Medizin
Refine
Year of publication
- 2012 (235) (remove)
Document Type
- Article (198)
- Part of Periodical (11)
- Conference Proceeding (7)
- Doctoral Thesis (6)
- Contribution to a Periodical (4)
- Review (4)
- Book (3)
- Part of a Book (2)
Has Fulltext
- yes (235)
Is part of the Bibliography
- no (235)
Keywords
- apoptosis (3)
- inflammation (3)
- ADHD (2)
- BCR/ABL (2)
- Cognitive behavioral therapy (2)
- HIV (2)
- PARK2 (2)
- Philadelphia chromosome (2)
- Ubiquitination (2)
- acute lymphoblastic leukemia (2)
Institute
- Medizin (235)
- Präsidium (10)
- Biochemie und Chemie (8)
- Pharmazie (7)
- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES) (6)
- Biowissenschaften (5)
- Frankfurt Institute for Advanced Studies (FIAS) (4)
- Georg-Speyer-Haus (4)
- Sonderforschungsbereiche / Forschungskollegs (4)
- Psychologie (3)
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.
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.
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.
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.
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 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: 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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: 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.
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.
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.
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.
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.
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.
Visual perception is highly variable and can be influenced by the surrounding world. Previous research has revealed that body perception can be biased due to adaptation to thin or fat body shapes. The aim of the present study was to show that adaptation to certain body shapes and the resulting perceptual biases transfer across different identities of adaptation and test stimuli. We designed two similar adaptation experiments in which healthy female participants adapted to pictures of either thin or fat bodies and subsequently compared more or less distorted pictures of their own body to their actual body shape. In the first experiment (n = 16) the same identity was used as adaptation and test stimuli (i.e. pictures of the participant’s own body) while in the second experiment (n = 16) we used pictures of unfamiliar thin or fat bodies as adaptation stimuli. We found comparable adaptation effects in both experiments: After adaptation to a thin body, participants rated a thinner than actual body picture to be the most realistic and vice versa. We therefore assume that adaptation to certain body shapes transfers across different identities. These results raise the questions of whether some type of natural adaptation occurs in everyday life. Natural and predominant exposure to certain bodily features like body shape – especially the thin ideal in Western societies – could bias perception for these features. In this regard, further research might shed light on aspects of body dissatisfaction and the development of body image disturbances in terms of eating disorders.
Introduction: In this article the personal study and life situation of parents who are also medical students at the Medical School of the Goethe University Frankfurt am Main is discussed. There is a special focus on the topics "studying with children" and "family-friendly university", which have been present in discussions about university development and in the daily life of academics, especially during the last decade. The workgroup "Individual Student Services" at the medical faculty at the Goethe University tries to meet the necessities of the individual study courses and to support the study success with a new counselling and student service concept.
Methods: The experience of parents studying medicine was recorded in semi-structured interviews (Date: April 2010), which were held as part of the sponsored pilot project on part-time medical studies ("Pilot Project Part-time Medical Studies"). Additionally, study results from the Medical School of the Goethe University Frankfurt am Main were integrated as well as a literature analysis.
Results: It was found that the teaching demands and support services, which have been suggested and needed for years now, have been partially implemented and are without sufficient support at the faculty level to date. Thus the current situation of medical students with children is still difficult and seems a big challenge for everyone involved.
Solution: As part of the "Individual Student Services" a new pilot project on part-time medical studies was established in November 2009. Only the use of new, unconventional and innovative ideas allows universities to adequately support the changing and heterogeneous student population and support them to successfully completing their medical studies.
Einleitung: In dem vorliegenden Artikel wird die persönliche Studien- und Lebenssituation von Studierenden mit Kindern am Fachbereich Medizin der Goethe-Universität Frankfurt am Main beleuchtet. Der spezielle Fokus liegt auf den Themen "Studium mit Kind" sowie "familiengerechte Hochschule", welche in Deutschland in der letzten Dekade sehr präsent sowohl in der Hochschulforschung als auch im akademischen Alltag sind. Hier versucht die Arbeitsstelle "Individuelle Studienbegleitung" am FB Medizin der Goethe-Universität durch ein neues Beratungs- und Servicekonzept mit wissenschaftlicher Begleitforschung den individuellen Studienverläufen gerecht zu werden und den Studienerfolg zu fördern.
Methoden: In erster Linie stammen die hier beschriebenen Erfahrungen der Medizinstudierenden mit Kind aus qualitativen Leitfadeninterviews (Stand April 2010), die im Rahmen des Modellprojektes Teilzeitstudium Medizin durchgeführt wurden. Ergänzend wurden die Studienergebnisse des Fachbereichs Medizin der Goethe-Universität Frankfurt am Main herangezogen und eine Literaturanalyse einbezogen.
Ergebnisse: Als Ergebnis kann festgehalten werden, dass die bereits seit mehreren Jahren erhobenen Forderungen und gewünschten Unterstützungsleistungen zwar zum Teil umgesetzt, aber noch nicht ausreichend an allen Fachbereichen implementiert wurden. Somit ist die aktuelle Situation von Studierenden mit Kindern in der Medizin nach wie vor schwierig und stellt für alle Beteiligten eine große Herausforderung dar.
Lösungsansatz: Im Rahmen der Arbeitsstelle "Individuelle Studienbegleitung" konnte im November 2009 das "Modellprojekt Teilzeitstudium Medizin" aufgebaut werden. Nur durch neue, unkonventionelle und innovative Ideen kann man die Sicherstellung der medizinischen Ausbildung erreichen und die heutige heterogene Studierendenschaft adäquat ansprechen sowie Studierende erfolgreich durch ihr Studium begleiten.
Objective: Betahistine is a histamine H1-receptor agonist and H3-receptor antagonist that is administered to treat Menière’s disease. Despite widespread use, its pharmacological mode of action has not been entirely elucidated. This study investigated the effect of betahistine on guinea pigs at dosages corresponding to clinically used doses for cochlear microcirculation.
Methods: Thirty healthy Dunkin-Hartley guinea pigs were randomly assigned to five groups to receive betahistine dihydrochloride in a dose of 1,000 mg/kg b. w. (milligram per kilogram body weight), 0.100 mg/kg b. w., 0.010 mg/kg b. w., 0.001 mg/kg b. w. in NaCl 0.9% or NaCl 0.9% alone as placebo. Cochlear blood flow and mean arterial pressure were continuously monitored by intravital fluorescence microscopy and invasive blood pressure measurements 3 minutes before and 15 minutes after administration of betahistine.
Results: When betahistine was administered in a dose of 1.000 mg/kg b. w. cochlear blood flow was increased to a peak value of 1.340 arbitrary units (SD: 0.246; range: 0.933–1.546 arb. units) compared to baseline (p<0.05; Two Way Repeated Measures ANOVA/Bonferroni t-test). The lowest dosage of 0.001 mg/kg b. w. betahistine or NaCl 0.9% had the same effect as placebo. Nonlinear regression revealed that there was a sigmoid correlation between increase in blood flow and dosages.
Conclusions: Betahistine has a dose-dependent effect on the increase of blood flow in cochlear capillaries. The effects of the dosage range of betahistine on cochlear microcirculation corresponded well to clinically used single dosages to treat Menière’s disease. Our data suggest that the improved effects of higher doses of betahistine in the treatment of Menière’s disease might be due to a corresponding increase of cochlear blood flow.
Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04–4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54–7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33–7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11–5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.
Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.
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.
Background: The diagnostic and pathophysiological relevance of antibodies to aquaporin-4 (AQP4-Ab) in patients with neuromyelitis optica spectrum disorders (NMOSD) has been intensively studied. However, little is known so far about the clinical impact of AQP4-Ab seropositivity.
Objective: To analyse systematically the clinical and paraclinical features associated with NMO spectrum disorders in Caucasians in a stratified fashion according to the patients' AQP4-Ab serostatus.
Methods: Retrospective study of 175 Caucasian patients (AQP4-Ab positive in 78.3%).
Results: Seropositive patients were found to be predominantly female (p < 0.0003), to more often have signs of co-existing autoimmunity (p < 0.00001), and to experience more severe clinical attacks. A visual acuity of ≤ 0.1 during acute optic neuritis (ON) attacks was more frequent among seropositives (p < 0.002). Similarly, motor symptoms were more common in seropositive patients, the median Medical Research Council scale (MRC) grade worse, and MRC grades ≤ 2 more frequent, in particular if patients met the 2006 revised criteria (p < 0.005, p < 0.006 and p < 0.01, respectively), the total spinal cord lesion load was higher (p < 0.006), and lesions ≥ 6 vertebral segments as well as entire spinal cord involvement more frequent (p < 0.003 and p < 0.043). By contrast, bilateral ON at onset was more common in seronegatives (p < 0.007), as was simultaneous ON and myelitis (p < 0.001); accordingly, the time to diagnosis of NMO was shorter in the seronegative group (p < 0.029). The course of disease was more often monophasic in seronegatives (p < 0.008). Seropositives and seronegatives did not differ significantly with regard to age at onset, time to relapse, annualized relapse rates, outcome from relapse (complete, partial, no recovery), annualized EDSS increase, mortality rate, supratentorial brain lesions, brainstem lesions, history of carcinoma, frequency of preceding infections, oligoclonal bands, or CSF pleocytosis. Both the time to relapse and the time to diagnosis was longer if the disease started with ON (p < 0.002 and p < 0.013). Motor symptoms or tetraparesis at first myelitis and > 1 myelitis attacks in the first year were identified as possible predictors of a worse outcome.
Conclusion: This study provides an overview of the clinical and paraclinical features of NMOSD in Caucasians and demonstrates a number of distinct disease characteristics in seropositive and seronegative patients
Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice [1]. But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis.
Methods: Ninj1 was repressed by transfecting HMEC-1 cells, a human dermal microvascular endothelial cell line with siRNA targeting Ninj1 (siNinj1) or a negative control (siC). Subsequently, cells were stimulated with 100 ng/ml LPS (TLR4 agonist), 3 μg/ml LTA (TLR2 agonist) or 100 n/ml poly(I:C) (TLR3 agonist) for 3 hours. The inflammatory response was analyzed by real-time PCR. In addition, transmigration of neutrophils across a HMEC-1 monolayer was measured using transwell plates (pore size 3 μm).
Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% (Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% (Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 (Figure 1C,D) while poly(I:C)-induced expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells (Figure 1E).
Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation. In vivo experiments will clarify its impact on systemic inflammation.
Background: Chronic renal disease is a serious complication of long-term intravenous drug use (IVDU). Recent reports have postulated a changing pattern of underlying nephropathy over the last decades.
Methods: Retrospective investigation including all patients with prior or present IVDU that underwent renal biopsy because of chronic kidney disease between 01.04.2002 and 31.03.2012 in the city of Frankfurt/Main, Germany.
Results: Twenty four patients with IVDU underwent renal biopsy because of progressive chronic kidney disease or proteinuria. Renal AA-amyloidosis was the predominant cause of renal failure in 50% of patients. Membranoproliferative glomerulonephritis (GN) was the second most common cause found in 21%. Patients with AA-amyloidosis were more likely to be HIV infected (67 vs.17%; p=0.036) and tended to have a higher rate of repeated systemic infections (92 vs. 50%; p=0.069). Patients with AA-amyloidosis presented with progressive renal disease and nephrotic-range proteinuria but most patients had no peripheral edema or systemic hypertension. Development of proteinuria preceded the decline of GFR for approximately 1--2 years.
Conclusions: AA-amyloidosis was the predominant cause of progressive renal disease in the last 10 years in patients with IVDU. The highest rate of AA-amyloidosis observed was seen in HIV infected patients with IVDU. We speculate that chronic HIV-infection as well as the associated immunosuppression might promote development of AA-amyloidosis by increasing frequency and duration of infections acquired by IVDU.
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.
Background: In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR.
Methods: Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment.
Results: Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients.
Conclusions: A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.
Lipid-laden alveolar macrophages and pH monitoring have been used in the diagnosis of chronic aspiration in children with gastroesophageal reflux (GER). This study was conducted to prove a correlation between the detection of alimentary pulmonary fat phagocytosis and an increasing amount of proximal gastroesophageal reflux. It was assumed that proximal gastroesophageal reflux better correlates with aspiration than distal GER. Patients from 6 months to 16 years with unexplained recurrent wheezy bronchitis and bronchial hyperreactivity, or recurrent pneumonia with chronic cough underwent 24-hour double-channel pH monitoring and bronchoscopy with bronchoalveolar lavage (BAL). Aspiration of gastric content was determined by counting lipid laden alveolar macrophages from BAL specimens. There were no correlations between any pH-monitoring parameters and counts of lipid-laden macrophages in the whole study population, even when restricting analysis to those with abnormal reflux index expressing clinically significant GER. Quantifying lipid-laden alveolar macrophages from BAL in children with gastroesophageal-related respiratory disorders does not have an acceptable specificity to prove chronic aspiration as an underlying etiology. Therefore, research for other markers of pulmonary aspiration is needed.
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.
Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone.
Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.
Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.
Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.
5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.