovuetrade mark (Bracco, Milano, Italy). Ten systematic biopsies were obtained in a standard spatial distribution. Cancer detection rates and Gleason score were compared. RESULTS: Prostate cancer was identified in 221 of 690 subjects (32%) with a mean prostate specific antigen of 4.6 ng/ml (range 1.4 to 35.0). Prostate cancer was detected in 180 of 690 subjects (26%) with contrast enhanced color Doppler targeted biopsy and in 166 of 690 patients (24%) with systematic ultrasound biopsy. The Gleason score of all 180 cancers detected on contrast enhanced color Doppler targeted biopsy was 6 or higher (mean 6.8). The Gleason score of all 166 cancers detected on systematic biopsy ranged from 4 to 6 and mean Gleason score was 5.4. Contrast enhanced color Doppler targeted biopsy detected significantly higher Gleason scores compared to systematic biopsy (Wilcoxon rank sum test p <0.003). CONCLUSIONS: Contrast enhanced color Doppler targeted biopsy detected cancers with higher Gleason scores and more cancer than systematic biopsy. Therefore, contrast enhanced color Doppler seems to be helpful in the grading of prostate cancer, which is important for defining prognosis and deciding treatment.

[Transrectal three dimensional sonography. Techniques and indications]. / Transrektale dreidimensionale Sonographie. Technik und Indikationen.

In 3-D transrectal ultrasound it is possible, for the first time, to investigate the region of interest in three planes simultaneously. Exact examination of the organs of the small pelvis as well as of pathologic changes in the region of the pelvic floor can be performed with this new imaging technique. The bulbourethral glands can be investigated routinely, which enables the diagnosis of cysts of these glands. The prostatic zones, their relations as well as the growth of the transitional zone during the development of benign prostatic hyperplasia can be visualized. Furthermore, 3-D transrectal ultrasound allows investigation of morphology and function of the rhabdosphincter. The contractility of the muscle can be quantified. 3-D ultrasound guided puncture and drainage of prostatic abscesses represents a minimally invasive therapeutic modality. This technique can be used to place needles as well as implants in the lower urinary tract. Generally, 3-D transrectal ultrasound offers new diagnostic and therapeutic possibilities.

[Stem cell therapy for urinary incontinence]. / Stammzelltherapie der Harninkontinenz.

Experimental and clinical studies investigated whether urinary incontinence can be effectively treated with transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts.This new therapy was performed in eight female pigs. It could be shown that the injected cells survived well and that new muscle tissue was formed. Next, 42 patients (29 women, 13 men) suffering from urinary stress incontinence were treated. The fibroblasts were mixed with a small amount of collagen as carrier material and injected into the urethral submucosa to treat atrophies of the mucosa. The myoblasts were directly injected into the rhabdosphincter to reconstruct the muscle and to heal morphological and functional defects. In 35 patients urinary incontinence could be completely cured. In seven patients who had undergone multiple surgical procedures and radiotherapy urinary incontinence improved. No side effects or complications were encountered postoperatively. The experimental as well as the clinical data clearly demonstrate that urinary incontinence can be treated effectively with autologous stem cells. The present data support the conclusion that this new therapeutic concept may represent a very promising treatment modality in the future.

Three-dimensional transrectal ultrasound of the male urethral rhabdosphincter.

The rhabdosphincter of the male urethra is an omega-shaped loop of striated muscle fibers that surrounds the membranous urethra at its lateral and anterior aspects. We investigated whether this muscle can be visualized by means of three-dimensional ultrasound to define morphological and dynamic ultrasound criteria. We examined the rhabdosphincter of the male urethra in 77 patients by means of this new imaging technique; 37 patients presented with urinary stress incontinence after transurethral resection of the prostate or radical prostatectomy while 40 were fully continent after radical prostatectomy and served as a control group. Contractility of the muscle was quantified by a specially defined parameter (rhabdosphincter-urethra distance). The anatomical arrangement and the contractions of the rhabdosphincter-loop could be clearly visualized in three-dimensional transrectal and transurethral ultrasound; during contraction the rhabdosphincter retracts the urethra, pulling it towards the rectum. We detected defects and postoperative scarrings in the majority of the patients with postoperative urinary stress incontinence. Furthermore, the patients presented with thinnings in parts of the muscle and atrophies of the rhabdosphincter. The rhabdosphincter-urethra distance was significantly lower in the incontinent group than in the continent group (59 vs. 1.42 mm). Our study shows that the rhabdosphincter of the male urethra can be visualized by means of three-dimensional transrectal ultrasound. The sonographic pathomorphological findings of postoperative urinary stress incontinence are well correlated well with the clinical symptoms.

[Anatomic basis for the innervation of the male pelvis]. / Anatomische Grundlagen zur Innervation des männlichen Beckens.

To reach an optimal outcome after radical prostatectomy or cystoprostatectomy, minimal bleeding from the dorsal vein complex, meticulous apical dissection without distraction of the rhabosphincter and preservation of the cavernous nerves are necessary.A total of 19 male cadavers were used, ranging in age from 52-78 years and including 8 fetuses. Fourteen complete pelves and five tissue blocks of prostate, rectum, membranous urethra and the rhabdosphincter were investigated. Besides anatomical preparations, the lower urinary tract was studied by means of serial histological sections. The cavernous nerves and the pudendal nerves as well as their branches were dissected. For this reason, the pubic symphysis was severed. Subsequently the right or left hip bone was removed, whereas the pelvic organs were left intact to achieve a complete exposure of the lower urinary tract and the pudendal nerves. The rhabdosphincter presents as a vertical structure extending from the bulb of the penis to the region of the bladder neck along the prostate and the membranous urethra. It is supplied by branches of the pudendal nerve after leaving the pudendal canal. The membranous urethra is innervated by branches of the autonomic pelvic plexus. The nervous branches that are responsible for erection travel along the lateral aspects of the prostate in the so called "neurovascular bundle" and traverse through the urogenital hiatus to reach the corpora cavernosa.

[Advantages of nerve-sparing pelvic surgery. Animal experiments and clinical results]. / Vorteile der nerverhaltenden Beckenchirurgie. Tier experimentelle und klinische Ergebnisse.

Traditionally, oncological factors have been the only ones used in the evaluation of treatment outcome for urological tumor patients. With increased diagnoses of early, curable tumors in younger individuals, health-related quality of life and functional aspects are gaining importance. Sexual and urinary function are significant aspects of quality of life, which are especially vulnerable in urological patients. New insights into the anatomy and physiology of the pelvic organs have resulted in an improvement in surgical therapy. In this article, we present the results of current experimental and clinical studies, which underline the importance of nerve sparing techniques for maintaining a satisfying urinary and sexual function in this patient population.

Late urologic effects after adjuvant irradiation in stage I endometrial carcinoma.

OBJECTIVES: To evaluate the incidence and type of incontinence after external beam radiotherapy (RT) and brachytherapy. Distinct late effects on the urinary bladder can occur and are frequently mild after adjuvant RT for Stage I endometrial carcinoma. Not all side effects that impair quality of life (eg, urinary incontinence) are classified in the commonly used grading system. METHODS: Forty-one patients were evaluated for newly occurred urinary incontinence after adjuvant RT. The mean follow-up was 64.8 months, and the mean age was 62.1 years. The validated incontinence score from Gaudenz was used. Additionally, quality-of-life questions were asked. RESULTS: Overall, 22 (53.7%) of 41 patients complained of urinary incontinence. Urge incontinence was classified in 45.5% (10 of 22 patients) and stress urinary incontinence in 54.5% (12 of 22 patients). CONCLUSIONS: The onset of stress urinary incontinence after brachytherapy can be explained by anatomic findings, such as adverse affects to the nerve supply of the rhabdosphincter. According to our results, the exposure to additional external beam RT can cause urge incontinence. Patients and doctors must be aware that urinary incontinence, with an occurrence rate of more than 50%, represents the most common side effect after surgery and RT for Stage I endometrial carcinoma. We conclude that, depending on the type of RT, a stress incontinence rate of 24.4% and an urge incontinence rate of 29.2% is possible.

Temporary hearing threshold shifts and restitution after energy-equivalent exposures to industrial noise and classical music.

In order to investigate whether the energy-equivalence principle is at least acceptable for exposures with a duration in the range of hours and in order to disclose the actual physiological responses to exposures which varied with respect to the time structure and the semantic quality of sounds, a series of tests was carried out where physiological costs associated with varying exposures were measured audiometrically. In a cross-over test design, 10 Subjects (Ss) participated in test series with 3 energetically equal sound exposures on different days. The exposures corresponded with a tolerable rating level of 85 dB / 8 h. In a first test series (TS I), the Ss were exposed to a prototype of industrial noise with a sound pressure level of 94 dB(A) / 1 h. In a second test series (TS II), the same type of noise was applied, but the exposure time of a reduced level of 91 dB(A) was increased to 2 hours. In a third test series (TS III), classical music was provided also for 2 h at a mean level of 91 dB(A). The physiological responses to the 3 exposures were recorded audiometrically via the temporary threshold shift TTS2, the restitution time t(0 dB), and the IRTTS-value. IRTTS is the integrated restitution temporary threshold shift which is calculated by the sum of all threshold shifts. It represents the total physiological costs the hearing must "pay" for the sound exposure. Physiological responses of the hearing to the industrial noise exposures in TS I and TS II, all in all, were identical in the 3 parameters. Maximum threshold shifts of approximately 25 dB occurred which did not dissipate completely until 2.5 h after the end of the exposure and IRTTS-values of about 800 dBmin were calculated. Therefore, at least for exposure times in the range of hours, the equilibration of intensity and duration of sound exposures according to the energy-equivalence principle seems to have no influence on the hearing. Classical music was associated with the least severe TTS of less than 10 dB which disappeared much more quickly. IRTTS added up to just about 100 dBmin and, in comparison with 800 dBmin as specific responses to industrial noise, amounted to only about 12%. The substantially lower physiological costs of classical music apparently indicate a decisive influence of the type of sound exposures. Making inferences from the results of the study, the conventional approach of rating sound exposures exclusively by the principle of energy equivalence can lead to gravely misleading assessments of their actual physiological costs.

Ureteroileal anastomosis in orthotopic urinary diversion: how much or how little is necessary?

PURPOSE: An increasing number of patients with an orthotopic neobladder and their expanding life expectancy necessitate an effective antireflux protection of the upper urinary tract. During the evolution of urinary diversion in the 20th century, several techniques to perform an ureteroileal anastomosis have been introduced. Those techniques most commonly used for orthotopic neobladders are discussed. MATERIALS AND METHODS: Vascularization of both the ureteral ends and the recipient bowel as well as a meticulous surgical technique are necessary to reduce the reported postoperative stricture rate, which ranges between 3% and 30%. Variations such as preservation of additional periureteral adventitial tissue and its use for coverage of the ureterointestinal suture line are described. High-pressure urinary reflux may lead to mechanical damage of the renal parenchyma, whereas low-pressure occasional reflux in conjunction with chronic bacteriuria will lead to pyelonephritic changes and eventually deterioration of renal function. An intraluminal valve mechanism with increasing efficiency during pouch filling is clearly the most physiological form of antireflux mechanism. Interposition of an afferent ileal limb in addition to the antireflux valve seems advantageous due to the favorable vascularization in the dissected midureter. CONCLUSIONS: Increased life expectancy in patients with orthotopic neobladders will increase the number of cases where access to the upper urinary tract becomes necessary due to a benign or malignant disease. Any technique that simultaneously provides an efficient antireflux mechanism and facilitates retrograde manipulation of the ureters (via the pouch) will be greatly appreciated by both patients and treating physicians.

Human rhabdosphincter cell culture: a model for videomicroscopy of cell contractions.

BACKGROUND: Physiology of the human rhabdosphincter and its innervation are still a subject to controversy. A better understanding of rhabdosphincter function and anatomy might help to solve important urological problems like urinary incontinence. It was the aim of the present study to develop a human sphincter cell culture model for investigation of contraction mechanisms in vitro. METHODS AND RESULTS Cells were isolated from human rhabdosphincter tissue obtained from prostatectomy and cystoprostatectomy specimens. Cultured cells expressed typical features of striated muscle cells. By means of videomicroscopy with a time lapse videosystem cell contractions could be documented. Under control conditions without any contractile stimulant 8% of the cells were seen to contract. Cholinergic stimulation with 10 mM of acetylcholine induced a significant increase in contraction rate to 49%. CONCLUSIONS: These results demonstrate that cholinergic stimulation triggers contraction of cultured human rhabdosphincter cells. This model might help to understand external urethral sphincter physiology and to establish new therapies for the treatment of sphincter dysfunctions. Prostate 47:189-193, 2001.

VI--choosing the right reconstruction for your female patients.

Like in male patients, selection of female patients for an orthotopic neobladder is important from an oncological and functional standpoint. The anatomy and the behavior of the isolated urethra in women had to be restudied because of the shorter urethra, the absence of the prostate, an undefined level of urethral dissection, and the different anatomy of the urethral sphincter-system. Retrospective long-term observations of female patients with primary bladder cancer revealed a 2% incidence of secondary urethral tumors in women. The only significant risk factor for urethral tumor involvement was primary bladder cancer at the bladder neck. Any type of gastrointestinal segment may be used for creating a pouch in women. As a result of an approximately 15% possibility of urinary retention or larger post-void residuals, the use of an efficient antireflux valve mechanism seems prudent. Clinical experience in an increasing number of women confirms the preliminary favorable results. Despite the selection criteria outlined in this article, most women can safely and rewardingly be offered an orthotopic neobladder.

Anatomic and functional studies of the male and female urethral sphincter.

A total of 28 human specimens (14 male, 14 female) was used to perform macro- and microscopic studies on the morphologic basis of the urethral continence mechanism. Furthermore, functional studies were performed in six sheep, with the aim of looking at the pudendal and autonomic innervation of the urethra and the rhabdosphincter, as well as the changes of autonomic innervation after selective denervation. Transurethral ultrasound was performed in 34 continent patients, in order to visualize the contractions of the rhabdosphincter. The membranous urethra is innervated by branches of the autonomic pelvic plexus. The rhabdosphincter is an omega-shaped loop of striated muscle fibers that is innervated by the pudendal nerves. These results are supported by the results of animal experiments that show that the autonomic nerves predominantly innervate and regulate the upper part of the urethra, whereas stimulation of the pudendal nerves leads to a contraction of the lower part of the sheep urethra. In electron-microscopy, marked degeneration of the smooth muscle cells could be seen in the sheep with bilateral denervation.

Age dependent apoptosis and loss of rhabdosphincter cells.

PURPOSE: To our knowledge the exact age dependent morphological and functional changes of the sphincter mechanism have not been investigated. Therefore, cell densities of the urethra and the urethral rhabdosphincter across various age groups, and the appearance of apoptosis were examined to explore the changes in these structures during the aging process. MATERIALS AND METHODS: Specimens were obtained from 16 male and 7 female cadavers 5 weeks to 92 years old. Histological sections were taken from 3 different levels of the rhabdosphincter and urethra. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method was used to detect apoptosis in the urethra and rhabdosphincter. In all specimens relative volume densities of the striated muscle fibers, apoptotic indexes and diameters of the rhabdosphincter and urethra were determined. RESULTS: An age dependent increase of apoptosis of the striated muscle fibers of the rhabdosphincter led to a dramatic decrease in the number of striated muscle cells. In the 5-week-old neonate 87.6% and in the 91-year-old woman 34.2% of the rhabdosphincter consisted of striated muscle cells. Overall, a direct linear correlation between the age of the specimens and decrease in volume densities of the striated muscle cells was evident. CONCLUSIONS: The dramatic decrease in the number of striated muscle cells in the rhabdosphincter of the elderly due to apoptosis represents the morphological basis for the high incidence of stress incontinence in this population.

Contextual control of long-term habituation in rats.

This study examined contextual control of long-term habituation and whether such effects are dependent on the habituating response system. Habituation of the acoustic startle response transferred from the home cage to the testing context, whereas habituation of lick suppression was context specific (Experiments 1 and 2). Contextual control of habituation was demonstrated between 2 experimental contexts for lick suppression to a tone (Experiment 3) and bar-press suppression to a light (Experiment 4). Experiment 5 extinguished habituation of lick suppression and the orienting response to a tone with 27 exposures to the habituation context. Context specificity of both responses also was found. Previous failures to demonstrate contextual control of habituation may be due to the choice of response system and to less sensitive procedures to detect response recovery. The habituation mechanism for startle is independent from the process or processes that underlie habituation in other response systems, but the nature of these mechanisms is not yet known.

Reconstruction of the lower urinary tract using autologous muscle transfer and cell seeding: current status and future perspectives.

Functioning free-muscle transfer has established itself for the treatment of skeletal muscle deficiency over the last two decades. The capability of skeletal muscle to empty a spherical reservoir has been shown to be lessened if the muscle has contracted due to dissection. Currently there is no established curative treatment for bladder acontractility. Experimental data and preliminary clinical results have shown that innervated free latissimus dorsi muscle (LD) may serve as a substitute for a dysfunctional detrusor. In a clinical protocol, latissimus dorsi detrusor myoplasty (LDDM) was applied in 11 patients (age 9-68 years) with bladder acontractility due to spinal cord injury (seven patients), congenital malformations (two patients), detrusor myopathy (one patient), and idiopathic causes (one patient) who had required catheterization for bladder emptying for a minimum of 2 years. In all, 10 of 11 patients were capable of voiding volitionally, with eight of them no longer requiring catheterization, throughout the follow-up period of 12-46 months. Transplanted LD activity was confirmed by ultrasonography and flow-mode computerized tomography. It can thus be concluded that emptying of the bladder can be induced through the contractility of reinnervated free LD that has been wrapped around the bladder. An innervated free-LD flap does not undergo the severe muscle fibrosis, contracture, and atrophy that occur after transfer of completely or partially denervated pedicled muscle. Apart from the restoration of deficient detrusor function, microneurovascular transfer of a free-LD flap may be combined with tissue engineering to serve as a basis for bladder augmentation and substitution.