Longitudinal stability of molecular alterations and drug response profiles in tumor spheroid cell lines enables reproducible analyses.

The utility of patient-derived tumor cell lines as experimental models for glioblastoma has been challenged by limited representation of the in vivo tumor biology and low clinical translatability. Here, we report on longitudinal epigenetic and transcriptional profiling of seven glioblastoma spheroid cell line models cultured over an extended period. Molecular profiles were associated with drug response data obtained for 231 clinically used drugs. We show that the glioblastoma spheroid models remained molecularly stable and displayed reproducible drug responses over prolonged culture times of 30 in vitro passages. Integration of gene expression and drug response data identified predictive gene signatures linked to sensitivity to specific drugs, indicating the potential of gene expression-based prediction of glioblastoma therapy response. Our data thus empowers glioblastoma spheroid disease modeling as a useful preclinical assay that may uncover novel therapeutic vulnerabilities and associated molecular alterations.

[Treatment of an osteo-fascio-cutaneous defect of the femur with a serial connected flow-through free-flap]. / Therapie eines osteo-fascio-cutanen Femurdefektes mit in Reihe geschalteter Durchflusslappenplastik.

Covering large osteo-fascio-cutaneous defects after debridement often calls for elaborate soft tissue reconstruction. Large tissue loss or structural damage that requires distinct repair is rarely coverable by a single conventional flap. Here, we report the case of serial flap coverage using sequentially connected fibular and latissimus dorsi free flaps.

The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for γ-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual γ-H2AX foci and a delayed decline of γ-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual γ-H2AX foci. Nuclear AhR was found in complex with γ-H2AX, DNA-PK, ATM and Lamin A. AhR and γ-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair.

[An immobilising malignant phyllodes tumour of the breast]. / Immobilisierendes malignes Cystosarcoma phylloides der Brust.

Phyllodes tumours of the breast are rare occurrences, but they can reach huge dimensions. Descriptions of tumours whereby the women are immobilised as a consequence of the size of the tumour, are hard to find in the literature. In this presentation we show a case of a woman in otherwise healthy condition with a giant phyllodes tumour of her left breast. Because of the weight of the tumour, the patient could not leave her bed for more than 6 months.

[Long-term outcome after surgical treatment of breast asymmetry: analysis of the patient's perspective in 51 cases]. / Langzeitresultate nach chirurgischer Behandlung von Brustasymmetrien aus Sicht der Patientinnen: Eine Analyse von 51 Fällen.

INTRODUCTION: Literature provides 3 studies only investigating the long-term outcome after surgical correction of breast asymmetry. The goal of this study was to analyse from a patient's perspective, which factors influence postoperative satisfaction most. PATIENTS AND METHODS: All patients undergoing surgical treatment for breast asymmetry between 2000 and 2009 were included. With help of the visual analogue scale the patients conducted a subjective assessment of their own long-term result using the following parameters: overall satisfaction, symmetry, size, shape, scarring and sensitivity. Anthropometric measurements of the breasts followed. RESULTS: 51 patients (80% follow-up) were seen 2-11 (mean 5±2.5) years postoperatively. The following mean values were recorded for overall satisfaction 8.31 (±1.91), symmetry 7.86 (±2.25), size 8.42 (±1.93), shape 8.12 (±2.03), scarring 7.82 (±1.94) and sensitivity 7.92 (±2.19). Overall satisfaction increased significantly with good scores for the parameters symmetry [p=0.01] and shape [p=0.048]. Neither size [p=0.46] nor scarring [p=0.69] nor sensitivity [p=0.34] had a statistically significant influence on overall satisfaction. Furthermore, overall satisfaction did not depend on the surgical technique, preoperative size, preoperative asymmetry, age of the patient at time of surgery, period of time between the operation and the assessment, resected weight (absolute and difference between left and right) or on postoperative symmetry of the nipple areola complex. CONCLUSION: In our patients, long-term overall satisfaction after surgical correction of breast asymmetry was primarily dependent on symmetry and shape. Size, scarring and sensitivity did not have a statistically significant influence on postoperative overall satisfaction. This also applied to preoperative size, preoperative extent of asymmetry, age of the patient at time of surgery, surgical technique and the time span between the operation and the assessment.

[The interesting case: unilateral hypoplasia of the breast in Becker nevus syndrome]. / Der interessante Fall: einseitige Hypoplasie der Brust bei Becker Nävus Syndrom.

The Becker nevus syndrome is defined by the association of a Becker's nevus with ipsilateral breast hypoplasia and/or musculoskeletal disorders. There are only a few dozen case reports in the literature. We here present the case of a 20-year-old female patient who was treated in our clinic due to a breast asymmetry.

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1.

Exposure of keratinocytes (KC) to ultraviolet (UV) radiation results in the initiation of apoptosis, a protective mechanism that eliminates cells harboring irreparable DNA damage. Hence, a manipulation of UV-induced apoptosis may significantly influence photocarcinogenesis. We have discovered that the aryl hydrocarbon receptor (AHR), a key regulator of drug metabolism and an UVB-sensitive transcription factor, serves an anti-apoptotic function in UVB-irradiated human KC. Chemical and shRNA-mediated inhibition of AHR signaling sensitized KC to UVB-induced apoptosis by decreasing the expression of E2F1 and its target gene checkpoint kinase 1 (CHK1). The decreased expression of these cell-cycle regulators was due to an enhanced expression of p27(KIP1) and an associated decrease in phosphorylation of both cyclin-dependent kinase 2 and its substrate molecule retinoblastoma protein. The subsequent inhibition of E2F1 autoregulation and downstream CHK1 expression resulted in an enhanced susceptibility of damaged cells to undergo apoptosis. Accordingly, ectopic overexpression of either E2F1 or CHK1 in AHR-knockdown KC attenuated the observed sensitization to UVB-induced apoptosis. Using an AHR-knockout SKH-1 hairless mouse model, we next demonstrated the physiological relevance of the anti-apoptotic function of AHR. In contrast to their AHR-proficient littermates, the constitutive expression of E2F1 and CHK1 was significantly reduced in the skin of AHR-knockout mice. Accordingly, a single exposure of the animals to UVB resulted in an enhanced cleavage of caspase-3 in the skin of AHR-knockout mice. These results identify for the first time the AHR-E2F1-CHK1 axis as a novel anti-apoptotic pathway in KC, which may represent a suitable target for chemoprevention of non-melanoma skin cancer.

[One-stage reconstruction of facial defects after tumor resection with the Integra system]. / Integra zur einzeitigen Defektdeckung nach Tumorresektion im Gesichtsbereich.

BACKGROUND: In 2010 excellent aesthetic results after basal cell carcinoma excision and one-stage coverage with Integra without split thickness skin graft (STSG) were published in a series of 10 Asian patients. Our aim in this study was to verify these results in a series of Caucasian patients and evaluate this procedure as a possible new standard. PATIENTS AND METHODS: 6 patients with facial basal cell carcinoma were treated by regular excision with 3 mm safety margins and one-stage coverage with Integra without STSG, followed by a clinical evaluation and fotodocumentation. RESULTS: In 3 patients local infection occurred with a complete loss of the Integra. 2 out of these 3 patients showed an unaesthetic scar and are considering another surgical approach for correction. The other 3 patients had an uneventful course, unfortunately 2 out of these patients (67%) developed an unaesthetic scar as well and are also considering surgical correction. CONCLUSION: Because of aesthetically unsatisfactory results and high infection rates we abandoned this procedure after 6 patients only. Our standard remains excision with 3 mm safety margins, histological analysis and one-stage repair with local facial flaps.

Activation of the aryl hydrocarbon receptor sensitises human keratinocytes for CD95L- and TRAIL-induced apoptosis.

In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[a]pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by ß-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure.

Automated neurosphere sorting and plating by the COPAS large particle sorter is a suitable method for high-throughput 3D in vitro applications.

Existing guidelines for testing developmental neurotoxicity (DNT) propose investigations in rodents, which are ethically questionable as well as time and cost intensive. Thus, there is international agreement that predictive in vitro methods are needed to increase efficiency of testing and limit the number of animals used. One of a variety of novel approaches for DNT testing utilizes neurospheres, three-dimensional aggregate cultures of primary normal neural progenitor cells (NPCs). Because sorting and plating of single neurospheres is one of the most time-consuming steps within the assay, the aim of this study was to evaluate if the complex object parametric analyzer and sorter (COPAS PLUS(TM), Union Biometrica Inc.) is a suitable tool for automated sorting and plating of neurospheres. The results of the comparison of NPC viability, proliferation, migration, differentiation and intracellular oxidative stress between manually and COPAS sorted and plated neurospheres of different species show that the automation by the COPAS instrument does not influence the basic performance of neurospheres. Therefore, we consider the COPAS instrument as a useful tool for higher throughput neurosphere research in toxicology, neuroregeneration, brain development, drug development and brain aging research.

Interleukin-7 (IL-7) and IL-7 splice variants affect differentiation of human neural progenitor cells.

Alternative splicing of pre-mRNA increases proteomic diversity, a crucial mechanism in defining tissue identity. We demonstrate differentially spliced interleukin (IL)-7 in distinct anatomic areas in the adult, in developing human brains and in normal human neuronal progenitor (NHNP) cells. IL-7c (c, the canonical form spanning all six exons) or its variants IL-7 delta 5, delta 4 or delta 4/5 were cloned and expressed as recombinant proteins. IL-7 and splice variants were able to shift the differentiation of NHNP cells as compared with the diluent control (P<0.01) defined by anti-beta (III)-tubulin and glial fibrillary acidic protein expression, with different degrees (IL-7c>delta 4/5>IL-7 delta 5); IL-7 delta 4 exhibited a significantly weaker potency. Differentiation was confirmed by transcriptome analysis of IL-7c-stimulated neural NHNP cells, resulting in 58 differentially expressed genes; some of these are involved in neural differentiation, for example, the developmentally regulated transcription factor krüppel-like factor 12, musashi 2, a translational regulator of cell fate or the sonic hedgehog receptor patch 1. This suggests that IL-7 influences neural development at a molecular level by participating in human brain architecture through glia cell formation: a paradigm that alternative splicing in cytokines, for example, for IL-7, has a physiological role in human organ development and progenitor cell differentiation.

[Sensory nerve function after unilateral digital vascular-nerve injury: nerve repair with and without arterial repair]. / Sensible Nervenfunktion nach unilateraler digitaler Gefäss-Nerven-Verletzung: Nervennaht mit und ohne Arterienanastomose.

PURPOSE: This study compares the clinical results of microsurgical nerve repairs in unilateral digital arterial-nerve-injuries with and without repair of the finger artery. PATIENTS AND METHODS: Between January 2000 and May 2007 a total of 81 patients with unilateral digital vascular nerve bundle lesions, including concomitant soft-tissue tendon lesions, were operated on the emergency day. Forty of the 56 patients treated with a nerve repair alone took part in a follow-up after an average of 47 (7-87) months. Twenty of the 25 patients treated with a microsurgical arterial and nerve repair took part in a follow-up after an average of 12 (6-66) months; 3 patients were excluded due to a negative digital Allen-test. In addition to anamnestic data, peripheral nerve function was evaluated by the static and the moving two-point discrimination test, and by Semmes-Weinstein pressure aesthesiometer in the autonomous zone of the affected side of the injured finger. Stereognosis also was examined. The patency of the reconstructed digital artery was tested by a digital Allen-test. RESULTS: No statistically significantly worse results were found in patients with a nerve repair alone compared to patients with additional repair of the finger artery. CONCLUSION: Repair of the finger artery therefore appears to offer no improvement of the clinical outcome following nerve repair in unilateral injury of a digital arterial-nerve bundle.

Internal thoracic vessels as recipient vessels for free flap reconstruction in head and neck surgery.

In rare cases the usage of the internal thoracic vessels as recipient vessels in reconstructive surgery of the head and neck region with free tissue transfer is a challenging but valid alternative if local recipient vessels are unusable.

Cavernous haemangioma in the hand mimicking subacute tenosynovitis.

A case of cavernous haemangioma arising from the superficial palmar arch is described. The initial symptoms were those of a subacute tenosynovitis. Surgical exploration showed that the tumor was not affecting the flexor tendons. It was completely resected and the patient had full recovery of hand function.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Functional characterization of cyclooxygenase-2 polymorphisms.

Cyclooxygenases (COX)-1 and -2 are the key enzymes in the conversion of arachidonic acid to prostaglandins. COX-2 appears to play an emerging role in inflammation and carcinogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of numerous diseases and reduce the risk of developing colorectal cancer. Polymorphisms in the COX-2 gene could alter enzyme expression, function, and/or the response to NSAIDs. Therefore, they could modify individual risks for developing cancer and other diseases or the occurrence of side effects or sensitivity toward selective or nonselective COX inhibitors. We sequenced the COX-2 gene of 72 individuals and identified rare polymorphisms in the promoter and the coding region. A COX-2 molecular model was used to locate the coding region polymorphisms relative to functional sites in the protein, and the COX-2 V511A polymorphism was very near to the active site. This variant protein was expressed, and function was evaluated, but no difference was detected in metabolism of the COX-2 substrates, arachidonic acid, linoleic acid, and 2-arachidonyl glycerol, compared with the wild type. The Km values for arachidonic acid showed no differences between the COX-2 wild type and V511A mutant. Inhibition with selective or nonselective COX inhibitors was essentially the same for the two enzymes. The absence of functionally important polymorphisms in the COX-2 gene may suggest that there has been selective pressure against those single nucleotide polymorphisms because of the critical role of this enzyme in maintenance of homeostasis.

[Mallet-thumb--presentation and discussion of 3 cases of a rare lesion]. / Der Mallet-Daumen--Vorstellung und Diskussion von drei Fällen einer seltenen Läsion.

Three cases of closed extensor tendon ruptures without osseous involvement in Verdan's zone 1 of the thumb (Mallet thumb) are presented and the various treatment options discussed based on the pertinent literature. As a consequence of the special anatomy of the thumb's extensor tendons which differs from that in the fingers, we recommend surgical treatment of this rare lesion by transosseous refixation of the ruptured tendon. Thus, early postoperative motion can be initiated resulting in rapid recovery of complete function.

[Reduction of breasts ... Hans Schaller and the first mammaplasty in 1561. Contribution to history of medicine]. / Vom Amputieren der Brüste ... Hanns Schaller und die erste Mammaplastik 1561. Beitrag zur Medizingeschichte.

In comparison with other surgical procedures concerning the breast, the history of reduction mammaplasty is relatively short. Some authors have mistaken Paulos of Aegina for the pioneer in this field, although he occupied himself exclusively with gynaecomastia. Since some decades Hanns Schaller, the so-called "barber" of Augsburg, is considered to be the first surgeon to have performed a reduction mammaplasty by breast amputation in 1561. However, exact references have not been available so far. We found the original text containing the description of the procedure written by a contemporary in a rather unexpected place as well as some details about the surgeon. We conclude that Hanns Schaller was the first surgeon to undertake a reduction mammaplasty in an otherwise healthy woman in order to relieve her physical symptoms. Undoubtedly, his intentions were purely functional without any further aesthetic considerations.

Xenobiotic metabolism, genetic polymorphisms and male infertility.

Male reproductive function may be impaired by various occupational and environmental chemical agents. The majority of these xenobiotics, however, require metabolic activation in order to exert adverse effects via covalent interactions between intermediate metabolites and cellular macromolecules such as DNA or protein. In addition, metabolization may alter endocrine-disrupting properties of xenobiotics. Thus tissue-specific expression and regulation of multiple xenobiotic-metabolizing enzymes are likely to play an important role in chemically induced disorders of male reproductive organs. Recent studies suggest that genetic polymorphisms underlying inter-individual and inter-ethnic variability of xenobiotic metabolism modulate susceptibility to male reproductive disorders. For cytochrome P450 1A1 (CYP1A1), a key enzyme in extra-hepatic metabolic activation of lipophilic xenobiotics, increased frequencies of two genetically linked polymorphisms have been found among infertile men.

Simultaneous avulsion fracture of the insertion of the ulnar and radial collateral ligaments of the metacarpophalangeal joint of the thumb.

We present a case of simultaneous avulsion fracture of the insertion on the volar base of the proximal phalanx of the ulnar and radial collateral ligaments of the metacarpophalangeal joint of the thumb. To our knowledge this combination has never been published before. The mechanism of this injury is not clearly understood.