Knut Albrecht

Adiposity and Age are Statistically Related to Enhanced RASSF1A Tumor Suppressor Gene Promoter Methylation in Normal Autopsy Kidney Tissue

Age, adiposity, and smoking are risk factors for the development of renal cell carcinoma. Hypermethylation of the RAS association domain family 1A gene (RASSF1A) promoter belongs to the most frequently detected epigenetic alterations in human cancers including renal cell carcinoma. RASSF1A is functionally involved in cell cycle control in normal cells and depletion promotes a number of cellular changes increasing the risk for neoplastic growth. We investigated the hypothesis that age, modulated by the factors adiposity and anthracosis as a surrogate for smoking, is a predictor of RASSF1A promoter methylation in normal kidney tissue. Using a cross-sectional study design, we quantitatively analyzed RASSF1A methylation in 78 normal autopsy kidney tissues by quantitative combined bisulfite and restriction analysis and bisulfite sequencing, and statistically evaluated the degree of relative methylation for a relationship with the predictor age and study factors adiposity and state of anthracosis. Statistical analysis showed that age (regression analysis; P < 0.001), adiposity (univariate analysis; P = 0.016), and state of anthracosis (t test; P = 0.005) are each significantly associated with an increase of RASSF1A promoter methylation in normal kidney tissue. However, only age (P = 0.008) and adiposity (P = 0.008) were identified as independent predictors of RASSF1A promoter methylation using covariance analysis. This study provides statistical evidence that the common cancer risk factors age and adiposity enhance RASSF1A promoter methylation in nonmalignant kidney tissue. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2526–32)

Femoral Press-Fit Fixation of the Hamstring Tendons for Anterior Cruciate Ligament Reconstruction

Background Press-fit fixation of patellar tendon–bone anterior cruciate ligament autografts is an interesting technique because no hardware is necessary. For hamstring tendon grafts, no biomechanical data exist of a press-fit procedure. Hypothesis Press-fit femoral fixation of hamstring tendons is mechanically equivalent to press-fit patellar tendon–bone fixation. Study Design Controlled laboratory study. Methods Patellar and hamstring tendons of 30 human cadavers (age, 53.8 ± 18.0 years) were used. An outside-in press-fit fixation with a knot in the semitendinosus and gracilis tendons and an inside-out and outside-in fixation with the tendons wrapped around a bone block were compared with patellar tendon–bone press-fit fixation in 30 ovine femora. Constructs were cyclically strained and then loaded until failure. Maximum load to failure, stiffness, and elongation during failure testing and cyclical loading were investigated. Results The maximum load to failure was 561 ± 309 N for the patellar tendon, 599 ± 234 N for the semitendinosus/gracilis tendons knot construct, 678 ± 231 for the semitendinosus/gracilis tendons bone construct inserted outside in, and 339 ± 236 for the semitendinosus/gracilis tendons bone construct inserted inside out (inferior to the others; analysis of variance, Dunn test, P < .01). Stiffness of the constructs averaged 134 ± 32 N/mm for the patellar tendon, 124 ± 21 N/mm for the knot construct, 118 ± 27 N/mm for the outside-in fixation, and 117 ± 23 N/mm for inside-out fixation. Elongation during initial cyclical loading was 0.7 ± 0.6 mm for the patellar tendon, 1.6 ± 0.5 mm for the knot construct, 1.9 ± 1.2 mm for the outside-in fixation, and 1.9 ± 0.9 mm for the inside-out fixation (significantly larger for all semitendinosus/gracilis tendon techniques, P < .05). Conclusions Failure loads for the semitendinosus/gracilis tendons bone construct inserted outside in and the semitendinosus/gracilis tendons knot construct were within the confidence interval of the patellar tendon press-fit fixation. All semitendinosus/gracilis tendon graft techniques exhibited larger elongation during initial cyclical loading than the patellar tendon graft. There was no difference in stiffness between all techniques. Clinical Relevance Two of the 3 hamstring press-fit fixation techniques showed loads to failure similar to the patellar tendon fixation. Preconditioning of the constructs is critical. These results must be interpreted with care because of high standard deviations.

Avanafil for the treatment of erectile dysfunction: initial data and clinical key properties

Orally active, selective inhibitors of phosphodiesterase type 5 (PDE 5, cyclic GMP PDE), such as sildenafil, tadalafil and vardenafil, are currently the first-choice treatment options for the clinical management of erectile dysfunction (ED) of various etiologies and severities. However, a significant number of patients remain dissatisfied with the available therapies due a lack of efficacy or discomfort arising from adverse events. Several new PDE5 inhibitors, among which are avanafil (TA-1790), lodenafil, mirodenafil, udenafil, SLX-2101, JNJ-10280205 and JNJ-10287069, have recently been approved and introduced into the market or are in the final stages of their clinical development. Avanafil (marketed in the US under the brand name STENDRA™) has been developed by VIVUS Inc. (Mountain View, CA, USA) and has recently received approval from the US Food and Drug Administration (FDA) for use in the treatment of male ED. The drug has demonstrated improved selectivity for PDE5, is rapidly absorbed after oral administration with a fast onset of action and a plasma half-life that is comparable to sildenfil and vardenafil. In phase II and phase III clinical trials that included a large number of patients, avanafil has been shown to be effective and well tolerated. Owing to its favorable pharmacodynamic and pharmacokinetic profile, avanafil is considered as a promising new option in the treatment of ED. The present article summarizes the initial data and clinical key properties of avanafil.

Anatomy of the sural nerve in a computer-assisted model: implications for surgical minimal-invasive Achilles tendon repair

Background: Sural nerve injuries are an evident risk especially of minimal-invasive surgical Achilles tendon repair. However, detailed anatomical studies focusing on the relationship of the sural nerve with the Achilles tendon at various levels are scarce, even pending in two planes. Aim: To determine the position and course of the sural nerve in relation to the Achilles tendon in two planes after trans-section and computer-assisted determination. Methods: The exact course of the sural nerve was determined in 10 cadavers (55.3 years, 19–89 years), using a computer-assisted method in two planes (transversal/sagittal). Results: The sural nerve crossed the Achilles tendon at 11 (8.7–12.4) cm proximal to the tuber calcanei. The distance between the lateral crossing and the proximal musculotendineus junction was 35 (20–58) mm. Starting from the tuber calcanei, the distance was 2/2 mm (transversal/sagittal plane) at 11 cm proximal to the tuber calcanei, 4/4 mm at 10 cm proximal, 5/6 mm at 9 cm, 8/10 mm at 5 cm and 11/18 mm at the tuber calcanei. Conclusion: In the lateral crossing region of the sural nerve and the lateral proximal Achilles tendon 9–12 cm proximal to the tuber calcanei, a close relationship of both anatomical structures can be visualised using computer-assisted measurements; caution is suggested to prevent sural nerve entrapment in either open or percutaneous Achilles tendon repair.

Immunohistochemical description of cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the human labia minora

INTRODUCTION Up until now, only minimal research has been carried out on those female genital organs known to contribute to the normal cycle of sexual arousal and orgasm. Some findings indicated that there might be a significance of cyclic nucleotide-mediated pathways in the control of the normal function of female genital tissues. AIM To elucidate, by means of immunohistochemistry, the distribution of the phosphodiesterase (PDE) isoenzymes 1, 3, 4, 5, 10, and 11 in the human labia minora. MAIN OUTCOME MEASURES The amount of immunohistochemical staining specific for cyclic adenosine monophosphate (cAMP)- and/or cyclic guanosine monophosphate (cGMP)-degrading PDE isoenzymes was detected. METHODS Human labial tissue was obtained from four female cadavers (age at death: 18-42 years). Vibratome sections prepared from formaldehyde-fixated tissue specimens were incubated with primary antibodies directed against the respective PDE isoenzymes. Sections were then incubated with fluorochrome (fluorescein isothiocyanate, Texas Red)-labeled secondary antibodies. Visualization was commenced by means of a laser fluorescence microscope. RESULTS Immunostaining indicating the expression of PDE4 and PDE5 was abundantly observed in the smooth musculature of vessels interspersing the tissue. Immunoreactions specific for PDE3 were recognized in epithelial and subepithelial layers, sebaceous glands, and interstitial or neuroendocrine-like single cells located in the epithelium. Signals related to PDE10 and PDE11 were limited to the epithelium or glandular-like structures, respectively. CONCLUSIONS Our results, for the first time, demonstrate the presence of cAMP- and cGMP-PDE isoenzymes in the human labia minora and give a hint to a significance of PDE4 and PDE5 in the control of labial vascular tissue function.

Melanocortin receptor agonists in the treatment of male and female sexual dysfunctions: results from basic research and clinical studies

Introduction: Over the last 20 years, basic and clinical research activities studying the male and female sexual responses have led to several pharmacological options to treat male erectile dysfunction (ED) and female arousal and orgasmic disorders. While some strategies exclusively focus on peripheral mechanisms – such as nitric oxide/cyclic GMP signaling, which is known to play a role in the control of genital vascular and nonvascular smooth muscle – others have considered the central pathways involved in mediating arousal and orgasmic functions in females as well as the induction of penile erection in males. Aside from dopaminergic agonists, drugs known to target the central melanocortin system have also been assumed to have a promising potential in the treatment of female and male sexual dysfunctions. Areas covered: The present review summarizes the achievements that have been made in the clinical development of melanocortin receptor (MCR) agonists (melanotan I, melanotan II, bremelanotide) for the treatment of symptoms of sexual arousal and orgasmic disorders in adult females and ED in males. Expert opinion: The data available at present have facilitated our understanding of how the melanocortin pathway regulates both the male and female sexual functions. Indeed the data warrant further investigation to demonstrate the impact of the activation of MCRs by specific agonists on penile erection and female arousal and orgasm function.

ORIGINAL RESEARCH—BASIC SCIENCEORIGINAL RESEARCH—BASIC SCIENCE: Immunohistochemical Description of Cyclic Nucleotide Phosphodiesterase (PDE) Isoenzymes in the Human Labia Minora

INTRODUCTION Up until now, only minimal research has been carried out on those female genital organs known to contribute to the normal cycle of sexual arousal and orgasm. Some findings indicated that there might be a significance of cyclic nucleotide-mediated pathways in the control of the normal function of female genital tissues. AIM To elucidate, by means of immunohistochemistry, the distribution of the phosphodiesterase (PDE) isoenzymes 1, 3, 4, 5, 10, and 11 in the human labia minora. MAIN OUTCOME MEASURES The amount of immunohistochemical staining specific for cyclic adenosine monophosphate (cAMP)- and/or cyclic guanosine monophosphate (cGMP)-degrading PDE isoenzymes was detected. METHODS Human labial tissue was obtained from four female cadavers (age at death: 18-42 years). Vibratome sections prepared from formaldehyde-fixated tissue specimens were incubated with primary antibodies directed against the respective PDE isoenzymes. Sections were then incubated with fluorochrome (fluorescein isothiocyanate, Texas Red)-labeled secondary antibodies. Visualization was commenced by means of a laser fluorescence microscope. RESULTS Immunostaining indicating the expression of PDE4 and PDE5 was abundantly observed in the smooth musculature of vessels interspersing the tissue. Immunoreactions specific for PDE3 were recognized in epithelial and subepithelial layers, sebaceous glands, and interstitial or neuroendocrine-like single cells located in the epithelium. Signals related to PDE10 and PDE11 were limited to the epithelium or glandular-like structures, respectively. CONCLUSIONS Our results, for the first time, demonstrate the presence of cAMP- and cGMP-PDE isoenzymes in the human labia minora and give a hint to a significance of PDE4 and PDE5 in the control of labial vascular tissue function.

Expression and distribution of key enzymes of the cyclic GMP signaling in the human clitoris: relation to phosphodiesterase type 5 (PDE5)

The clitoris contributes to the normal female sexual response cycle. A significance of cyclic guanosine monophosphate (GMP) has been assumed in the control of clitoral vascular smooth muscle. As only a few investigations on the physiology of the vascular and non-vascular clitoral tissue have been carried out, knowledge on the mechanisms controlling this particular female genital organ is still vague. It has been suggested that human clitoral corpus cavernosum smooth muscle is regulated by nitric oxide (NO)/cyclic GMP and related key enzymes, such as NO synthases (NOSs) and the phosphodiesterase type 5 (PDE5). The present study evaluated in the human clitoris, by means of immunohistochemistry, the expression and distribution of key enzymes of the cyclic GMP pathway, such as the endothelial NOS, PDE2, PDE11 and cyclic GMP-dependent protein kinase type I (cGKI) in relation to the PDE5. Immunohistochemistry revealed the presence of PDE2, PDE5 and cGKI in the smooth muscle wall of blood vessels transversing the supepithelial and stromal space. Immunosignals specific for PDE2 were also identified in interstitial-like cells located in the basal epithelial layer. Staining for PDE11A was observed in single nerve trunks located in the clitoral stroma. The results are in favor of a role of the cyclic GMP signaling in the control of clitoral blood flow. It seems likely that PDE2 and PDE11 are also involved in the mechanism of local (neuro)transmission in the clitoris.

Expression and distribution of cyclic AMP- and cyclic GMP-binding protein kinases in the human vagina- an immunohistochemical study

INTRODUCTION In contrast to research findings describing the localization of nitric oxide synthases (NOS), guanylyl cyclases, and cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-degrading phosphodiesterase isoenzymes in the human vagina, the distribution of proteins known as major targets for cyclic nucleotides has not yet been evaluated. cAMP- and cGMP-dependent protein kinases (cAK, cGKI) have been identified as important receptors for cyclic nucleotides downstream the signaling cascades. AIM To investigate, by means of immunohistochemistry, the expression of cAK and cGKI in relation to endothelial NOS (eNOS), vasoactive intestinal polypeptide (VIP), and protein gene product 9.5 (PGP 9.5) in the human vagina. MAIN OUTCOME MEASURES Expression and distribution of cAK and cGKI(alpha,beta) in relation to eNOS, VIP, and PGP 9.5 in human vaginal tissue. METHODS Immunohistochemical techniques were applied to sections of human vaginal full wall specimens in order to evaluate the presence of cAK and cGKI(alpha,beta) in relation to VIP, PGP 9.5, and eNOS, respectively. Western blot analyses were conducted using cytosolic supernatants of homogenized specimens of the vaginal wall and epithelium. RESULTS Immunostaining specific for cGKIbeta was observed in vascular and nonvascular smooth muscle of the vagina. In the endothelial layer, cGKIbeta was found colocalized with eNOS. In contrast, no signals indicating cGKIalpha were registered. cAK-positive subepithelial vessels were found to be innervated by a dense meshwork of PGP-containing varicose nerve fibers, some of which presented expression of VIP. The expression of cAK and cGKIbeta was confirmed by Western blotting. CONCLUSIONS Our results demonstrate the expression of cAK and cGKIbeta in the human vagina. The colocalization with VIP and eNOS underlines the significance of both the cAMP and GMP pathway in the control of human vaginal vascular and nonvascular smooth muscle.

Ethanol-Induced Malfunction of Neutrophils Respiratory Burst on Patients Suffering From Alcohol Dependence

BACKGROUND Polymorphonuclear, neutrophil granulocytes (PMN) play a major role in the control of infections, and people who abuse alcohol are susceptible to infections. Resistance against infections ensues intracellularly following initial phagocytosis of microorganisms with the oxygen-dependent respiratory burst, the key enzyme of which is the respiratory burst oxidase, whereby oxygen radicals are produced for microbial destruction. To date there is insufficient information available in connection with the process of impaired defence against infection in patients suffering from alcohol dependence. Therefore, our investigation was carried out to determine the influence of alcohol exposition on the formation of oxygen radicals and the respiratory burst. METHODS 4.5 ml of whole blood was taken from 10 healthy adults and 10 patients suffering from alcohol dependence. An additional 3.5 ml of whole blood was taken from the alcoholic patients for determination of the blood alcohol concentration. The respiratory burst of PMN was tested using the Four-Colour-Continuous Flow Cytometer. Each experimental procedure consisted of 4 test samples [negative controls, Escherichia coli, FMLP-supplement (N-formyl-l-methionyl-l-leucyl-l-phenylalanin), PMA-supplement (phorbol-12-myristate-13-acetate)]. Differing concentrations of ethanol were also introduced to each of the tests performed (0.20 to 4.00 g/l). RESULTS Ethanol revealed a marked decrease of burst activity in those patients suffering from alcoholism with increased alcohol concentration. A dependence between the burst activity and the ethanol concentration was seen to be statistically significant. This effect was only evident after stimulation with E. coli and FMLP in those patients with alcohol dependence. CONCLUSION The results presented in this study show an impairment in the function of PMN in those patients addicted to alcohol due to the decrease in burst activity. In view of the results of the different stimuli, the second-messenger effects were not evident. A clarification of this phenomenon could well be assumed as an allosteric receptor effect on the burst oxidase, namely, a direct effect on the phagocytosis interaction between circulating granulocytes and causative organisms.