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Institute
- Medizin (500) (remove)
Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.
Background: Transplantation of human corneal tissue is associated with the potential risk of transmittance of viral infections. In accordance with European directives and federal laws, in Germany each tissue donor has to be tested for infectious diseases such as hepatitis B and C virus (HBV and HCV) and human immunodeficiency virus (HIV) infection. However, most of the currently available CE-marked serologic and nucleic acid screening systems are only validated for antemortem blood. Methods: Twenty related and paired ante- and postmortem blood samples from cornea donors were obtained and subsequently analyzed for hepatitis B surface antigen (HBsAg), hepatitis B antibody (anti-HBc), anti-HCV, HCV RNA, anti-HIV-1/2, and HIV p24 Ag using Abbott test systems. The sera were also spiked with reference materials in concentrations giving low and high positivity for HBV, HCV, and HIV markers. Results: The spiked ante- and postmortem sera from related donors showed similar results for HBsAg, anti-HBc, anti-HCV, HCV RNA, anti-HIV, and HIV p24 Ag, indicating a high stability of viral markers in cadaveric specimens. Three cornea donors had a medical history of HBV infection and revealed anti-HBc at similar levels in the ante- and postmortem sera. In addition, there was a single postmortem sample demonstrating a weak signal of anti-HIV-1 and HIV-1 p24 Ag. False-positive or false-negative results were not detected. The results obtained with the Abbott ARCHITECT analyzer and Abbott RealTime HCV PCR showed no significant differences. Conclusion: The analyzed screening assays are suitable for the detection of infectious markers of HBV, HCV, and HIV at similar levels in spiked ante- and postmortem sera from cornea donors.
Background: Gait kinematics after total hip replacement only partly explain the differences in the joint moments in the frontal plane between hip osteoarthritis patients after hip replacement and healthy controls. The goal of this study was to determine if total hip replacement surgery affects radiological leg alignment (Hip-Knee-Shaft-Angle, femoral offset, Neck-Shaft-Angle and varus/valgus alignment) and which of these parameters can explain the joint moments, additionally to the gait kinematics.
Methods: 22 unilateral hip osteoarthritis patients who were scheduled for total hip replacement were included in the study. Preoperatively and 1 year postoperatively all patients had biplanar radiographic examinations and 3D gait analysis.
Results: The operated leg showed significantly (P < 0.05) more varus (1.1°) as well as a larger femoral offset (+ 8 mm) and a larger Hip-Knee-Shaft-Angle (+ 1.3°) after total hip replacement; however no significant differences in the joint moments in the frontal plane compared to healthy controls were found. The hip moment (first half of stance) and the knee moments (first and second half of stance) were mostly determined by the varus/valgus alignment (29% and respectively 36% and 35%). The combination with a kinematic parameter (knee range of motion, foot progression angle) increased the predictive value for the knee moments.
Conclusion: In our patient group the joint moments after total hip replacement did not differ from healthy controls, whereas radiological leg alignment parameters changed significantly after the total hip replacement. A combination of these radiological leg parameters, especially the varus alignment, and the deviating kinematics explain the joint moments in the frontal plane during gait after total hip replacement surgery. For surgeons it is important not to create too much of a structural varus alignment by implanting the new hip joint as varus alignment can increase the knee adduction moment and the risk for osteoarthritis of the medial knee compartment.
Trial registration: This study was retrospectively registered with DRKS (German Clinical Trials Register) under the number DRKS00015053. Registered 1st of August 2018.
Treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia (Ph+ ALL) has been revolutionized with the advent of tyrosine kinase inhibitors (TKIs). Most patients with CML achieve long-term survival similar to individuals without CML due to treatment with TKIs not only in frontline but also in further lines of therapy. The third-generation TKI ponatinib has demonstrated efficacy in patients with refractory CML and Ph+ ALL. Ponatinib is currently the most potent TKI in this setting demonstrating activity against T315I mutant clones. However, ponatinib’s safety data revealed a dose-dependent, increased risk of serious cardiovascular (CV) events. Guidance is needed to evaluate the benefit–risk profile of TKIs, such as ponatinib, and safety measures to prevent treatment-associated CV events. An expert panel of German hematologists and cardiologists summarize current evidence regarding ponatinib’s efficacy and CV safety profile. We propose CV management strategies for patients who are candidates for ponatinib.
Das adaptive Immunsystem CRISPR (engl. „clustered regularly interspaced short palindromic repeats“) revolutioniert die medizinische Grundlagenforschung. Die Einfachheit, Präzision und Vielseitigkeit der CRISPR-Technologie ermöglicht es nicht nur, Gene gezielt aus- oder einzuschalten, sondern auch zu korrigieren. Die Hoffnung richtet sich auf eine CRISPR-vermittelte Gentherapie, um krebsverursachende Mutationen gezielt zu korrigieren und somit Tumorwachstum zu verhindern oder therapieren zu können. Technisch ist dies zeitnah vorstellbar, doch ethische und rechtliche Rahmenbedingungen sollten dringend vorab geklärt werden. Die durch Gene Editing aufgeworfenen ethischen und rechtlichen Fragen werden zwar schon seit vielen Jahren diskutiert; durch die nun eingetretene rapide technische Entwicklung stellen sie sich jedoch in neuer Dringlichkeit. Eine umfassende ethische Bewertung der Erforschung und möglichen Anwendung ist daher geboten, einschließlich Fragen der Wissenschaftsethik und -kultur sowie längerfristiger potenzieller sozialer Konsequenzen der CRISPR-Technologie. Rechtlich unterliegt die Gentherapie den allgemeinen arzneimittelrechtlichen Regelungen, die Keimbahntherapie dagegen ist in Deutschland verboten. Auf Dauer und angesichts der erwartbaren weltweiten Entwicklung ist dieses Verbot jedoch zu hinterfragen. In der vorliegenden Arbeit erläutern die Autoren technische, ethische und rechtliche Aspekte des Gene Editing in der Krebsforschung und -therapie und diskutieren die daraus resultierenden Fragen: „Was kann, soll und darf gemacht werden?“.
RITA, the RBP‐J interacting and tubulin‐associated protein, has been reported to be related to tumor development, but the underlying mechanisms are not understood. Since RITA interacts with tubulin and coats microtubules of the cytoskeleton, we hypothesized that it is involved in cell motility. We show here that depletion of RITA reduces cell migration and invasion of diverse cancer cell lines and mouse embryonic fibroblasts. Cells depleted of RITA display stable focal adhesions (FA) with elevated active integrin, phosphorylated focal adhesion kinase, and paxillin. This is accompanied by enlarged size and disturbed turnover of FA. These cells also demonstrate increased polymerized tubulin. Interestingly, RITA is precipitated with the lipoma‐preferred partner (LPP), which is critical in actin cytoskeleton remodeling and cell migration. Suppression of RITA results in reduced LPP and α‐actinin at FA leading to compromised focal adhesion turnover and actin dynamics. This study identifies RITA as a novel crucial player in cell migration and invasion by affecting the turnover of FA through its interference with the dynamics of actin filaments and microtubules. Its deregulation may contribute to malignant progression.
Objective: Excessive inflammation in the central nervous system (CNS) and the periphery can result in neurodegeneration and parkinsonism. Recent evidence suggests that immune responses in Parkinson disease patients are dysregulated, leading to an increased inflammatory reaction to unspecific triggers. Although α‐synuclein pathology is the hallmark of Parkinson disease, it has not been investigated whether pathologic α‐synuclein is a specific trigger for excessive inflammatory responses in Parkinson disease.
Methods: We investigated the immune response of primary human monocytes and a microglial cell line to pathologic forms of α‐synuclein by assessing cytokine release upon exposure.
Results: We show that pathologic α‐synuclein (mutations, aggregation) results in a robust inflammatory activation of human monocytes and microglial BV2 cells. The activation is conformation‐ dependent, with increasing fibrillation and early onset mutations having the strongest effect on immune activation. We also found that activation of immune cells by extracellular α‐synuclein is potentiated by extracellular vesicles, possibly by facilitating the uptake of α‐synuclein. Blood extracellular vesicles from Parkinson disease patients induce a stronger activation of monocytes than blood extracellular vesicles from healthy controls. Most importantly, monocytes from Parkinson disease patients are dysregulated and hyperactive in response to stimulation with pathologic α‐synuclein. Furthermore, we demonstrate that α‐synuclein pathology in the CNS is sufficient to induce the monocyte dysregulation in the periphery of a mouse model.
Interpretation: Taken together, our data suggest that α‐synuclein pathology and dysregulation of monocytes in Parkinson disease can act together to induce excessive inflammatory responses to α‐synuclein. ANN NEUROL 2019;86:593–606
The Masquelet technique for the treatment of large bone defects is a two‐stage procedure based on an induced membrane. The size of a scaffold is reported to be a critical factor for bone healing response. We therefore aimed to investigate the influence of the granule size of a bone graft substitute on bone marrow derived mononuclear cells (BMC) supported bone healing in combination with the induced membrane. We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) with or without BMC in vivo in a rat femoral critical size defect. A 10 mm defect was made in 126 rats and a membrane induced by a PMMA‐spacer. After 3 weeks, the spacer was taken out and membrane filled with different granule sizes. After 8 weeks femurs were taken for radiological, biomechanical, histological, and immunohistochemical analysis. Further, whole blood of the rat was incubated with granules and expression of 29 peptide mediators was assessed. Smallest granules showed significantly improved bone healing compared to larger granules, which however did not lead to an increased biomechanical stability in the defect zone. Small granules lead to an increased accumulation of macrophages in situ which could be assigned to the inflammatory subtype M1 by majority. Increased release of chemotactic respectively proangiogenic active factors in vitro compared to syngenic bone and beta‐TCP was observed. Granule size of the bone graft substitute Herafill® has significant impact on bone healing of a critical size defect in combination with Masquelet's technique in terms of bone formation and inflammatory.
Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.
Quantitative T1 mapping indicates tumor infiltration beyond the enhancing part of glioblastomas
(2019)
The aim of this study was to evaluate whether maps of quantitative T1 (qT1) differences induced by a gadolinium‐based contrast agent (CA) are better suited than conventional T1‐weighted (T1w) MR images for detecting infiltration inside and beyond the peritumoral edema of glioblastomas. Conventional T1w images and qT1 maps were obtained before and after gadolinium‐based CA administration in 33 patients with glioblastoma before therapy. The following data were calculated: (i) absolute qT1‐difference maps (qT1 pre‐CA ‐ qT1 post‐CA), (ii) relative qT1‐difference maps, (iii) absolute and (iv) relative differences of conventional T1w images acquired pre‐ and post‐CA. The values of these four datasets were compared in four different regions: (a) the enhancing tumor, (b) the peritumoral edema, (c) a 5 mm zone around the pathology (defined as the sum of regions a and b), and (d) the contralateral normal appearing brain tissue. Additionally, absolute qT1‐difference maps (displayed with linear gray scaling) were visually compared with respective conventional difference images. The enhancing tumor was visible both in the difference of conventional pre‐ and post‐CA T1w images and in the absolute qT1‐difference maps, whereas only the latter showed elevated values in the peritumoral edema and in some cases even beyond. Mean absolute qT1‐difference values were significantly higher (P < 0.01) in the enhancing tumor (838 ± 210 ms), the peritumoral edema (123 ± 74 ms) and in the 5 mm zone around the pathology (81 ± 31 ms) than in normal appearing tissue (32 ± 35 ms). In summary, absolute qT1‐difference maps—in contrast to the difference of T1w images—of untreated glioblastomas appear to be able to visualize CA leakage, and thus might indicate tumor cell infiltration in the edema region and beyond. Therefore, the absolute qT1‐difference maps are potentially useful for treatment planning.