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Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.
Mutations in the PINK1 gene cause autosomal recessive familial Parkinson’s disease (PD). The gene encodes a mitochondrial protein kinase that plays an important role in maintaining mitochondrial function and integrity. However, the pathophysiological link between mutation-related bioenergetic deficits and the degenerative process in dopaminergic neurons remains to be elucidated. We performed phosphorous (31P) and proton (1H) 3-T magnetic resonance spectroscopic imaging (MRSI) in 11 members of a German family with hereditary PD due to PINK1 mutations (PARK6) compared to 23 age-matched controls. All family members had prior 18-Fluorodopa (FDOPA) positron emission tomography (PET). The striatal FDOPA uptake was correlated with quantified metabolic brain mapping in MRSI. At group level, the heterozygous PINK1 mutation carriers did not show any MRSI abnormalities relative to controls. In contrast, homozygous individuals with manifest PD had putaminal GPC, PCr, HEP and β-ATP levels well above the 2SD range of controls. Across all subjects, the FDOPA Ki values correlated positively with MI (r = 0.879, p<0.001) and inversely with β-ATP (r = −0.784, p = 0.008) and GPC concentrations (r = −0.651, p = 0.030) in the putamen. Our combined imaging data suggest that the dopaminergic deficit in this family with PD due to PINK1 mutations relates to osmolyte dysregulation, while the delivery of high energy phosphates was preserved. Our results corroborate the hypothesis that PINK1 mutations result in reduced neuronal survival, most likely due to impaired cellular stress resistance.
In der vorliegenden prospektiven Studie in der neurochirurgischen Klinik an der Johann Wolfgang Goethe-Universität in Frankfurt am Main wurden Oktober 2001 bis August 2006 dreiunddreißig Patienten mit intraduralen Raumforderungen der zervikalen und thorakalen Wirbelsäule unter Betrachtung eines möglichen prädiktiven Wertes der Serumkonzentration des Gliaproteins S100B und des intraoperativen Monitorings untersucht.
Bei jedem Patienten erfolgte das intraoperative Monitoring mittels MEPs und SEPs, außerdem wurde prä- und mehrfach postoperativ die S100B-Serumkonzentration bestimmt. Anschließend wurden die Patienten mindestens 1 Jahr nach dem Eingriff in Bezug auf die noch bestehenden körperlichen Folgen sowie auch die Einschränkungen im alltäglichen Leben und im psychischen Wohlbefinden untersucht.
Der Zusammenhang zwischen dem neurologischen Langzeitoutcome, den Teilergebnissen im intraoperativen Monitoring und im postoperativen S100B-Verlauf zeigte sich deutlich und war statistisch signifikant. Die gemeinsame Betrachtung der Serumkonzentration des Gliaproteins und des IOM unterstrich diese Ergebnisse nochmals, die Spezifität für ein gutes Outcome bei unkompliziertem Monitoring und ausbleibenden Anstieg des S100B lag zum Beispiel bei 100%.
Auch in der Befragung zum alltäglichen Leben anhand des SF-36 zeigte sich ein klarer Zusammenhang zwischen dem klinischen Outcome und den subjektiv aus Patientensicht geschilderten Problemen in der Bewältigung des Alltags. Interessanterweise korrelierte aber die psychische Summenskala des Scores nicht mit dem körperlichen Befund. Die Gruppe des schlechten Outcomes zeigte ein besseres psychisches Wohlbefinden als die Gruppe des guten Outcomes, die aber mit der Normstichprobe vergleichbare Ergebnisse erzielte.
Meeting abstract : Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2012), 23.10.-26.10.2012, Berlin.
Fragestellung: Die Behandlung langstreckiger Knochendefekte ist eine große Herausforderung. Die Masquelet-Technik zur Behandlung solcher Defekte ist eine zweizeitige Operationstechnik. Zuerst erfolgt die Insertion eines PMMA (Polymethylmethacrylat)-Zementspacers in den Knochendefekt, der die Bildung einer Membran induziert. Diese Membran enthält Wachstumsfaktoren und regenerative Zellen, die möglicherweise die Knochenheilung unterstützen. Nach einigen Wochen wird der Zementspacer entfernt und der induzierte Membranschlauch mit Beckenkammspongiosa aufgefüllt. Im weiteren Verlauf kommt es zu einer kompletten Knochenheilung. Ziele dieser Untersuchung waren die Etablierung der Masquelettechnik am Rattenmodell und die Definition eines Zeitpunkts, an welchem die Membran eine ausreichende Festigkeit sowie einen signifikanten Gehalt von Vorläuferzellen aufweist.
Methodik: Nach Genehmigung der Experimente wurden die Femura von 24 männlichen SD-Ratten osteotomiert. Die Lücke (10 mm) wurde mit PMMA-Zement aufgefüllt und mittels Miniplatte stabilisiert. Parallel wurden den Versuchstieren gleich große PMMA-Spacer subcutan unter die Rückenhaut implantiert. Nach 2, 4, bzw. 6 Wochen (W) erfolgte die Entnahme der Membranen. Ein Teil der Membran wurde für (immun)histologische Untersuchungen aufbereitet (CD34, vWF, van Giesson), ein Teil für die in vitro Kultur. Auswachsende Vorläuferzellen in vitro wurden über CD34 und STRO-1-Färbung nachgewiesen. Statistik: Mediane, Kruskal-Wallis-Test, p<0,05 ist signifikant.
Ergebnisse und Schlussfolgerungen: Im zeitlichen Verlauf nahmen die Vaskularisierung (vWF-positive Fläche [%]: 2 W: 1,8; 4 W:1.6 vs 6 W: 6,4), die Dicke der Membran ([µm]: 2 W: 350 vs 4W: 517, 6 W: 592) und der Bindegewebsanteil ([µm]: 2W: 201 vs 4W: 324, 6W: 404) signifikant zu. Der Hauptanteil elastischer Fasern war auf der dem Zement zugewandten Seite, Vaskularisierung war eher auf der Weichteil zugewandten Seite zu finden. Der Anteil CD34 positiver Zellen nahm signifikant ab (2W: 5%, 4 W: 4% vs 6 W: 1%). Auswachsende STRO-1 positive Zellen konnten nur in zweiwöchigen Membranen nachgewiesen werden. Ältere Membranen wiesen einen zunehmenden Anteil seneszenter Zellen auf. Subcutan induzierte Membranen waren vergleichbar, wiesen jedoch tendentiell eine geringere Dicke und keine STRO-1 positiven Zellen auf.
Mit dieser Studie wurde erstmalig die Induktion einer Membran nach Masquelet im Rattenmodell etabliert. Es konnte nachgewiesen werden, dass der strukturelle Aufbau sowie die zellulären Komponenten zeitlichen Änderungen unterliegen und der Ort der Induktion Einfluss auf die zellulären Komponenten der Membran hat. Junge Membranen (2W) enthielten CD34 und STRO-1 positive Zellen. 4W-Membranen enthielten nur CD34 positive Zellen wiesen aber einen signifikanten Bindegewebsanteil auf, der für eine erhöhte mechanische Stabilität spricht. Ob 2 bzw. 4 Wochen alte Membranen den Knochenheilungsprozess fördern, muss in weiterführenden Studien untersucht werden.
Background: Repetitive transcranial magnetic stimulation (rTMS) allows non-invasive stimulation of the human brain. However, no suitable marker has yet been established to monitor the immediate rTMS effects on cortical areas in children.
Objective: TMS-evoked EEG potentials (TEPs) could present a well-suited marker for real-time monitoring. Monitoring is particularly important in children where only few data about rTMS effects and safety are currently available.
Methods: In a single-blind sham-controlled study, twenty-five school-aged children with ADHD received subthreshold 1 Hz-rTMS to the primary motor cortex. The TMS-evoked N100 was measured by 64-channel-EEG pre, during and post rTMS, and compared to sham stimulation as an intraindividual control condition.
Results: TMS-evoked N100 amplitude decreased during 1 Hz-rTMS and, at the group level, reached a stable plateau after approximately 500 pulses. N100 amplitude to supra-threshold single pulses post rTMS confirmed the amplitude reduction in comparison to the pre-rTMS level while sham stimulation had no influence. EEG source analysis indicated that the TMS-evoked N100 change reflected rTMS effects in the stimulated motor cortex. Amplitude changes in TMS-evoked N100 and MEPs (pre versus post 1 Hz-rTMS) correlated significantly, but this correlation was also found for pre versus post sham stimulation.
Conclusion: The TMS-evoked N100 represents a promising candidate marker to monitor rTMS effects on cortical excitability in children with ADHD. TMS-evoked N100 can be employed to monitor real-time effects of TMS for subthreshold intensities. Though TMS-evoked N100 was a more sensitive parameter for rTMS-specific changes than MEPs in our sample, further studies are necessary to demonstrate whether clinical rTMS effects can be predicted from rTMS-induced changes in TMS-evoked N100 amplitude and to clarify the relationship between rTMS-induced changes in TMS-evoked N100 and MEP amplitudes. The TMS-evoked N100 amplitude reduction after 1 Hz-rTMS could either reflect a globally decreased cortical response to the TMS pulse or a specific decrease in inhibition.
Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade.
Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1.
Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism.
Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.
Validierung einer neuen Messmethode zur direkten Bestimmung der Heparin-Konzentration im Blut
(2012)
In dieser Arbeit wurde ein neues Verfahren zur Heparin-Bestimmung (LiSA-H, light scattering assay of heparin) evaluiert. Dieses wurde an der Universität Frankfurt a. M. am Institut für Biophysik entwickelt und ermittelt erstmals die direkte Heparin-Konzentration im Blutplasma. Durch die Analyse der Lichtstreuung einer Plasmaprobe wird die Bildung von Nanopartikeln aus Heparin und Protamin verfolgt. Die Lichtstreuintensität ist dabei proportional zu der in der Probe enthaltenen Heparin-Plasmakonzentration (Heparin-PK). Das Antikoagulans Heparin wird bei Herz-OPs mit Einsatz der Herz-Lungen-Maschine (HLM) verwendet und soll perioperativ eine lutgerinnselbildung in der HLM, sowie Thromboembolien im Patienten verhindern. Am OP-Ende wird die Wirkung durch Protamin antagonisiert, um eine suffiziente Gerinnung wieder herzustellen. Das derzeitige Gerinnungsmanagement basiert auf einem indirekten Messverfahren, der ACT (activated clotting time), welches starken Störeinflüssen, wie z.B. der Hypothermie, Hämodilution und bestimmten Medikamenten unterliegt. Durch die mögliche Falschdosierung der beiden Medikamente, steigt die Gefahr einer Blutung und Thrombose. LiSA-H soll in Zukunft eine zuverlässigere und kostengünstige „point of care― Analyse der Gerinnung und hierdurch eine gezielte Dosierung von Heparin und Protamin ermöglichen, die die Komplikationsrate verringert. In der vorliegenden Studie handelt es sich um eine offene, nicht kontrollierte, prospektive Multicenter-Studie, die mit 50 Patienten am Universitätsklinikum Frankfurt a.M. und 30 Patienten am Kinderherzzentrum Gießen durchgeführt wurde. Es wurde gezeigt, dass die durchschnittliche perioperative Heparin-PK bei Erwachsenen und Kindern bei ca. 5,4 I.E./ml liegt. Es wurde nachgewiesen, dass die Heparin-Clearance bei Kindern (~113 Min.) um das zweifache im Vergleich zu Erwachsenen (~254 Min.), erhöht ist. Besonders hervorzuheben ist die hohe Fehlerquote der ACT-Messung, die bei Erwachsenen in bis zu 1,8 % und bei Kindern in bis zu 20 % der Messungen keinen aussagekräftigen Wert lieferte. Das bedeutet, dass bei Kindern während einer OP bis zu zwei und bei Erwachsenen bis zu drei Stunden keine Information über den aktuellen Gerinnungszustand vorlag. Um eine Validierung der Messergebnisse vorzunehmen, wurden Rückstellproben mit dem Standardlaborverfahren PiCT (Prothrombinase induced clotting time) gemessen. Die Daten aus dem PiCT korrelieren mit den Ergebnissen aus der LiSA-H-Messung wesentlich besser (r² = 0,80), als mit der herkömmlichen ACT-Messmethode (r² = 0,57). Die ermittelten Heparin-PK und die ACT-Werte während einer OP wurden in Chronogrammen dargestellt. Es wurde gezeigt, dass in 30 % der OPs bei Erwachsenen und in 60 % bei Kindern die Messdaten aus der ACT und LiSA-H nur unzureichend synchron bei Nachdosierung mit Heparin anstiegen oder entsprechend der Heparin-Clearance im OP-Verlauf abfielen. Dies zeigte sich besonders kritisch während langandauernder, komplikationsreicher OPs, die einen erhöhten Blutverlust oder sogar Rethorakotomien nach sich zogen, in denen der ACT-Wert eine suffiziente Gerinnung nahe legte, die LiSA-HMessung aber eine noch hohe Heparin-PK nachwies. Erfahrungen aus den klinischen Studien zeigten, dass die Kombination aus der Messung der Heparin-PK und einer Gerinnungsanalyse bei einem ATIII-Mangel von Vorteil ist. Erst die Kombination aus einerseits mehrfach niedrig gemessener ACT-Werte, trotz ggf. Nachdosierungen von Heparin und andererseits ausreichend gemessener Heparin-PK im LiSA-H, kann einen ATIII-Mangelzustand aufdecken. Dadurch können Nach- bzw. Überdosierungen vermieden und damit die Wahrscheinlichkeit für postoperative Komplikationen verringert werden. Der wichtigste Einflussfaktor auf die LiSA-H-Messung ist die Hämodilution, die durch Einbeziehung des Patienten-Blutvolumens (z.B. mit der Nadler-Formel) durch mathematische Korrektur berücksichtigt werden kann. Patientenindividuelle Reaktionen auf gleiche Heparin- und Protamin-Dosierungen sowie eine patientenspezifische Heparin-Clearance zeigten in diesen Studien auf, dass das derzeitige Antikoagulationsmanagement mit den Dosierungsempfehlungen (Körpergewichtsbezogene Dosierung, 1:1 Dosierung von Protamin zur initialen Heparin-Dosis oder der summierten Heparin-Dosis, „pauschale― Nachdosierungen von 5.000 oder 10.000 I.E. Heparin bei ACT < 480) für eine optimale Dosierung der Medikamente unzureichend ist. In Outcome-Studien soll mit der LiSA-H-Messung Dosierungsempfehlungen von Heparin und Protamin ausgearbeitet werden. Außerhalb der Herz-Thorax-Chirurgie eröffnen sich weitere Möglichkeiten, wie z.B. in Dialysezentren und in der Neurochirurgie, für die bereits Studien geplant sind.
Background: Increased glycolytic activity is a hallmark of cancer, allowing staging and restaging with 18F-fluorodeoxyglucose-positron-emission-tomography (PET). Since interim-PET is an important prognostic tool in Hodgkin lymphoma (HL), the aim of this study was to investigate the expression of proteins involved in the regulation of glucose metabolism in the different HL subtypes and their impact on clinical outcome.
Methods: Lymph node biopsies from 54 HL cases and reactive lymphoid tissue were stained for glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA) and lactate exporter proteins MCT1 and MCT4. In a second series, samples from additional 153 HL cases with available clinical data were stained for GLUT1 and LDHA.
Results: Membrane bound GLUT1 expression was frequently observed in the tumor cells of HL (49% of all cases) but showed a broad variety between the different Hodgkin lymphoma subtypes: Nodular sclerosing HL subtype displayed a membrane bound GLUT1 expression in the Hodgkin-and Reed-Sternberg cells in 56% of the cases. However, membrane bound GLUT1 expression was more rarely observed in tumor cells of lymphocyte rich classical HL subtype (30%) or nodular lymphocyte predominant HL subtype (15%). Interestingly, in both of these lymphocyte rich HL subtypes as well as in progressively transformed germinal centers, reactive B cells displayed strong expression of GLUT1. LDHA, acting downstream of glycolysis, was also expressed in 44% of all cases. We evaluated the prognostic value of different GLUT1 and LDHA expression patterns; however, no significant differences in progression free or overall survival were found between patients exhibiting different GLUT1 or LDHA expression patterns. There was no correlation between GLUT1 expression in HRS cells and PET standard uptake values.
Conclusions: In a large number of cases, HRS cells in classical HL express high levels of GLUT1 and LDHA indicating glycolytic activity in the tumor cells. Although interim-PET is an important prognostic tool, a predictive value of GLUT1 or LDHA staining of the primary diagnostic biopsy could not be demonstrated. However, we observed GLUT1 expression in progressively transformed germinal centers and hyperplastic follicles, explaining false positive results in PET. Therefore, PET findings suggestive of HL relapse should always be confirmed by histology.
Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi.
Malignant melanoma of the urethra is a rare tumour that is difficult to diagnose and treat, resulting in a poor prognosis. In this paper, we present the case of a 65-year-old woman who was referred to a gynaecologist because of a urethral mass that mimicked a caruncle. The tumour was removed by local excision, and a pathological analysis revealed a malignant melanoma. Distal urethrectomy was performed after three months with no evidence of residual tumour. There was no evidence of disease at a six-year followup. In this paper, we compare the epidemiology, treatment, staging, and prognosis of vulvar cancer in general to malignant melanoma of the vulva in particular.
Treatment specific competence predicts outcome in cognitive therapy for social anxiety disorder
(2012)
Several studies have demonstrated a positive relationship between competence and outcome in CBT for depression but studies of CBT for anxiety disorders are lacking. The present study explores the relationship between competence and outcome in cognitive therapy (CT) for social anxiety disorder, using hierarchical linear modeling analyses (HLM). Data were drawn from a multicenter randomized controlled trial. Five trained raters evaluated videotapes of two therapy sessions per patient using the Cognitive Therapy Competence Scale for Social Phobia (CTCS-SP). Overall adherence to the treatment manual and patient difficulty were also assessed. Patient outcome was rated by other assessors using the Clinical Global Impression Improvement Scale (CGI-I) and the Liebowitz Social Anxiety Scale (LSAS). Results indicated that competence significantly predicted patient outcome on the CGI-I (β = .79) and LSAS (β = .59). Patient difficulty and adherence did not further improve prediction. The findings support the view that competence influences outcome and should be a focus of training programs. Further research is needed to compare different ways of assessing competence and to understand the complex relationships between competence and other therapy factors that are likely to influence outcome.
AIM: To investigate morphological changes of intestinal smooth muscle contractile fibres in small bowel atresia patients. METHODS: Resected small bowel specimens from small bowel atresia patients (n = 12) were divided into three sections (proximal, atretic and distal). Standard histology hematoxylin-eosin staining and enzyme immunohistochemistry was performed to visualize smooth muscle contractile markers α-smooth muscle actin (SMA) and desmin using conventional paraffin sections of the proximal and distal bowel. Small bowel from age-matched patients (n = 2) undergoing Meckel's diverticulum resection served as controls.
RESULTS: The smooth muscle coat in the proximal bowel of small bowel atresia patients was thickened compared with control tissue, but the distal bowel was unchanged. Expression of smooth muscle contractile fibres SMA and desmin within the proximal bowel was slightly reduced compared with the distal bowel and control tissue. There were no major differences in the architecture of the smooth muscle within the proximal bowel and the distal bowel. The proximal and distal bowel in small bowel atresia patients revealed only minimal differences regarding smooth muscle morphology and the presence of smooth muscle contractile filament markers.
CONCLUSION: Changes in smooth muscle contractile filaments do not appear to play a major role in postoperative motility disorders in small bowel atresia.
Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.
One challenge of squamous cell carcinoma of the head and neck (SCCHN) chemotherapy is a small percentage of tumor cells that arrest in the G0 phase of the cell cycle and are thus not affected by chemotherapy. This could be one reason for tumor recurrence at a later date. The recruitment of these G0-arresting cells into the active cell cycle and thus, proliferation, may increase the efficacy of chemotherapeutic agents. The aim of this study was to investigate whether stimulation with recombinant epidermal growth factor (EGF) or serotonin leads to an increased tumor cell proliferation in xenografts. Detroit 562 cells were injected into NMRI-Foxn1nu mice. Treatment was performed with 15 µg murine or human EGF, or 200 µg serotonin. The control mice were treated with Lactated Ringer's solution (5 mice/group). Tumor size was measured on days 4, 8 and 12 after tumor cell injection. The EGF stimulated mice showed a significantly higher tumor growth compared to the serotonin-stimulated mice and the untreated controls. In the present study, we show that it is possible to stimulate tumor cells in xenografts by EGF and thus, enhance cell proliferation, resulting in a higher tumor growth compared to the untreated control group. In our future investigations, we plan to include a higher number of mice, an adjustment of the EGF dosage and cell subanalysis, considering the heterogeneity of SCCHN tumors.
VEGF (vascular endothelial growth factor) promotes vascularization and remodeling of bone substitutes. The aim of this study was to examine the effect of distinct resorbable ceramic carriers on bone forming capacities of VEGF transfected bone marrow stromal cells (BMSC). A critical size defect of the radius in rabbits was filled either by a low surface scaffold called beta-TCP (tricalciumphsphate) or the high surface scaffold CDHA (calcium deficient hydroxy-apatite) loaded with autologous BMSC, which were either transfected with a control plasmid or a plasmid coding for phVEGF165. They were compared to unloaded scaffolds. Thus, six treatment groups (n = 6 in each group) were followed by X-ray over 16 weeks. After probe retrieval, the volume of new bone was measured by micro-CT scans and vascularization was assessed in histology. While only minor bone formation was found in both carriers when implanted alone, BMSC led to increased osteogenesis in both carriers. VEGF promoted vascularization of the scaffolds significantly in contrast to BMSC alone. Bone formation was increased in the beta-TCP group, whereas it was inhibited in the CDHA group that showed faster scaffold degradation. The results indicate that the interaction of VEGF transfected BMSC with resorbable ceramic carrier influences the ability to promote bone healing.
Speech production involves widely distributed brain regions. This MEG study focuses on the spectro-temporal dynamics that contribute to the setup of this network. In 21 participants performing a cue-target reading paradigm, we analyzed local oscillations during preparation for overt and covert reading in the time-frequency domain and localized sources using beamforming. Network dynamics were studied by comparing different dynamic causal models of beta phase coupling in and between hemispheres. While a broadband low frequency effect was found for any task preparation in bilateral prefrontal cortices, preparation for overt speech production was specifically associated with left-lateralized alpha and beta suppression in temporal cortices and beta suppression in motor-related brain regions. Beta phase coupling in the entire speech production network was modulated by anticipation of overt reading. We propose that the processes underlying the setup of the speech production network connect relevant brain regions by means of beta synchronization and prepare the network for left-lateralized information routing by suppression of inhibitory alpha and beta oscillations.
Background: In Emergency and Medical Admission Departments (EDs and MADs), prompt recognition and appropriate infection control management of patients with Highly Infectious Diseases (HIDs, e.g. Viral Hemorrhagic Fevers and SARS) are fundamental for avoiding nosocomial outbreaks.
Methods: The EuroNHID (European Network for Highly Infectious Diseases) project collected data from 41 EDs and MADs in 14 European countries, located in the same facility as a national/regional referral centre for HIDs, using specifically developed checklists, during on-site visits from February to November 2009.
Results: Isolation rooms were available in 34 facilities (82,9%): these rooms had anteroom in 19, dedicated entrance in 15, negative pressure in 17, and HEPA filtration of exhausting air in 12. Only 6 centres (14,6%) had isolation rooms with all characteristics. Personnel trained for the recognition of HIDs was available in 24 facilities; management protocols for HIDs were available in 35.
Conclusions: Preparedness level for the safe and appropriate management of HIDs is partially adequate in the surveyed EDs and MADs.
Background: To improve and assess the effectiveness of disease management programs (DMPs), it is critical to understand how many people drop out of disease management programs and why.
Methods: We used routine data provided by a statutory health insurance fund from the regions North Rhine, North Wurttemberg and Hesse. As part of the German DMP for type 2 diabetes, the insurance fund received regular documentation of all members participating in the program. We followed 10,989 patients who enrolled in the DMP between July 2004 and December 2005 until the end of 2007 to study how many patients dropped out of the program. Dropout was defined based on the discontinuation of program documentation on a particular patient, excluding situations in which the patient died or left the insurance fund. Predictors of dropout, assessed at the time of program enrolment, were explored using logistic regression analysis.
Results: 5.5% of the patients dropped out of the disease management program within the observation period. Predictors of dropout at the time of enrolment were: region; retirement status; the number of secondary diseases; presence of a disabling secondary disease; doctors recommendations to stop smoking or to seek nutritional counselling; and the completion and outcome of the routine foot and eye exams. Different trends of dropout were observed among retired and employed patients: retired patients of old age, who possibly drop out of the program due to other health care priorities and employed people of younger age who have not yet developed many secondary diseases, but were recommended to change their lifestyle.
Conclusions: Overall, dropout rates for the German disease management programs for type 2 diabetes were low compared to other studies. Factors assessed at the time of program enrolment were predictive of later dropout and should be further studied to provide information for future program improvements.
Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications
(2012)
Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
Resistance of rhabdomyosarcoma to current therapies remains one of the key issues in pediatric oncology. Since the success of most cytotoxic therapies in the treatment of cancer, for example, chemotherapy, depends on intact signaling pathways that mediate programmed cell death (apoptosis), defects in apoptosis programs in cancer cells may result in resistance. Evasion of apoptosis in rhabdomyosarcoma may be caused by defects in the expression or function of critical mediators of apoptosis or in aberrant expression of antiapoptotic proteins. Therefore, the identification of the molecular mechanisms that confer primary or acquired resistance to apoptosis in rhabdomyosarcoma presents a critical step for the rational development of molecular targeted drugs. This approach will likely open novel perspectives for the treatment of rhabdomyosarcoma.
Signaling via the intrinsic (mitochondrial) pathway of apoptosis represents one of the critical signal transduction cascades that control the regulation of cell death. This pathway is typically altered in human cancers, thereby providing a suitable target for therapeutic intervention. Members of the Bcl-2 family of proteins as well as cell survival signaling cascades such as the PI3K/Akt/mTOR pathway are involved in the regulation of mitochondria-mediated apoptosis. Therefore, further insights into the molecular mechanisms that form the basis for the control of mitochondria-mediated apoptosis will likely open new perspectives to bypass evasion of apoptosis and treatment resistance in human cancers.
ADAM15 protein amplifies focal adhesion kinase phosphorylation under genotoxic stress conditions
(2012)
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
Allein die Texte zur Abwicklung des Frankfurter Instituts für Sexualwissenschaft 2006 füllen ein Viertel dieses Sammelbandes. Doch Wundenlecken ist nicht die Sache des Autors und ehemaligen Institutsleiters Volkmar Sigusch, der als einer der renommiertesten Sexualwissenschaftler weltweit gilt. Sein facettenreiches Buch enthält neben zeithistorischen Dokumentationen höchst unterschiedliche, allerdings auch unterschiedlich gut lesbare Artikel zu aktuellen die Sexualität als gesamtgesellschaftliches Phänomen betreffenden Fragen. Es kulminiert in scharfer sexualwissenschaftlich fundierter Gesellschaftskritik.
Purpose: To investigate the prognostic value of survivin expression in pretreatment specimens from patients with anal cancer treated with concurrent 5-FU and mitomycin C-based chemoradiation (CRT).
Material and methods: Immunohistochemical staining for survivin was performed in pretreatment biopsies of 62 patients with anal carcinoma. Survivin expression was correlated with clinical and histopathological characteristics as well as local failure free- (LFFS), distant metastases free- (DMFS), cancer specific- (CSS), and overall survival (OS).
Results: Survivin staining intensity was weak in 10%, intermediate in 48% and intense in 42% of the patients. No association between survivin expression and clinicopathologic factors (tumor stage, age and HIV status) could be shown. In univariate analysis, the level of survivin staining was significantly correlated with DMFS (low survivin vs. high survivin: 94% vs. 74%, p=0.04). T-stage, N-stage and the tumor grading were significantly associated with OS and CSS and with DMFS and LFFS, respectively. In multivariate analysis, survivin was confirmed as independent prognostic parameter for DMFS (RR, 0.04; p=0.02) and for OS (RR, 0.27; p=0.04).
Conclusion: Our results demonstrated that the level of pretreatment survivin is correlated with the clinical outcome in patients with anal carcinoma treated with concurrent CRT. Further studies are warranted to elucidate the complex role of survivin for the oncologic treatment and to exploit the protein as a therapeutic target in combined modality treatment of anal cancer.
Introduction: Erectile dysfunction (ED) is common in men with systemic sclerosis (SSc) but the demographics, risk factors and treatment coverage for ED are not well known.
Method: This study was carried out prospectively in the multinational EULAR Scleroderma Trial and Research database by amending the electronic data-entry system with the International Index of Erectile Function-5 and items related to ED risk factors and treatment. Centres participating in this EULAR Scleroderma Trial and Research substudy were asked to recruit patients consecutively.
Results: Of the 130 men studied, only 23 (17.7%) had a normal International Index of Erectile Function-5 score. Thirty-eight per cent of all participants had severe ED (International Index of Erectile Function-5 score ≤ 7). Men with ED were significantly older than subjects without ED (54.8 years vs. 43.3 years, P < 0.001) and more frequently had simultaneous non-SSc-related risk factors such as alcohol consumption. In 82% of SSc patients, the onset of ED was after the manifestation of the first non-Raynaud's symptom (median delay 4.1 years). ED was associated with severe cutaneous, muscular or renal involvement of SSc, elevated pulmonary pressures and restrictive lung disease. ED was treated in only 27.8% of men. The most common treatment was sildenafil, whose efficacy is not established in ED of SSc patients.
Conclusions: Severe ED is a common and early problem in men with SSc. Physicians should address modifiable risk factors actively. More research into the pathophysiology, longitudinal development, treatment and psychosocial impact of ED is needed.
Fettleibigkeit, Bluthochdruck, erhöhte Blutfettwerte und Insulin-Resistenz – diese als »Metabolisches Syndrom« bezeichnete Kombination von Risikofaktoren ist auch als »tödliches Quartett« bekannt. Typ-II-Diabetes und Herz-Kreislauf-Erkrankungen sind die Folge. Doch wie hängen diese auf den ersten Blick recht unterschiedlichen Phänomene zusammen? Neuere Untersuchungen zeigen, dass Diabetes und ein gestörter Fettstoffwechsel mehr gemeinsam haben, als man bisher annahm.
Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the ‘mitochondrial infectious damage adaptation’ (MIDA) model according to which a deceleration of fusion–fission cycles reflects a systemic adaptation increasing life span.
Background: Patients with chronic kidney disease (CKD) are at increased risk for inappropriate or potentially harmful prescribing. The aim of this study was to examine whether a multifaceted intervention including the use of a software programme for the estimation of creatinine clearance and recommendation of individual dosage requirements may improve correct dosage adjustment of relevant medications for patients with CKD in primary care.
Methods: A cluster-randomized controlled trial was conducted between January and December 2007 in small primary care practices in Germany. Practices were randomly allocated to intervention or control groups. In each practice, we included patients with known CKD and elderly patients (>=70 years) suffering from hypertension. The practices in the intervention group received interactive training and were provided a software programme to assist with individual dose adjustment. The control group performed usual care. Data were collected at baseline and at 6 months. The outcome measures, analyzed across individual patients, included prescriptions exceeding recommended maximum daily doses, with the primary outcome being prescriptions exceeding recommended standard daily doses by 30% or more.
Results: Data from 44 general practitioners and 404 patients are included. The intervention was effective in reducing prescriptions exceeding the maximum daily dose per patients, with a trend in reducing prescriptions exceeding the standard daily dose by more than 30%.
Conclusions: A multifaceted intervention including the use of a software program effectively reduced inappropriately high doses of renally excreted medications in patients with CKD in the setting of small primary care practices.
The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer metastasis. It is highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. The nuclear transcript MALAT-1 has been functionally associated with gene regulation and alternative splicing and its regulation has been shown to impact proliferation, apoptosis, migration and invasion.
Here, we have developed a human and a mouse knockout system to study the loss-of-function phenotypes of this important ncRNA. In human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases. Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation nor cell cycle progression nor nuclear architecture in human lung or liver cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not give rise to any obvious phenotype or histological abnormalities in Malat1-null compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA MALAT1 is compatible with cell viability and normal development.
Background: The posterior cruciate ligament (PCL) plays an important role in maintaining physiological kinematics and function of the knee joint. To date mainly in-vitro models or combined magnetic resonance and fluoroscopic systems have been used for quantifying the importance of the PCL. We hypothesized, that both tibiofemoral and patellofemoral kinematic patterns are changed in PCL-deficient knees, which is increased by isometric muscle flexion. Therefore the aim of this study was to simultaneously investigate tibiofemoral and patellofemoral 3D kinematics in patients suffering from PCL deficiency during different knee flexion angles and under neuromuscular activation.
Methods: We enrolled 12 patients with isolated PCL-insufficiency as well as 20 healthy volunteers. Sagittal MR-images of the knee joint were acquired in different positions of the knee joint (0[degree sign], 30[degree sign], 90[degree sign] flexion, with and without flexing isometric muscle activity) on a 0.2 Tesla open MR-scanner. After segmentation of the patella, femur and tibia local coordinate systems were established to define the spatial position of these structures in relation to each other.
Results: At full extension and 30[degree sign] flexion no significant difference was observed in PCL-deficient knee joints neither for tibiofemoral nor for patellofemoral kinematics. At 90[degree sign] flexion the femur of PCL-deficient patients was positioned significantly more anteriorly in relation to the tibia and both, the patellar tilt and the patellar shift to the lateral side, significantly increased compared to healthy knee joints. While no significant effect of isometric flexing muscle activity was observed in healthy individuals, in PCL-deficient knee joints an increased paradoxical anterior translation of the femur was observed at 90[degree sign] flexion compared to the status of muscle relaxation.
Conclusions: Significant changes in tibiofemoral and patellofemoral joint kinematics occur in patients with isolated PCL-insufficiency above 30 degrees of flexion compared to healthy volunteers. Since this could be one reasonable mechanism in the development of OA our results might help to understand the long-term development of tibiofemoral and/or patellofemoral osteoarthritis in PCL-insufficient knee joints.
The objective of this pilot clinical study was to assess the safety, technical feasibility, pharmacokinetic (PK) profile and tumour response of DC Bead™ with irinotecan (DEBIRI™) delivered by intra-arterial embolisation for the treatment of metastatic colorectal cancer. Eleven patients with unresectable liver metastases from CRC, tumour burden <30% of liver volume, adequate haematological, liver and renal function, performance status of <2 were included in this study. Patients received up to 4 sessions of TACE with DEBIRI at 3-week intervals. Feasibility of the procedure, safety and tumour response were assessed after each cycle. PK was measured after the first cycle. Patients were followed up to 24 weeks. Only mild to moderate adverse events were observed. DEBIRI is a technically feasibile procedure; no technical complications were observed. Average Cmax for irinotecan and SN-38 was 194 ng/ml and 16.7 ng/ml, respectively, with average t½ of 4.6 h and 12.4 h following administration of DEBIRI. Best overall response during the study showed disease control in 9 patients (2 patients with partial response and 7 with stable disease, overall response rate of 18%). Our study shows that transarterial chemoembolisation with irinotecan-loaded DC beads (DEBIRI) is safe, technically feasible and effective with a good PK profile.
During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.
Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells
(2012)
For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.
Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissuespecific effects, which may be partially alleviated by the induction of FBXW8.
Background: The European Centres of Reference Network for Cystic Fibrosis (ECORN-CF) established an Internet forum which provides the opportunity for CF patients and other interested people to ask experts questions about CF in their mother language. The objectives of this study were to: 1. develop a detailed quality assessment tool to analyze quality of expert answers, 2. evaluate the intra- and inter-rater agreement of this tool, and 3. explore changes in the quality of expert answers over the time frame of the project.
Methods: The quality assessment tool was developed by an expert panel. Five experts within the ECORN-CF project used the quality assessment tool to analyze the quality of 108 expert answers published on ECORN-CF from six language zones. 25 expert answers were scored at two time points, one year apart. Quality of answers was also assessed at an early and later period of the project. Individual rater scores and group mean scores were analyzed for each expert answer.
Results: A scoring system and training manual were developed analyzing two quality categories of answers: content and formal quality. For content quality, the grades based on group mean scores for all raters showed substantial agreement between two time points, however this was not the case for the grades based on individual rater scores. For formal quality the grades based on group mean scores showed only slight agreement between two time points and there was also poor agreement between time points for the individual grades. The inter-rater agreement for content quality was fair (mean kappa value 0.232+/-0.036, p<0.001) while only slight agreement was observed for the grades of the formal quality (mean kappa value 0.105+/-0.024, p<0.001). The quality of expert answers was rated high (four language zones) or satisfactory (two language zones) and did not change over time.
Conclusions: The quality assessment tool described in this study was feasible and reliable when content quality was assessed by a group of raters. Within ECORN-CF, the tool will help ensure that CF patients all over Europe have equal possibility of access to high quality expert advice on their illness.
Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[DPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.
Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK) like c-Jun N-terminal kinase (JNK) 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R) was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT) and JNK2 deficient (KO) mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P < 0.05). Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123), indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT). By contrast, KO mice displayed less depolarization after H/R (23%, P < 0.05). In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R.
Patients that survive hemorrhage and resuscitation (H/R) may develop a systemic inflammatory response syndrome (SIRS) that leads to dysfunction of vital organs (multiple organ dysfunction syndrome, MODS). SIRS and MODS may involve mitochondrial dysfunction. Under pentobarbital anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer’s solution containing minocycline, tetracycline (both 10 mg/kg body weight) or vehicle. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R with vehicle or tetracycline, ALT increased to 4538 U/L and 3999 U/L, respectively, which minocycline decreased to 1763 U/L (P<0.01). Necrosis and TUNEL also decreased from 24.5% and 17.7 cells/field, respectively, after vehicle to 8.3% and 8.7 cells/field after minocycline. Tetracycline failed to decrease necrosis (23.3%) but decreased apoptosis to 9 cells/field (P<0.05). Minocycline and tetracycline also decreased caspase-3 activity in liver homogenates. Minocycline but not tetracycline decreased lipid peroxidation after resuscitation by 70% (P<0.05). Intravital microscopy showed that minocycline preserved mitochondrial polarization after H/R (P<0.05). In conclusion, minocycline decreases liver injury and oxidative stress after H/R by preventing mitochondrial dysfunction.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine.
Much is known about the computation in individual neurons in the cortical column. Also, the selective connectivity between many cortical neuron types has been studied in great detail. However, due to the complexity of this microcircuitry its functional role within the cortical column remains a mystery. Some of the wiring behavior between neurons can be interpreted directly from their particular dendritic and axonal shapes. Here, I describe the dendritic density field (DDF) as one key element that remains to be better understood. I sketch an approach to relate DDFs in general to their underlying potential connectivity schemes. As an example, I show how the characteristic shape of a cortical pyramidal cell appears as a direct consequence of connecting inputs arranged in two separate parallel layers.
To search for novel strategies to enhance the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis pathways in glioblastoma, we used the B-cell lymphoma 2/Bcl2-like 2-inhibitor ABT-737. Here we report that ABT-737 and TRAIL cooperate to induce apoptosis in several glioblastoma cell lines in a highly synergistic manner (combination index <0.1). Interestingly, the concerted action of ABT-737 and TRAIL to trigger the accumulation of truncated Bid (tBid) at mitochondrial membranes is identified as a key underlying mechanism. ABT-737 and TRAIL cooperate to cleave BH3-interacting domain death agonist (Bid) into its active fragment tBid, leading to increased accumulation of tBid at mitochondrial membranes. Coinciding with tBid accumulation, the activation of Bcl2-associated X protein (Bax), loss of mitochondrial membrane potential, release of cytochrome-c and second mitochondria-derived activator of caspase (Smac) into the cytosol and caspase activation are strongly increased in cotreated cells. Of note, knockdown of Bid significantly decreases ABT-737- and TRAIL-mediated Bax activation and apoptosis. Also, caspase-3 silencing reduces ABT-737- and TRAIL-induced Bid cleavage and apoptosis, indicating that a caspase-3-driven, mitochondrial feedback loop contributes to Bid processing. Importantly, ABT-737 profoundly enhances TRAIL-triggered apoptosis in primary cultured glioblastoma cells derived from tumor material, underlining the clinical relevance. Also, ABT-737 acts in concert with TRAIL to suppress tumor growth in an in vivo glioblastoma model. In conclusion, the rational combination of ABT-737 and TRAIL cooperates to trigger tBid mitochondrial accumulation and apoptosis. This approach presents a promising strategy for targeting the apoptosis pathways in glioblastoma, which warrants further investigation.
Background: Cognitive-behavioral therapy (CBT) is generally known to be efficacious in the treatment of social phobia when applied in RCT's, namely when the treatment manual is based on the Clark-Wells approach. However, little is known about the efficacy of manualized treatments in routine clinical practice (Phase IV of psychotherapy research). The present study (SOPHO-PRAX) is a continuation of a large multi-centre randomized clinical trial (SOPHO-NET) and analyses the extent to which additional training practitioners in manualized procedures enhances treatment effect.
Methods: N = 36 private practitioners will be included in three treatment centres and randomly designated to either training in manualized CBT or no specific training. The treatment effects of the therapies conducted by both groups of therapists will be compared. A total of 162 patients (N = 116 completers; N = 58 per condition) will be enrolled. Liebowitz Social Anxiety Scale (LSAS) will serve as primary outcome measure. Remission from social phobia is defined as LSAS total [less than or equal to] 30 points. Data will be collected at treatment begin, after 8, 15, and 25 sessions (50 mins. each), at treatment completion, as well at 6 and 12 months post-treatment.
Discussion: The present CBT trial combines elements of randomized-controlled trials and naturalistic studies in an innovative way. It will directly inform about the incremental effects of procedures established in a controlled trial into clinical practice. Study results are relevant to health care decisions and policy. They may serve to improve quality of treatment, and shorten the timeframe between the development and widespread dissemination of effective methods, thereby reducing health cost expenditures. The results of this study will not only inform about the degree to which the new methods lead to an improvement of treatment course and outcome, but also about whether the effects of routine psychotherapeutic treatment are comparable to those of the controlled, strictly manualized treatments of the SOPHO-NET study. Trial Registration: ClinicalTrials.gov identifier: NCT01388231. This study was funded by the German Federal Ministry of Education and Research (SOPHO-NET: BMBF 01GV0607; SOPHO-PRAX: BMBF 01GV1001).
Hereditary Angioedema (HAE) is a rare disease and for this reason proper diagnosis and appropriate therapy are often unknown or not available for physicians and other health care providers. For this reason we convened a group of specialists that focus upon HAE from around the world to develop not only a consensus on diagnosis and management of HAE, but to also provide evidence based grades, strength of evidence and classification for the consensus. Since both consensus and evidence grading were adhered to the document meets criteria as a guideline. The outcome of the guideline is to improve diagnosis and management of patients with HAE throughout the world and to help initiate uniform care and availability of therapies to all with the diagnosis no matter where the residence of the individual with HAE exists.
Background: Hodkin s lymphoma is one of the most frequent lymphoma in western world. Despite an overall good prognosis some patients suffer relapsing tumors which are difficult to cure. Over a long period Vitamin D has been shown to be a potential treatment for cancer. Vitamin D acts via the vitamin D receptor, a nuclear receptor, acting as an inducible transcription factor. We aimed to investigate the expression of vitamin D receptor as potential therapeutic target structure in Hodgkin s lymphoma as well as in non Hodgkin s lymphoma.
Methods: We used a panel of 193 formalin fixed tissues of lymphoma cases consisting of 55 cases of Hodgkin s lymphoma and 138 cases on several non Hodgkin s lymphoma entities.
Results: Vitamin D receptor is strongly expressed in Hodgkin s lymphoma, regardless of the subentity with an overall positivity of 80% of all Hodgkin lymphoma cases. In contrast, only about 17% of the analyzed non Hodgkin s lymphoma of B-cell origin showed positivity for vitamin D receptor. Predominant nuclear localization of vitamin D receptor in Hodgkin s lymphoma suggests activated status of the vitamin D receptor.
Conclusions: From this study, we conclude that vitamin D receptor plays a potentially important role in pathogenesis of Hodgkin s lymphoma but not in non Hodgkin s lymphoma. Further investigations of mutational status and functional studies may shed some light in functional relevance of vitamin D receptor signaling in Hodgkin s lymphoma.
Evaluation der SSRI-Medikation bei Kindern und Jugendlichen bezüglich ansteigender Suizidalität
(2012)
Zielsetzung dieser Arbeit war die Klärung der Frage, inwieweit eine SSRI-Medikation
bei Kindern und Jugendlichen mit dem Ansteigen der Suizidalität in Zusammenhang
steht. Es sollte untersucht werden, ob Suizidgedanken und Suizidverhalten im
Verlauf eines stationären Aufenthaltes allgemein ansteigen, diese nur bei
vorliegender Medikation mit SSRI ansteigen, etwa nur bei bereits vorliegender
Suizidalität ansteigen, oder ob suizidales Gedankengut allein unter einer
Wechselwirkung der Faktoren „SSRI-Medikation“ und „vorliegende Suizidalität“
entsteht.
Zur Klärung der Fragen wurden im Rahmen einer klinischen Studie Patienten
zwischen 12 und 19 Jahren über ein Fragebogeninventar befragt. Die Stichprobe
wurde konsekutiv im Zeitraum von Juni 2004 bis Dezember 2006 aus den
aufgenommen Patienten im Zentrum der Kinder- und Jugendpsychiatrie des
Klinikums der Johann Wolfgang Goethe-Universität in Frankfurt am Main rekrutiert.
Sie umfasste 44 Patienten. Ausgeschlossen wurden hierbei hochgradig geistig
retardierte Patienten, Patienten mit ausgeprägter Lese- und Rechtschreibschwäche,
sowie psychotisch erkrankte Patienten. Zur Teilnahme an der Studie war eine
Einverständniserklärung von Seiten der Eltern nötig. Zur Untersuchung der
Fragestellung wurde ein spezielles Fragebogeninventar erstellt zu dem der SBQ,
BSI, JTCI 12-18, YSR, EWL, die Achse V und die wöchentliche Erhebung externaler
Stressoren gehört. Die Suizidalität wurde so hinsichtlich eines Zusammenhangs mit
Depressivität, Impulsivität, Ängstlichkeit, Aggressivität, externalen sozialen Faktoren
und einer SSRI-Medikation in einem Gesamtzeitraum von fünf Wochen untersucht.
Das Gesamtkollektiv teilte sich in 14 männliche und 30 weibliche Patienten. Der
jüngste Patient war 12 Jahre, der älteste 19 Jahre (mittleres Alter 15,68).
Anhand des SBQ (d = 1,83) und des BSI (d = 2,09) Fragebogeninventars konnten
Suizidgedanken und -verhaltensweisen (Erhängen, Vergiften, etc.) sehr gut zwischen
„Suizidalen“ und „Nicht-Suizidalen“ trennen.
Die SSRI haben keinen signifikanten Effekt auf die Befindlichkeit (EWL), auf die
pathologiebezogene Aggressivität und Ängstlichkeit (YSR) und die
persönlichkeitsbezogene „Impulsivität“ und „Ängstlichkeit“ (unter 1%- Niveau).
Abschließend kann somit geschlossen werden, dass kein signifikanter
Zusammenhang zwischen der Gabe von SSRI und Suizidalität besteht.
The decision in September 2011 in the UK to accept blood donations from non-practicing men who have sex with men (MSM) has received significant public attention. Will this rule change substantially boost the number of blood donations or will it make our blood less safe? Clearly, most European countries have a blood procurement problem. Fewer young people are donating, while the population is aging and more invasive therapies are requiring more blood. Yet if that was the reason for allowing non-practicing MSM to donate, clearly re-admission of some other, much larger populations that are currently deferred from donation should likewise be considered. As far as risks for blood safety are concerned, evidence has been provided that the current quality of infectious disease marker testing significantly mitigates against, although does not completely eradicate, risks associated with admission of donors with a high risk of carrying certain blood-transmissible agents. However, it could be argued that more effective recruitment of the non-donor pool, which is substantially larger than the group of currently ineligible donors, would be a better strategy. Recruitment of this group will benefit the availability of blood without jeopardizing the current excellent safety profile of blood.
Introduction: In recent genome-wide association studies for psoriatic arthritis (PsA) and psoriasis vulgaris, common coding variants in the TRAF3IP2 gene were identified to contribute to susceptibility to both disease entities. The risk allele of p.Asp10Asn (rs33980500) proved to be most significantly associated and to encode a mutant protein with an almost completely disrupted binding property to TRAF6, supporting its impact as a main disease-causing variant and modulator of IL-17 signaling.
Methods: To identify further variants, exons 2-4 encoding both known TNF-receptor-associated factor (TRAF) binding domains were sequenced in 871 PsA patients. Seven missense variants and one three-base-pair insertion were identified in 0.06% to 1.02% of alleles. Five of these variants were also present in 931 control individuals at comparable frequency. Constructs containing full-length wild-type or mutant TRAF3IP2 were generated and used to analyze functionally all variants for TRAF6-binding in a mammalian two-hybrid assay.
Results: None of the newly found alleles, though, encoded proteins with different binding properties to TRAF6, or to the cytoplasmic tail of the IL-17-receptor α-chain, suggesting that they do not contribute to susceptibility.
Conclusions: Thus, the TRAF3IP2-variant p.Asp10Asn is the only susceptibility allele with functional impact on TRAF6 binding, at least in the German population.
Background: Hereditary angioedema (HAE) is a rare but serious disease marked by swelling attacks in the extremities, face, trunk, airway, or abdominal areas that can be spontaneous or the result of trauma and other triggers. It can be life-threatening due to the risk of asphyxiation. While there have been major advancements in our understanding of the immunogenetics of HAE, there are significant gaps in the literature regarding understanding of the humanistic and economic impact of the disease, particularly in Europe. The purpose of the HAE Burden of Illness Study-Europe (HAE-BOIS-Europe), the development and methodology of which is described here, is to better understand the management and impact of HAE from the patient perspective in Europe.
Methods/Design: This is a cross-sectional study in which retrospective data were also collected being conducted in Denmark, Germany and Spain. The study is open to patients ages 12 and older with a diagnosis of HAE-I or HAE-II. Data collection includes: (i) a survey on individuals’ health care resource use, direct and indirect medical costs, impact on work and school, treatment satisfaction, and emotional functioning (via the Hospital Anxiety and Depression Scale); and (ii) one-on-one interviews to collect detailed descriptive data and patient testimonials on the impact of HAE on patients’ health-related quality of life.
Discussion: The present manuscript describes the development and plans for implementing a multi-country European study with the aim of characterizing the humanistic and economic burden of HAE from the patient perspective. This study will help raise awareness of HAE as a rare but debilitating condition with wide-ranging impacts.
This report describes the clinical courses of two acute myeloid leukemia patients. Both had MLL translocations, the first a t(10;11)(p11.2;q23) with MLL-AF10 and the second a t(11;19)(q23;p13.1) with MLL-ELL fusion. They achieved a clinical remission under conventional chemotherapy but relapsed shortly after end of therapy. Both had a history of invasive mycoses (one had possible pulmonary mycosis, one systemic candidiasis). Because no HLA-identical donor was available, a haploidentical transplantation was performed in both cases. Using a specially designed PCR method for the assessment of minimal residual disease (MRD), based on the quantitative detection of the individual chromosomal breakpoint in the MLL gene, all patients achieved complete and persistent molecular remission after transplantation. The immune reconstitution after transplantation is described in terms of total CD3+/CD4+, CD3+/CD8+, CD19+, and CD16+/CD56+ cell numbers over time. The KIR and HLA genotypes of donors and recipients are reported and the possibility of a KIR-mediated alloreactivity is discussed. This report illustrates that haploidentical transplantation may offer a chance of cure without chronic graft-versus-host disease in situations where no suitable HLA-identical donor is available even in a high-risk setting and shows the value of MRD monitoring in the pre- and posttransplant setting.