[Adjustable transobturatoric sling system in men : diagnosis and therapy recommendations to persistent pain]. / Adjustierbare transobturatorische Schlingensysteme beim Mann : Diagnostik und Therapieempfehlungen bei persistierenden Schmerzen.

BACKGROUND: In addition to artificial sphincters, male slings are recommended in the current guidelines for the treatment of persistent male stress incontinence. Today, several sling systems are available. Well-known complications of all sling systems are infections, erosion, residual urine/urinary retention, de novo urgency, and postoperative pain. DISCUSSION: Compared to retropubic implanted adjustable sling systems or functional slings, pain is more common after transobturatoric implantation of adjustable sling systems. Early postoperative pain is very common. In contrast, persistent pain is rare. However, the treatment of persistent pain is a large challenge for urologists and patients. There are no recommendations for diagnostic workup or treatment. RESULTS: After pain classification, pain management should be started with nonsteroidal anti-inflammatory drugs and/or tricyclic antidepressive agents, if necessary treatment escalation with a weak opioid and if not effective interventional procedures should be performed. Sling explantation is only necessary in rare cases.

[Structure, use, and risks of biomaterial repositories of embryonal tumors]. / Biomaterialbanken für embryonale Tumoren: Struktur, Nutzen und Risiken.

Availability of statistically sufficient numbers of tumor samples and other biomaterials in high quality together with corresponding clinical data is crucial for biomedical research. Tumor repositories from individual scientists are mostly not sufficient to satisfy these criteria, especially since pediatric tumors are rare. In 2000 three centralized tumor repositories (neuroblastoma in Cologne, nephroblastoma in Würzburg, hepatoblastoma, brain tumors in Bonn) have been established by the "German Competence Net Pediatric Oncology und Hematology". The aim was to collect biomaterial including tumor samples, normal tissue, and blood in high quality for research and diagnostic purposes at a central institution. Informed consent of the parents or patients is a prerequisite for scientific use of the samples and is requested by the therapy trial. The samples are collected according to accepted standards and shipped in the specially designed Tumorbox. The tumor repository organizes the distribution of the samples to the cooperating diagnostic laboratories. The number of collected tumor samples has increased over the years. In 2000, samples from 200 patients were collected while the patient number increased to 321 in 2005. Over the years the tumor repositories collected more than 7,150 samples (fresh frozen tumor, fresh frozen normal tissue, and blood). Through links with clinical trial databases the samples can be connected with clinical data. 12 of 14 applications for tumor material to be used in specific scientific projects have been approved by an independent supervisory board. The establishment of central tumor repositories represents a major step for biomedical research activities and quality control in pediatric oncology.

Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters.

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.

[Botulinum toxin A in orthopedic pain therapy]. / Botulinumtoxin A in der orthopädischen Schmerztherapie.

Botulinum toxin A (BTX A) has been used for more than 20 years as a safe and effective treatment for numerous diseases characterized by pathological muscle hypertension. In patients suffering from dystonia or spasticity, it has been observed that use of BTX A results not only in muscle relaxation but also frequently relieves associated pain. This pain relief is often seen earlier and to a much greater extent than the muscular relaxation itself. This has led to extending the use of BTX A to treat various focal pain syndromes. The results of initial studies in specific musculoskeletal pain therapy suggest that BTX A infiltrations are effective in the treatment of chronic, therapy-resistant pain of the shoulder and back region. Furthermore, BTX A has been found to be a less invasive option for the treatment of chronic epicondylitis and similar tendonitis conditions. The healing process following rupture of tendons or muscle transfer operations may be improved. In adults with increased muscle tone and endoprostheses, the targeted relaxation of spastic muscles might increase the lifetime of the implant and diminish aseptic loosening. In children with cerebral palsy, prophylactic treatment of hip luxation appears possible. The doses used in pain therapy are low; if correctly applied, the tolerance and safety are high and the effect lasts for a number of weeks.

A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease.

Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations.

Gain of 1q is associated with adverse outcome in favorable histology Wilms' tumors.

Although several genes/genetic loci involved in the etiology of Wilms' tumor have been identified, little is known of the molecular changes associated with relapse. We therefore undertook an analysis by comparative genomic hybridization (CGH) of 58 tumor samples of favorable histology Wilms' tumor taken at initial diagnosis and/or relapse. Tumors with anaplastic histology were excluded as this is known to be associated with p53 mutation and a poor prognosis. A control group of 21 Wilms' tumors that did not relapse was also analyzed. The overall frequency of gains or losses of genetic material detected by CGH was similar in both groups (77% in relapsing tumors and 70% in the nonrelapse group) as was the median number of changes per tumor (relapse group: n = 4, range, 1 to 19; nonrelapse group: n = 3, range, 1 to 8). However, gain of 1q was significantly more frequent in the relapse series [27 of 46 (59%) versus 5 of 21 (24%), P: = 0.019]. In 12 matched tumor pairs, the CGH profiles, including 1q gain, were similar at diagnosis and relapse, with little evidence for further copy number changes being involved in clonal evolution. The results suggest that 1q gain at diagnosis could be used to identify patients with favorable histology Wilms' tumor at increased risk of relapse who might benefit from early treatment intensification.

Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors.

Vertebrate somitogenesis comprises the generation of a temporal periodicity, the establishment of anteroposterior compartment identity, and the translation of the temporal periodicity into the metameric pattern of somites. Molecular players at each of these steps are beginning to be identified. Especially, members of the Notch signaling cascade appear to be involved in setting up the somitogenesis clock and subsequent events. We had previously demonstrated specific expression of the mHey1 and mHey2 basic helix-loop-helix (bHLH) factors during somitogenesis. Here we show that perturbed Notch signaling in Dll1 and Notch1 knockout mutants affects this expression in the presomitic mesoderm (PSM) and the somites. In the caudal PSM, however, mHey2 expression is maintained and thus is likely to be independent of Notch signaling. Furthermore, we analysed the dynamic expression of the respective chicken c-Hey1 and c-Hey2 genes during somitogenesis. Not only is c-Hey2 rhythmically expressed across the chicken presomitic mesoderm like c-hairy1, but its transcription is similarly independent of de novo protein synthesis. In contrast, the dynamic expression of c-Hey1 is restricted to the anterior segmental plate. Both c-Hey genes are coexpressed with c-hairy1 in the posterior somite half. Further in vitro and in vivo interaction assays demonstrated direct homo- and heterodimerisation between these hairy-related bHLH proteins, suggesting a combinatorial action in both the generation of a temporal periodicity and the anterior-posterior somite compartmentalisation.

Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos.

In vertebrates Notch signaling regulates cell fate decisions and boundary formation and it underlies several murine and human diseases. Gene targeting experiments point to key roles of Notch receptors, ligands, modulators and downstream targets in somitogenesis, neurogenesis and vascular development. Here we report the embryonic expression of the hairy-related basic helix-loop-helix gene HeyL in wild-type and Notch pathway mutant mice. We show that HeyL is strongly expressed in the presomitic mesoderm, the somites, the peripheral nervous system and smooth muscle of all arteries. Loss of HeyL expression at the level of nascent somites in Notch1 and Delta-like1 knockout mutants implicates HeyL as a Notch effector during somite formation. Furthermore, HeyL expression in vascular smooth muscle cells and in the thymus strikingly overlaps with that of Notch3, mutations of which underlie the CADASIL vascular disorder.

Comparative analysis of the human and mouse Hey1 promoter: Hey genes are new Notch target genes.

Hey genes (Hey1, Hey2 and HeyL) encode a new group of basic helix-loop-helix transcription factors that are related to the hairy/Enhancer of split genes. In the present study, we cloned and characterized the promoter region of the human and mouse Hey1 gene. The transcription initiation site was located 138 nucleotides upstream of the start codon. There is a minimal sequence element (nt -30 to -247) that is essential and important for basal transcription in three different cell types. Further upstream, a highly conserved sequence block (nt -324 to -646; approximately 90% human/mouse similarity) could be identified that contains several putative binding sites for transcription factors and likely represents an important regulatory region for this gene. Cotransfection experiments demonstrated that the mHey1 promoter activity is up-regulated by the activated form of all four mammalian Notch receptors via two functional RBP-Jkappa binding sites. The other members of the Hey gene family, Hey2 and HeyL, also possess RBP-Jkappa binding sites and they are similarly responsive to Notch signaling. Thus, our data clearly demonstrate that Hey genes form a new class of Notch signal transducers that should prove to be relevant in various developmental processes.

Molecular analysis of E-cadherin and cadherin-11 in Wilms' tumours.

Different studies of Wilms' tumours have demonstrated a loss of heterozygosity (LOH) of chromosome 16q ranging from 17 to 25%. In order to search for a potential tumour suppressor gene on 16q, we chose the calcium-dependent cell adhesion molecules E-cadherin and cadherin-11 as candidate genes, which are both located on the long arm of chromosome 16. E-cadherin is known to be expressed in epithelial structures, whereas cadherin-11 is supposed to be expressed in mesenchymal structures and developing epithelium, including renal tubules. For the present study, fresh frozen tissue from 30 Wilms' tumours and corresponding non-tumour tissues were analysed. Single nucleotide polymorphisms of the E-cadherin and cadherin-11 genes were chosen and analysed for allelic inactivation by polymerase chain reaction (PCR) amplification and sequence analysis. Loss of expression of one E-cadherin allele was seen in 10% (2/20) of the informative cases. Two out of 11 informative cases (18%) showed loss of expression of one cadherin-11 allele. No length alterations of either the E-cadherin or the cadherin-11 messenger RNAs were identified using reverse transcription PCR and agarose gel electrophoresis in tumour tissue. Sequencing of the entire E-cadherin coding region in seven cases showed the wild-type sequence. These data imply that E-cadherin and cadherin-11 are not likely to play typical tumour suppressor roles in Wilms' tumour. Interestingly, the E-cadherin immunohistochemistry showed a deviation from the normal reaction pattern in 50% of the cases, with 27% (8/30) showing an apical or cytoplasmic reaction and 23% (7/30) being completely negative. Northern blot analysis revealed that the overall expression of cadherin-11 is much stronger than that of E-cadherin. In several cases, the expression levels of the two genes were inversely correlated, suggesting the existence of a regulatory mechanism. Analysis of differential expression of the various cadherins and their subsequent signal transduction pathways might contribute to a better understanding of the complexity of Wilms' tumour formation.

Characterization of the human and mouse HEY1, HEY2, and HEYL genes: cloning, mapping, and mutation screening of a new bHLH gene family.

Many basic helix-loop-helix (bHLH) transcription factors are known as key regulators of embryonic development or differentiation in various species. We have isolated and characterized three new hairy-related bHLH transcription factor genes from mouse and human (hairy and Enhancer-of-split related with YRPW motif; HEY1, HEY2, and HEYL). All three HEY genes have a similar genomic structure with five exons. Together with a highly related Drosophila homologue, they form a new bHLH gene subfamily that is different from both hairy and the known vertebrate Hes and Her genes. While the overall structure with the bHLH domain, Orange domain, and WRPW motif is similar, the last motif is changed to KPYRPWG in Hey1/2 and absent in HeyL. This and other sequence features suggest Hey proteins to have unique functional properties. The genes were mapped by fluorescence in situ hybridization and RH mapping to the following human chromosomes: (HEY1) 8q21, (HEY2) 6q21, and (HEYL) 1p34.3. Based on expression patterns and map location, HEY genes are candidates for several human or mouse disease loci. However, initial screening of DNA from affected individuals for two human disorders and four mouse mutants did not reveal any diagnostic alterations in the coding regions.

Activation of the Notch pathway in the hair cortex leads to aberrant differentiation of the adjacent hair-shaft layers.

Little is known about the mechanisms underlying the generation of various cell types in the hair follicle. To investigate the role of the Notch pathway in this process, transgenic mice were generated in which an active form of Notch1 (Notch(DeltaE)) was overexpressed under the control of the mouse hair keratin A1 (MHKA1) promoter. MHKA-Notch(DeltaE) is expressed only in one precursor cell type of the hair follicle, the cortex. Transgenic mice could be easily identified by the phenotypes of curly whiskers and wavy, sheen pelage hair. No effects of activated Notch on proliferation were detected in hair follicles of the transgenic mice. We find that activating Notch signaling in the cortex caused abnormal differentiation of the medulla and the cuticle, two neighboring cell types that did not express activated Notch. We demonstrate that these non-autonomous effects are likely caused by cell-cell interactions between keratinocytes within the hair follicle and that Notch may function in such interactions either by directing the differentiation of follicular cells or assisting cells in interpreting a gradient emanating from the dermal papilla.

Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations.

The hSNF5/INI1 gene which encodes a member of the SWI/SNF chromatin ATP-dependent remodeling complex, is a new tumor suppressor gene localized on chromosome 22q11.2 and recently shown to be mutated in malignant rhabdoid tumors. We have searched for hSNF5/INI1 mutations in 229 tumors of various origins using a screening method based on denaturing high-performance liquid chromatography. A total of 31 homozygous deletions and 36 point alterations were identified. Point mutations were scattered along the coding sequence and included 15 nonsense, 15 frameshift, three splice site, two missense and one editing mutations. Mutations were retrieved in most rhabdoid tumors, whatever their sites of occurrence, indicating the common pathogenetic origin of these tumors. Recurrent hSNF5/INI1 alterations were also observed in choroid plexus carcinomas and in a subset of central primitive neuroectodermal tumors (cPNETs) and medulloblastomas. In contrast, hSNF5/INI1 point mutations were not detected in breast cancers, Wilms' tumors, gliomas, ependymomas, sarcomas and other tumor types, even though most analyzed cases harbored loss of heterozygosity at 22q11.2 loci. These results suggest that rhabdoid tumors, choroid plexus carcinomas and a subset of medulloblastomas and cPNETs share common pathways of oncogenesis related to hSNF5/INI1 alteration and that hSNF5/INI1 mutations define a genetically homogeneous family of highly aggressive cancers mainly occurring in young children and frequently, but not always, exhibiting a rhabdoid phenotype.

A 7.5 Mb sequence-ready PAC contig and gene expression map of human chromosome 11p13-p14.1.

The region p13 of the short arm of human chromosome 11 has been studied intensely during the search for genes involved in the etiology of the Wilms' tumor, aniridia, genitourinary abnormalities, mental retardation (WAGR) syndrome, and related conditions. The gene map for this region is far from being complete, however, strengthening the need for additional gene identification efforts. We describe the extension of an existing contig map with P1-derived artificial chromosomes (PACs) to cover 7.5 Mb of 11p13-14.1. The extended sequence-ready contig was established by end probe walking and fingerprinting and consists of 201 PAC clones. Utilizing bins defined by overlapping PACs, we generated a detailed gene map containing 20 genes as well as 22 anonymous ESTs which have been identified by searching the RH databases. RH maps and our established gene map show global correlation, but the limits of resolution of the current RH panels are evident at this scale. Initial expression studies on the novel genes have been performed by Northern blot analyses. To extend these expression profiles, corresponding mouse cDNA clones were identified by database search and employed for Northern blot analyses and RNA in situ hybridizations to mouse embryo sections. Genomic sequencing of clones along a minimal tiling path through the contig is currently under way and will facilitate these expression studies by in silico gene identification approaches.

Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis.

We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

Screen for genes regulated during early kidney morphogenesis.

Kidney development starts with the reciprocal induction of mesenchymal and ureteric bud cells which leads to condensation, epithelialization, and nephron formation in the mesenchyme. To identify changes in gene expression during these processes, we compared differential display polymerase chain reaction (PCR) profiles of uninduced and induced mesenchymal cells. In vitro kidney development in the form of the transfilter organ culture system was used to generate homogeneous cell populations for this type of comparison. Here we describe the isolation of known and novel genetags from this screening. Among the known genes the ufo receptor tyrosine kinase, sFRP2, and the groucho related gene (grg) were verified as being upregulated upon induction. With four of eight novel genes tested, Northern blot analysis proved to be sensitive enough to confirm differential expression. To improve sensitivity and gain additional spatial information, in situ hybridization was performed. Expression analysis of two differential display PCR products, designated C0-5 and M2-4, demonstrated the cell-specific and dynamic expression of these novel genetags in the developing kidney and other tissues. C0-5 transcripts were expressed in the ureteric bud, S-shaped bodies, and in the collecting system. Signals for M2-4, a gene not detectable by Northern blot analysis, were only found in condensing mesenchymal cells and early differentiation stages, but not in the collecting ducts. The large fraction of novel genetags from the present screening that have not yet been analyzed provides a rich resource to clone genetic networks regulating early nephrogenesis.

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.