UVA-induced metabolic changes in non-malignant skin cells and the potential role of pyruvate as antioxidant.

The exposure to UVA (320-400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts-normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.

Material properties and bioactivity of a resin infiltrant functionalized with polyhedral oligomeric silsesquioxanes.

OBJECTIVES: To investigate the effect of methacrylate polyhedral oligomeric silsesquioxanes (POSS-8) on various material properties and mineral precipitation potential of a resin infiltrant. METHODS: A TEGDMA-based resin infiltrant was mixed with 0.5, 1, 3, 5 or 10 wt% POSS-8 or left unchanged (control). Degree of conversion (DC), water sorption (WS), viscosity, elastic modulus (E-modulus), flexural strength (FS), Knoop microhardness (KHN) and softening ratio (SR) were assessed. Growth of calcium phosphate (Ca/P) precipitates infiltrant-treated bovine enamel and dentin specimens immersed in artificial saliva or artificial dentinal fluid, respectively, for 28 days was analyzed by scanning electron microscopy and energy-dispersive X-ray spectroscopy. For viscosity assessment, pure TEGDMA filled with 0-10 wt% POSS-8 was used. Statistical analyses were performed using ANOVA and Tukey's post-hoc tests (p < 0.05). RESULTS: POSS-8 did not change the flexural strength, water sorption and softening ratio. The apparent degree of conversion was increased at lower concentrations only while E-modulus remained constant in almost all groups. The particles led to a slight decrease of KHN at concentrations below 3%. The effect on viscosity is comparable to the reinforcement effect. Ca/P precipitates formed on dentin specimens treated with POSS-8-filled infiltrant after 4 weeks of immersion, but were not detected on the control infiltrant. The mineral precipitation on enamel was not improved by POSS-8. SIGNIFICANCE: POSS-8 particles did not worsen the material properties of the resin infiltrant, while the Ca/P precipitation on dentin was stimulated.

Enhancing neurosteroid synthesis--relationship to the pharmacology of translocator protein (18 kDa) (TSPO) ligands and benzodiazepines.

INTRODUCTION: The treatment of anxiety disorders is still a challenge; novel pharmacological approaches that combine rapid anxiolytic efficacy with fewer side effects are needed. A promising target for such compounds is the mitochondrial translocator protein (18 kDa) (TSPO). TSPO plays an important role for the synthesis of neurosteroids, known to modulate GABAA receptors, thereby exerting anxiolytic effects. METHODS: We investigated the pharmacological profile of 2 well established TSPO ligands (XBD173 and etifoxine) compared to the benzodiazepine diazepam with regard to TSPO binding affinity, TSPO expression and neurosteroidogenesis. RESULTS: In BV-2 microglia and C6 glioma cells all compounds significantly enhanced TSPO protein expression. Radioligand binding assays revealed the highest binding affinity to TSPO for XBD173, followed by diazepam and etifoxine. Pregnenolone synthesis was most potently enhanced by etifoxine. DISCUSSION: Etifoxine turned out to be the most potent enhancer of neurosteroidogenesis, although its binding affinity to TSPO was lowest. These results indicate that the efficacy of TSPO ligands to stimulate neurosteroid synthesis, thereby leading to anxiolytic effects cannot be concluded from their binding affinity to TSPO.

Protein trafficking from synapse to nucleus in control of activity-dependent gene expression.

Long-lasting changes in neuronal excitability require activity-dependent gene expression and therefore the transduction of synaptic signals to the nucleus. Synaptic activity is rapidly relayed to the nucleus by membrane depolarization and the propagation of Ca(2+)-waves. However, it is unlikely that Ca(2+)-transients alone can explain the specific genomic response to the plethora of extracellular stimuli that control gene expression. In recent years a steadily growing number of studies report the transport of proteins from synapse to nucleus. Potential mechanisms for active retrograde transport and nuclear targets for these proteins have been identified and recent reports assigned first functions to this type of long-distance signaling. In this review we will discuss how the dissociation of synapto-nuclear protein messenger from synaptic and extrasynaptic sites, their transport, nuclear import and the subsequent genomic response relate to the prevailing concept behind this signaling mechanism, the encoding of signals at their site of origin and their decoding in the nucleus.

[Position Paper of the Youth Forum DGOU 2010 - part 2]. / Klausurtagung - Positionspapier Junges Forum der DGOU 2010 - Teil 2.

The aim of the "Youth Forum of the DGOU" during the Convention 2010 in Heidelberg was to place a statement concerning the professional politics in the field of Orthopaedic and Trauma Surgery. The Bologna Process realizes a standardization of the academic training within the European Union. For medicine this concept would raise the opportunity to opt out after three years with a bachelor degree applying for alternative occupations within the health care system. However, these alternative occupations are rarely defined and, in addition, the current structure of medical school in Germany provides the highest possible education for doctors in a direct and very well established way. Thus, reforming medical school in Germany into a Bachelor-master's system is an ambivalent approach, which considers a thorough reappraisal. There is currently no necessity for an speciality training in emergency medicine. The rapid and qualified treatment by the specific subspecialty provides a high standard of care for the patient. The high frequency exposure of the trauma and orthopaedic resident with emergency cases is an essential part of the current professional training.The "Junge Forum der DGOU" continues to understand the speciality training "Facharzt für Orthopädie und Unfallchirurgie" as the basic module of the profession. After that it should be possible to continuing subspecialty training and obtaining "spezielle Unfallchirurgie" bzw "spezielle orthopädische Chirurgie". After that further subspecialty training should be encouraged.

[Position Paper of the Youth Forum DGOU 2010 - part 1]. / Klausurtagung - Positionspapier Junges Forum der DGOU 2010 - Teil 1.

The aim of the "Youth Forum of the DGOU" during the Convention 2010 in Heidelberg was to place a statement concerning the professional politics in the field of Orthopaedic and Trauma Surgery. The emigration of young German physicians, the occupational image of the Physician Assistant and the quality of the German residency programs in Orthopaedic and Trauma Surgery we discussed as main topics. The main reason for young German physicians to go abroad is, besides better work conditions and less bureaucracy, the better structured education during residency. Therefore the "Youth Forum" generally supports the concept of the "physician assistant" as "a relief from non-physician duties rather than discussing the delegation of true physician duties". The "Youth Forum" is looking forward to collaborating on the improvement of the ongoing education of residents. In this regard, Orthopaedic and Trauma Surgery needs to become more attractive for young academics. We also support a uniform and nationwide curriculum, which guarantees a structured education to improve the theoretic, practical and academic skills of the future specialist in orthopaedic and trauma surgery. Additional surveys and interviews among the current generation of residents are needed to further specify the potential goals of such a curriculum. We would like to discuss the future of our speciality with our colleagues. Therefore different communication platforms including our website http://www.jf-dgou.de have been created.

Interleukin-10 promotes NK cell killing of autologous macrophages by stimulating expression of NKG2D ligands.

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

CD rats as an animal model in the experimental study of laparoscopic hernia repair.

PURPOSE: The aim of this study was to evaluate the technical feasibility and efficacy of a sutureless laparoscopic hernia repair in a small animal model. The objective was to occlude the processus vaginalis with biocompatible fleece and/or fibrin glue as an alternative to suturing. METHODS: Sixty-three male CD rats were randomly assigned to one of three groups. In group A (n=21), the internal inguinal ring was filled with 0.5 ml fibrin glue. The second group (B, n=21) also received fibrin glue, and a biocompatible fleece was placed on top. The third group consisted of control animals (C, n=21). Eleven rats in each group underwent laparoscopic surgery. The remaining rats were operated using an open technique, and the paraductal lipomas were resected in addition to inguinal ring closure. RESULTS: Complete closure of the internal hernia ring was not achieved in any of the rats, neither in the laparoscopic group nor in the open group or the control group. The paraductal lipoma grew back to its normal size, although resection of the lipoma was performed during the first procedure. CONCLUSION: The physiology of paraductal lipomas in this animal appears to make it an inadequate model for the study of laparoscopic inguinal hernia repair.

A comparison of the synaptic proteome in human chronic schizophrenia and rat ketamine psychosis suggest that prohibitin is involved in the synaptic pathology of schizophrenia.

Many studies in recent years suggest that schizophrenia is a synaptic disease that crucially involves a hypofunction of N-methyl-D-aspartate receptor-mediated signaling. However, at present it is unclear how these pathological processes are reflected in the protein content of the synapse. We have employed two-dimensional gel electrophoresis in conjunction with mass spectrometry to characterize and compare the synaptic proteomes of the human left dorsolateral prefrontal cortex in chronic schizophrenia and of the cerebral cortex of rats treated subchronically with ketamine. We found consistent changes in the synaptic proteomes of human schizophrenics and in rats with induced ketamine psychosis compared to controls. However, commonly regulated proteins between both groups were very limited and only prohibitin was found upregulated in both chronic schizophrenia and the rat ketamine model. Prohibitin, however, could be a new potential marker for the synaptic pathology of schizophrenia and might be causally involved in the disease process.

Expression of CD68 in non-myeloid cell types.

CD68, the human homologue of macrosialin, is commonly regarded as a selective marker for human monocytes and macrophages. Its expression is thought to be regulated by a macrophage-specific promoter. However, several immunohistochemical studies have indicated that CD68 antibodies also react with other haematopoietic and non-haematopoietic cell types. We investigated the expression of CD68 in various primary cells and carcinoma cell lines using immunohistochemistry, flow cytometry, Western blot analysis and qRT-PCR. Weak but significant immunoreactivity was detected in lymphocytes and several tumour cell lines whereas staining of primary fibroblasts and endothelial cells was comparable to macrophages. The intensity of CD68 staining in individual cell types depended on the antibody clone and the fixation technique. Anti-CD68 mAb KP1 should be used with great caution for frozen tissue sections due to its reactivity with a wide variety of cell types. Also, care should be taken when distinguishing macrophages from fibroblasts/stromal cells in paraffin sections after formalin fixation since both cell types are stained highly positive for CD68. In accordance, mRNA expression of CD68 was not only detected in macrophages and monocytes but also in fibroblasts as well as endothelial cells and tumour cells, although with a varying intensity. Cloning of full length 5'-sequences and determination of transcription start sites shows that macrophages and fibroblasts initiate transcription within the known promoter region; however, from different start sites, indicating alternative promoter architecture in myeloid versus non-myeloid cells. We suggest that CD68 is not a selective macrophage marker but rather a lysosomal protein that is enriched in macrophages.

Differentiation of human tumour-associated dendritic cells into endothelial-like cells: an alternative pathway of tumour angiogenesis.

Until recently, the only accepted mechanism of tumour vascularization was the sprouting of endothelial cells (EC) from pre-existing vessels, while recent studies suggest the contribution of stem cell-derived endothelial progenitors as well as cells from the myeloid lineage. Here, we show a new way of endothelial differentiation that involves the specific modulation of monocytes by the tumour environment. The tumour milieu is characterized by the presence of cytokines and lactate which induce the differentiation of tumour-invading monocytes into tumour-associated dendritic cells (DC). Additional incubation of tumour-associated DC with pro-angiogenic factors, such as vascular endothelial growth factor and oncostatin M, led to transdifferentiation into endothelial-like cells. The cells showed strong expression of von Willebrand factor and VE-Cadherin, both classical EC markers, while leukocytic markers were reduced. In addition, they were able to form network-like structures on matrigel, which could be blocked by the DNA-based drug Defibrotide. This finding may be of great therapeutic relevance for tumour therapy.

Decay of 1+ states as a new probe of the structure of 0+ shape isomers.

The nuclides 98Mo and 100Mo have been studied in photon-scattering experiments by using bremsstrahlung produced from electron beams with kinetic energies from 3.2 to 3.8 MeV. Six electromagnetic dipole transitions in 98Mo and 19 in 100Mo were observed for the first time in the energy range from 2 to 4 MeV. A specific feature in the two nuclides is the de-excitation of one state with spin J = 1 to the 0+ ground state as well as to the first excited 0+ state, which cannot be explained in standard models. We present a model that allows us to deduce the mixing coefficients for the two 0+ shape-isomeric states from the experimental ratio of the transition strengths from the J = 1 state to the 0+ ground state and to the 0+ excited state.

The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer's as compared to normal ageing brains.

The EF-hand calcium binding protein Calmyrin (also called CIB-1) was shown to interact with presenilin-2 (PS-2), suggesting that this interaction might play a role in the pathogenesis of Alzheimer's disease (AD). Here we have investigated the distribution of Calmyrin in normal human and AD brain. In normal brain Calmyrin immunoreactivity was unevenly distributed with immunostaining in pyramidal neurones and interneurones of the palaeo-cortex and neocortex, cerebellar granule cells and hypothalamic neurones of the paraventricular, ventromedial and arcuate nuclei. Moderate immunoreactivity was present in hippocampal pyramidal cells and stronger in dentate gyrus neurones. Thalamic and septal neurones were devoid of immunoreactivity. No apparent differences were visible between stainings of brain sections from younger and older nondemented patients. In AD brain a substantial loss of Calmyrin-immunopositive neurones was observed in all regions, especially in cortical areas. Still immunoreactive neurones, however, displayed stronger staining that was especially concentrated in perinuclear regions. Calmyrin immunosignals were in part associated with diffuse and senile plaques. Thus, although protein levels of Calmyrin are low in human forebrain, its cellular localization as well as its altered distribution in AD brain suggest that it may be involved in the pathogenesis of AD.

Delivering cognitive therapy to people with psychosis in a community mental health setting: an effectiveness study.

OBJECTIVE: Cognitive therapy (CT) has been shown to be an efficacious treatment for persistent psychotic symptoms. However, there is some debate regarding whether this is transportable to real life clinical settings. This study aimed to evaluate the effectiveness of CT for psychosis in a community mental health team (CMHT) setting. METHOD: Patients referred for CT for psychosis were naturalistically allocated (determined by the availability of a therapist) to CT or waiting-list (WL)/treatment-as-usual (TAU). Outcome assessments were performed at WL, pre-CT, post-CT and 1-year follow-up. Data from 59 patients were analysed. RESULTS: Random effects regression analyses showed there was a significant improvement, attributable to CT, on most outcome measures, and that many of the symptomatic improvements were maintained at follow-up. Wilcoxon signed ranks tests indicated that there was a significant reduction in psychiatric hospital use following CT. CONCLUSION: These results confirm that CT is an effective treatment for psychosis that is generalizable to a community setting.

Diagnostic and neural analysis of skin cancer (DANAOS). A multicentre study for collection and computer-aided analysis of data from pigmented skin lesions using digital dermoscopy.

BACKGROUND: Early detection of melanomas by means of diverse screening campaigns is an important step towards a reduction in mortality. Computer-aided analysis of digital images obtained by dermoscopy has been reported to be an accurate, practical and time-saving tool for the evaluation of pigmented skin lesions (PSLs). A prototype for the computer-aided diagnosis of PSLs using artificial neural networks (NNs) has recently been developed: diagnostic and neural analysis of skin cancer (DANAOS). OBJECTIVES: To demonstrate the accuracy of PSL diagnosis by the DANAOS expert system, a multicentre study on a diverse multinational population was conducted. METHODS: A calibrated camera system was developed and used to collect images of PSLs in a multicentre study in 13 dermatology centres in nine European countries. The dataset was used to train an NN expert system for the computer-aided diagnosis of melanoma. We analysed different aspects of the data collection and its influence on the performance of the expert system. The NN expert system was trained with a dataset of 2218 dermoscopic images of PSLs. RESULTS: The resulting expert system showed a performance similar to that of dermatologists as published in the literature. The performance depended on the size and quality of the database and its selection. CONCLUSIONS: The need for a large database, the usefulness of multicentre data collection, as well as the benefit of a representative collection of cases from clinical practice, were demonstrated in this trial. Images that were difficult to classify using the NN expert system were not identical to those found difficult to classify by clinicians. We suggest therefore that the combination of clinician and computer may potentially increase the accuracy of PSL diagnosis. This may result in improved detection of melanoma and a reduction in unnecessary excisions.

[The "classical" macrophage marker CD68 is strongly expressed in primary human fibroblasts]. / Der "klassische" Makrophagenmarker CD68 ist in primären humanen Fibroblasten stark exprimiert.

AIM: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker. METHODS: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11. RESULTS: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA. CONCLUSION: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53.

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.