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[Nachruf] Helmut Siefert
(2012)
Wolfgang Schlote : 80 Jahre
(2012)
Das Institut für Medizinische Virologie, untergebracht in dem 100 Jahre alten Gebäude des ehemaligen Frankfurter Gesundheitsamtes direkt neben dem Georg-Speyer-Haus, ist eine wunderbare Kombination von Funktionalität und Schönheit. Von der geschwungenen Holztreppe des historischen Treppenhauses geht der Blick immer wieder in hochmoderne biomedizinische Laboratorien, gesichert hinter dicken Glastüren – ein Blick in eine futuristisch anmutende Welt.
Hausherr ist hier seit April 2012 der mehrfach ausgezeichnete HIV/AIDS-Experte Professor Oliver T. Keppler. Unter seiner Leitung ist das Institut umgehend mit Wirkung zum 1. Oktober 2012 vom Präsidenten des Robert-Koch-Instituts zum „Nationalen Referenzzentrum für Retroviren“ berufen worden. Mit Retroviren sind zu 95 Prozent HI-Viren (Humanes Immundefizienz-Virus) gemeint – die restlichen fünf Prozent umfassen die HTL-Viren (Humanes T-lymphotropes-Virus), mit denen weltweit etwa 20 Millionen Menschen infiziert sind, das jedoch kaum in Deutschland vorkommt. Das Institut ist damit das deutsche Referenzlabor für die Routine- und Spezialdiagnostik von Retroviren, Schwerpunkt HIV-Infektionen, sowie für Stellungnahmen zu Fragen der Krankheitsentstehung und Behandlung.
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.
Volkmar Siguschs 1984 erschienenes Buch "Die Mystifikation des Sexuellen" bietet gute Anknüpfungspunkte, um die Eingebundenheit der Kategorien "Geschlecht" und "Sexualität" in die kapitalistische Produktionsweise verstehen und Ableitungen für emanzipatorisches Streiten treffen zu können. Zusammen mit weiteren – auch neueren – Arbeiten Siguschs ergeben sich Anschlussmöglichkeiten für die kapitalismuskritische und antikapitalistische Fortentwicklung feministischer und queer-feministischer Ansätze.
Macrophages are highly plastic leukocytes that differentiate from monocytes following their entry into extravascular tissues. Macrophages can enter various tissues under inflammatory or non-inflammatory conditions and assume different functions and phenotypes according to the cues they receive from the environment. The notion that inflammation in general and macrophage responses in particular affect physiological phenomena that were previously considered to be not immune-related has enhanced and broadened our understanding of macrophage function during inflammation and its resolution....
Background: Standardization in clinical practice may lead to improved outcomes. Unfortunately, little is known about the variability of non-pharmacological anti-infective measures in children with cancer.
Design and Methods: A web-based survey assessed institutional recommendations regarding restrictions of social contacts, pets and food and instructions on wearing face masks in public for children with standard- risk acute lymphoblastic leuk emia and acute myeloid leukemia during intensive chemotherapy.
Results: A total of 336 institutions in 27 countries responded to the survey (range, 1-76 institutions per country; overall response rate 61%). Most institutions recommend that patients with acute myeloid leukemia avoid indoor public places and daycare, kindergarten and school, whereas recommendations for patients with acute lymphoblastic leukemia differ considerably by institution. In terms of restrictions related to pets, there was a wide variability between institutions for both acute lymphoblastic and acute myeloid leukemia patients. Most, but not all institutions do not allow children with either acute lymphoblastic or acute myeloid leukemia to eat raw meat, raw seafood or unpasteurized milk. Whereas most institutions do not routinely recommend that patients with acute lymphoblastic leukemia wear face masks in public, advice on this matter varies for patients with acute myeloid leukemia.
Conclusions: The survey demonstrates that there is a wide variation in recommendations on non-pharmacological anti-infective measures between different institutions, countries and continents. This information may be used to encourage harmonization of supportive care practices and future clinical trials.
Development of single-port cholecystectomy : results of a case-control study matched to one surgeon
(2012)
Background: Single-port laparoscopic cholecystectomy is an evolving technique which is now widely established. Up until now, the safety of the procedure and a respective learning curve have not been adequately reported in most studies. The aim of this study was to demonstrate that single-port cholecystectomy is a safe procedure, with a positive learning curve from a case-control study matched to one surgeon.
Methods: One hundred single-port cholecystectomies performed by one surgeon (AB) were retrospectively matched to 100 patients who underwent conventional laparoscopic cholecystectomy carried out by the same surgeon. The two groups were matched in respect of surgical indication, gender, age, and body mass index. The groups were compared with respect to operation time, use of additional trocars, analgesics required in the post anesthesia care unit, postoperative complications, and duration of hospital stay.
Results: No significant difference was found between the two groups with respect to postoperative complications and stay in hospital. The operation time increased slightly in the single-port group. Directly after the operation, the analgesic use required in the post anesthesia care unit was higher in the single-port group. Consumption of analgesics on the surgical ward was very similar in each group. In respect to the learning curve, the operation time and use of additional trocars showed a positive trend, starting with the thirtieth operation.
Conclusion: Single-port cholecystectomy is a feasible and safe procedure in a specialist setting. The procedure can be done under the same safety rules as those for conventional laparoscopic cholecystectomy. Considering the learning curve, starting with the thirtieth operation, a positive trend was seen. Long-term studies will be needed to establish the incidence and rate of incisional hernias.
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.
The prognostic impact of epidermal growth factor receptor in patients with metastatic gastric cancer
(2012)
Background: The epidermal growth factor receptor (EGFR) is a potential target of anticancer therapy in gastric cancer. However, its prognostic role in metastatic gastric or gastroesophageal junction (GE) cancer has not been established yet.
Methods: EGFR status was analyzed by immunohistochemistry (IHC) in paraffin-embedded samples from 357 patients who received chemotherapy in 4 first-line trials. Automated RNA extraction from paraffin and RT-quantitative PCR were additionally used to evaluate EGFR mRNA expression in 130 patients.
Results: EGFR protein expression (any grade) and overexpression (3+) were observed in 43% and 11% of patients, respectively. EGFR positivity correlated with intestinal type histology (p = 0.05), but not with other clinicopathologic characteristics. Median follow-up was 18.2 months. Median overall survival (OS) was similar in patients with EGFR positive vs. those with EGFR negative tumors, regardless whether positivity was defined as ≥1+ (10.6 vs. 10.9 months, p = 0.463) or as 3+ (8.6 vs. 10.8 months, p = 0.377). The multivariate analysis indicated that EGFR status is not an independent prognostic factor (hazard ratio 0.85, 0.56 to 1.12, p = 0.247). There were also no significant differences in overall survival when patients were categorized according to median (p = 0.116) or quartile (p = 0.767) distribution of EGFR mRNA gene expression. Similar distributions of progression-free survival according to EGFR status were observed.
Conclusions: Unlike different cancer types where EGFR-positive disease is associated with an adverse prognostic value, EGFR positivity is not prognostic of patient outcome in metastatic gastric or GE cancer.
Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency ≤1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10(-4) after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756-162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10(-3) after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10(-2)). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.
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.
Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.
Background: The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins are encoded as a result of the translocation, depending on whether a "minor" or "major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL) exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia.
Design and methods: We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their resistance mutants.
Results: The inhibitory effects of GNF-2 differed constantly between p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring resistance mutations.
Conclusions: Our data provide the first evidence of a differential response of p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia.
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.
Hereditary angioedema (HAE) in children and adolescents : a consensus on therapeutic strategies
(2012)
Hereditary angioedema due to C1 inhibitor (C1 esterase inhibitor) deficiency (types I and II HAE-C1-INH) is a rare disease that usually presents during childhood or adolescence with intermittent episodes of potentially life-threatening angioedema. Diagnosis as early as possible is important to avoid ineffective therapies and to properly treat swelling attacks. At a consensus meeting in June 2011, pediatricians and dermatologists from Germany, Austria, and Switzerland reviewed the currently available literature, including published international consensus recommendations for HAE therapy across all age groups. Published recommendations cannot be unconditionally adopted for pediatric patients in German-speaking countries given the current approval status of HAE drugs. This article provides an overview and discusses drugs available for HAE therapy, their approval status, and study results obtained in adult and pediatric patients. Recommendations for developing appropriate treatment strategies in the management of HAE in pediatric patients in German-speaking countries are provided.Conclusion Currently, plasma-derived C1 inhibitor concentrate is considered the best available option for the treatment of acute HAE-C1-INH attacks in pediatric patients in German-speaking countries, as well as for short-term and long-term prophylaxis.
1. Introduction: The autosomal dominant cerebellar ataxias (ADCA) are a clinically, pathologically and genetically heterogeneous group of neurodegenerative disorders caused by degeneration of cerebellum and its afferent and efferent connections. The degenerative process may additionally involves the ponto- medullar systems, pyramidal tracts, basal ganglia, cerebral cortex, peripheral nerves (ADCA I) and the retina (ADCA II), or can be limited to the cerebellum (ADCA III) (Harding et al., 1993). The most common of these dominantly inherited autosomal ataxias, ADCA I, includes many Spinocerebellar Ataxias (SCA) subtypes, some of which are caused by pathological CAG trinucleotide repeat expansion in the coding region on the mutated gene. Such is the case for SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17 and Dentatorubral-pallidoluysian atrophy (DRPLA) (Matilla et al., 2006). Among the almost 30 SCAs, the variant SCA2 is the second most prevalent subtype worldwide, only surpassed by SCA3 (Schöls et al., 2004; Matilla et al., 2006; Auburger, 2011)...
Research over the past few years has provided fascinating results indicating that biglycan, besides being a ubiquitous structural component of the extracellular matrix (ECM), may act as a signaling molecule. Proteolytically released from the ECM, biglycan acts as a danger signal signifying tissue stress or injury. As a ligand of innate immunity receptors and activator of the inflammasome, biglycan stimulates multifunctional proinflammatory signaling linking the innate to the adaptive immune response. By clustering several types of receptors on the cell surface and orchestrating their downstream signaling events, biglycan is capable to autonomously trigger sterile inflammation and to potentiate the inflammatory response to microbial invasion. Besides operating in a broad biological context, biglycan also displays tissue-specific affinities to certain receptors and structural components, thereby playing a crucial role in bone formation, muscle integrity, and synapse stability at the neuromuscular junction. This review attempts to provide a concise summary of recent data regarding the involvement of biglycan in the regulation of inflammation and the musculoskeletal system, pointing out both a signaling and a structural role for this proteoglycan. The potential of biglycan as a novel therapeutic target or agent for the treatment of inflammatory diseases and skeletal muscular dystrophies is also addressed.
Background: The recommendation of artemisinin combination therapy (ACT) as first-line treatment for uncomplicated falciparum malaria is supported by a plethora of high quality clinical trials. However, their recommendation for the treatment of mixed-species malaria and the large-scale use for the treatment of non-falciparum malaria in endemic regions is based on anecdotal rather than systematic clinical evidence.
Methods: This study prospectively observed the efficacy of artemether-lumefantrine for the treatment of uncomplicated non-falciparum or mixed-species malaria in two routine district hospitals in the Central African country of Gabon.
Results: Forty patients suffering from uncomplicated Plasmodium malariae, Plasmodium ovale or mixed-species malaria (including Plasmodium falciparum) presenting at the hospital received artemether-lumefantrine treatment and were followed up. All evaluable patients (n = 38) showed an adequate clinical and parasitological response on Day 28 after oral treatment with artemether-lumefantrine (95% confidence interval: 0.91,1). All adverse events were of mild to moderate intensity and completely resolved by the end of study.
Conclusions: This first systematic assessment of artemether-lumefantrine treatment for P. malariae, P. ovale and mixed-species malaria demonstrated a high cure rate of 100% and a favourable tolerability profile, and thus lends support to the practice of treating non-falciparum or mixed-species malaria, or all cases of malaria without definite species differentiation, with artemether-lumefantrine in Gabon.
Trial Registration: ClinicalTrials.gov Identifier: NCT00725777
Recent research suggests that the brain routinely binds together information from gesture and speech. However, most of this research focused on the integration of representational gestures with the semantic content of speech. Much less is known about how other aspects of gesture, such as emphasis, influence the interpretation of the syntactic relations in a spoken message. Here, we investigated whether beat gestures alter which syntactic structure is assigned to ambiguous spoken German sentences. The P600 component of the Event Related Brain Potential indicated that the more complex syntactic structure is easier to process when the speaker emphasizes the subject of a sentence with a beat. Thus, a simple flick of the hand can change our interpretation of who has been doing what to whom in a spoken sentence. We conclude that gestures and speech are integrated systems. Unlike previous studies, which have shown that the brain effortlessly integrates semantic information from gesture and speech, our study is the first to demonstrate that this integration also occurs for syntactic information. Moreover, the effect appears to be gesture-specific and was not found for other stimuli that draw attention to certain parts of speech, including prosodic emphasis, or a moving visual stimulus with the same trajectory as the gesture. This suggests that only visual emphasis produced with a communicative intention in mind (that is, beat gestures) influences language comprehension, but not a simple visual movement lacking such an intention.
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.
The study of acupuncture-related sensations, like deqi and propagated sensations along channels (PSCs), has a long tradition in acupuncture basic research. The phenomenon itself, however, remains poorly understood. To study the connection between PSC and classical meridians, we applied a geographic information system (GIS) to analyze sketches of acupuncture sensations from healthy volunteers after laser acupuncture. As PSC can be subtle, we aimed at reducing the confounding impact of external stimuli by carrying out the experiment in a floatation tank under restricted environmental stimulation. 82.4% of the subjects experienced PSC, that is, they had line-like or 2-dimensional sensations, although there were some doubts that these were related to the laser stimulation. Line-like sensations on the same limb were averaged to calculate sensation mean courses, which were then compared to classical meridians by measuring the mean distance between the two. Distances ranged from 0.83 cm in the case of the heart (HT) and spleen (SP) meridian to 6.27 cm in the case of the kidney (KI) meridian. Furthermore, PSC was observed to “jump” between adjacent meridians. In summary, GIS has proven to be a valuable tool to study PSC, and our results suggest a close connection between PSC and classical meridians.
The cellular and molecular mechanisms of tumor angiogenesis and its prospects for anti-angiogenic cancer therapy are major issues in almost all current concepts of both cancer biology and targeted cancer therapy. Currently, (1) sprouting angiogenesis, (2) vascular co-option, (3) vascular intussusception, (4) vasculogenic mimicry, (5) bone marrow-derived vasculogenesis, (6) cancer stem-like cell-derived vasculogenesis and (7) myeloid cell-driven angiogenesis are all considered to contribute to tumor angiogenesis. Many of these processes have been described in developmental angiogenesis; however, the relative contribution and relevance of these in human brain cancer remain unclear. Preclinical tumor models support a role for sprouting angiogenesis, vascular co-option and myeloid cell-derived angiogenesis in glioma vascularization, whereas a role for the other four mechanisms remains controversial and rather enigmatic. The anti-angiogenesis drug Avastin (Bevacizumab), which targets VEGF, has become one of the most popular cancer drugs in the world. Anti-angiogenic therapy may lead to vascular normalization and as such facilitate conventional cytotoxic chemotherapy. However, preclinical and clinical studies suggest that anti-VEGF therapy using bevacizumab may also lead to a pro-migratory phenotype in therapy resistant glioblastomas and thus actively promote tumor invasion and recurrent tumor growth. This review focusses on (1) mechanisms of tumor angiogenesis in human malignant glioma that are of particular relevance for targeted therapy and (2) controversial issues in tumor angiogenesis such as cancer stem-like cell-derived vasculogenesis and bone-marrow-derived vasculogenesis.
We among others have recently demonstrated that normal cells produce “fusion mRNAs”. These fusion mRNAs do not derive from rearranged genomic loci, but rather they are derived from “early-terminated transcripts” (ETTs). Premature transcriptional termination takes place in intronic sequences that belong to “breakpoint cluster regions”. One important property of ETTs is that they exhibit an unsaturated splice donor site. This results in: (1) splicing to “cryptic exons” present in the final intron; (2) Splicing to another transcript of the same gene (intragenic trans-splicing), resulting in “exon repetitions”; (3) splicing to a transcript of another gene (intergenic trans-splicing), leading to “non-genomically encoded fusion transcripts” (NGEFTs). These NGEFTs bear the potential risk to influence DNA repair processes, since they share identical nucleotides with their DNA of origin, and thus, could be used as “guidance RNA” for DNA repair processes. Here, we present experimental data about four other genes. Three of them are associated with hemato-malignancies (ETV6, NUP98 and RUNX1), while one is associated with solid tumors (EWSR1). Our results demonstrate that all genes investigated so far (MLL, AF4, AF9, ENL, ELL, ETV6, NUP98, RUNX1 and EWSR1) display ETTs and produce transpliced mRNA species, indicating that this is a genuine property of translocating genes.
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.
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.
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.
Purpose: Student circus artists train as both artists and athletes with their bodies holding the key to professional success. The daily training load of student circus artists is often associated with maximum physical and psychological stress with injuries posing a threat to a potential professional career. The purpose of this study is the differentiated analysis and evaluation of work accidents in order to initiate the development of injury preventive programs.
Methods: The 17 years of data were obtained from standardized anonymous work accident records of the Berlin State Accident Insurance (UKB) as well as a State Artist Educational School (n = 169, Male: 70; Female: 99) from student artists. Evaluation and descriptive statistics were conducted with Excel 2007 and PASW Statistics 18.
Results: The injury risk seems to be relatively low (0.3 injuries/1000h). There are gender specific differences as to the location of injuries. Only 7% of the accidents demand a break of more than 3 days. Injury patterns vary depending on the activity and the employment of props/equipment. 75.2% of work accidents have multifactorial and 24.8% exogenous causes.
Conclusions: Because physical fitness is all important in the circus arts there are numerous options for injury prevention programs that should be realized subject to gender-specific differences. Follow-ups on chronic complaints and a more individual approach are indispensable due to the very specific activities in the circus arts.
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.
Fragestellung: Beurteilung der Korrektur des Astigmatismus mit der multifokalen torischen Intraokularlinsen (IOL) ReSTOR Toric (Alcon, Ft. Worth, USA) bei Kataraktoperation.
Methodik: Die Multicenterstudie umfasste Kataraktepatienten mit präoperativem Astigmatismus von ≥0,75 bis ≤2,5 dpt. die Patienten wurden einer bilateralen Implantation einer torischen multifokalen IOL zur Korrektur der Hornhautverkrümmung unterzogen. Die OP erfolgte ohne relaxierende limbale Inzisionen durch eine clear-cornea Inzision <3,0 mm. Prä- und postoperativ wurden für diese Subanalyse Autokeratometrie sowie subjektiver Astigmatismus von 39 Augen von 40 Patienten im Alter von 59,8±7,0 Jahren analysiert.
Ergebnisse: Präoperativ betrug der mittlere keratometrische Astigmatismus 1,43±0,57 dpt. Die mittlere Inzisionsgröße betrug 2,59±0,41 mm. 1 Monat postoperativ betrug der mittlere keratometrische Astigmatismus 1,51±0,95 dpt (25 Augen). Der Unterschied im keratometrischen Astigmatismus zwischen präoperativ und 1-Monat-postop betrug 0,57±0,96 dpt. Der präoperative subjektive Astigmatismus wurde signifikant von 0,32±0,33 dpt (25 Augen) auf 0,99±0,70 dpt reduziert. (39 Augen, p <0,0001).
Schlussfolgerung: Die Implantation der multifokale torischen IOL zeigt vorhersehbare postoperative Ergebnisse bei der Korrektur des Astigmatismus nach kataraktoperation.
Die exakte Positionierung von Intraokularlinsen in den Kapselsack ist von entscheidender Bedeutung für das postoperative visuelle Ergebnis nach Kataraktoperation oder refraktivem Linsenaustausch. Schon leichte Dezentrierungen können optische Aberrationen hervorrufen welche das Sehen der Patienten negativ beeinflussen. Besonders kommt dieses Phänomen bei sog. Premium Intraokularlinsen mit speziellen Optiken (asphärisch, torisch oder multifokal, sowie Kombinationen dieser) zum Tragen. Diese Optiken können Ihre gewünschte Wirkung nur bei exakter Positionierung entfalten. Eine postoperative Feinpositionierung ist nicht möglich, was die Ansprüche an den Operateur bei Implantation der Linsen erhöht. Minimalinvasive microinzisionelle Operationstechniken bieten heute gute Möglichkeiten, die Implantate exakt zu positionieren. Neben den Dezentrierungen können Intraokularlinsen auch dislozieren, beispielweise durch intra- oder postoperative Kapselrupturen, Linsenverziehungen oder auch Rotation der Implantate. Hier ist ein weiterer chirurgischer Eingriff von Nöten. Der Vortrag stellt dementsprechend verschiedene Videos und praktische Hinweise zur Handhabung postoperativer Linsendislokationen vor.
Uncalibrated semi-invasive continous monitoring of cardiac index (CI) has recently gained increasing interest. The aim of the present study was to compare the accuracy of CI determination based on arterial waveform analysis with transpulmonary thermodilution. Fifty patients scheduled for elective coronary surgery were studied after induction of anaesthesia and before and after cardiopulmonary bypass (CPB), respectively. Each patient was monitored with a central venous line, the PiCCO system, and the FloTrac/Vigileo-system. Measurements included CI derived by transpulmonary thermodilution and uncalibrated semi-invasive pulse contour analysis. Percentage changes of CI were calculated. There was a moderate, but significant correlation between pulse contour CI and thermodilution CI both before (r(2) = 0.72, P < 0.0001) and after (r(2) = 0.62, P < 0.0001) CPB, with a percentage error of 31% and 25%, respectively. Changes in pulse contour CI showed a significant correlation with changes in thermodilution CI both before (r(2) = 0.52, P < 0.0001) and after (r(2) = 0.67, P < 0.0001) CPB. Our findings demonstrated that uncalibrated semi-invasive monitoring system was able to reliably measure CI compared with transpulmonary thermodilution in patients undergoing elective coronary surgery. Furthermore, the semi-invasive monitoring device was able to track haemodynamic changes and trends.
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.
Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination–dependent inactivation of Rac1 signaling.
While it is apparent that rare variation can play an important role in the genetic architecture of autism spectrum disorders (ASDs), the contribution of common variation to the risk of developing ASD is less clear. To produce a more comprehensive picture, we report Stage 2 of the Autism Genome Project genome-wide association study, adding 1301 ASD families and bringing the total to 2705 families analysed (Stages 1 and 2). In addition to evaluating the association of individual single nucleotide polymorphisms (SNPs), we also sought evidence that common variants, en masse, might affect the risk. Despite genotyping over a million SNPs covering the genome, no single SNP shows significant association with ASD or selected phenotypes at a genome-wide level. The SNP that achieves the smallest P-value from secondary analyses is rs1718101. It falls in CNTNAP2, a gene previously implicated in susceptibility for ASD. This SNP also shows modest association with age of word/phrase acquisition in ASD subjects, of interest because features of language development are also associated with other variation in CNTNAP2. In contrast, allele scores derived from the transmission of common alleles to Stage 1 cases significantly predict case status in the independent Stage 2 sample. Despite being significant, the variance explained by these allele scores was small (Vm< 1%). Based on results from individual SNPs and their en masse effect on risk, as inferred from the allele score results, it is reasonable to conclude that common variants affect the risk for ASD but their individual effects are modest.
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.
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.
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.
Immunomodulatory properties and molecular effects in inflammatory diseases of low-dose X-irradiation
(2012)
Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice.
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.
ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation.
METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs.
RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity.
CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.
Rho GTPases are involved in homing and mobilization of hematopoietic stem and progenitor cells due to their impact on cytoskeleton remodeling. We have previously shown that inhibition of Rho, Rac and Cdc42 clearly impairs adhesion of normal and leukemic hematopoietic progenitor cells (HPC) to fibronectin and migration in a three-dimensional stromal cell model. Here, we identified the Ras GTPase-Activating Protein SH3 Domain-Binding Protein (G3BP) as a target gene of Rho GTPases and analysed its role in regulating HPC motility. Overexpression of G3BP significantly enhanced adhesion of murine 32D HPC to fibronectin and human umbilical vein endothelial cells, increased the proportion of adherent cells in a flow chamber assay and promoted cell migration in a transwell assay and a three-dimensional stromal cell model suggesting a strong impact on the cytoskeleton. Immunofluorescent staining of G3BP-overexpressing fibroblasts revealed a Rho-like phenotype characterized by formation of actin stress fibers in contrast to the Rac-like phenotype of control fibroblasts. This is the first report implicating a role for G3BP in Rho GTPase-mediated signalling towards adhesion and migration of HPC. Our results may be of clinical importance, since G3BP was found overexpressed in human cancers.
Renal perfusion in scleroderma patients assessed by microbubble-based contrast-enhanced ultrasound
(2012)
OBJECTIVES: Renal damage is common in scleroderma. It can occur acutely or chronically. Renal reserve might already be impaired before it can be detected by laboratory findings. Microbubble-based contrast-enhanced ultrasound has been demonstrated to improve blood perfusion imaging in organs. Therefore, we conducted a study to assess renal perfusion in scleroderma patients utilizing this novel technique.
MATERIALS AND METHODOLOGY: Microbubble-based contrast agent was infused and destroyed by using high mechanical index by Siemens Sequoia (curved array, 4.5 MHz). Replenishment was recorded for 8 seconds. Regions of interests (ROI) were analyzed in renal parenchyma, interlobular artery and renal pyramid with quantitative contrast software (CUSQ 1.4, Siemens Acuson, Mountain View, California). Time to maximal Enhancement (TmE), maximal enhancement (mE) and maximal enhancement relative to maximal enhancement of the interlobular artery (mE%A) were calculated for different ROIs.
RESULTS: There was a linear correlation between the time to maximal enhancement in the parenchyma and the glomerular filtration rate. However, the other parameters did not reveal significant differences between scleroderma patients and healthy controls.
CONCLUSION: Renal perfusion of scleroderma patients including the glomerular filtration rate can be assessed using microbubble-based contrast media.
The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.
Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.
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.