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The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, “3Y”) as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.
Background: Self-myofascial release (SMR) aims to mimic the effects of manual therapy and tackle dysfunctions of the skeletal muscle and connective tissue. It has been shown to induce improvements in flexibility, but the underlying mechanisms are still poorly understood. In addition to neuronal mechanisms, improved flexibility may be driven by acute morphological adaptations, such as a reduction in passive tissue stiffness or improved movement between fascial layers. The aim of the intended study is to evaluate the acute effects of SMR on the passive tissue stiffness of the anterior thigh muscles and the sliding properties of the associated fasciae.
Methods: In a crossover study de sign, 16 participants will receive all of the following interventions in a permutated random order: (1) one session of 2 × 60 s of SMR at the anterior thigh, (2) one session of 2 × 60 s of passive static stretching of the anterior thigh and (3) no intervention. Passive tissue stiffness, connective tissue sliding, angle of first stretch sensation, as well as maximal active and passive knee flexion angle, will be evaluated before and directly after each intervention.
Discussion: The results of the intended study will allow a better understanding of, and provide further evidence on, the local effects of SMR techniques and the underlying mechanisms for flexibility improvements.
Background: Caloric restriction is associated with broad therapeutic potential in various diseases and an increase in health and life span. In this study, we assessed the impact of caloric restriction on acute and inflammatory nociception in mice, which were either fed ad libitum or subjected to caloric restriction with 80% of the daily average for two weeks.
Results: The behavioral tests revealed that inflammatory nociception in the formalin test and in zymosan-induced mechanical hypersensitivity were significantly decreased when mice underwent caloric restriction. As potential mediators of the diet-induced antinociception, we assessed genes typically induced by inflammatory stimuli, AMP-activated kinase, and the endocannabinoid system which have all already been associated with nociceptive responses. Zymosan-induced inflammatory markers such as COX-2, TNFα, IL-1β, and c-fos in the spinal cord were not altered by caloric restriction. In contrast, AMPKα2 knock-out mice showed significant differences in comparison to C57BL/6 mice and their respective wild type littermates by missing the antinociceptive effects after caloric restriction. Endocannabinoid levels of anandamide and 2-arachidonyl glyceroldetermined in serum by LC-MS/MS were not affected by either caloric restriction alone or in combination with zymosan treatment. However, cannabinoid receptor type 1 expression in the spinal cord, which was not altered by caloric restriction in control mice, was significantly increased after caloric restriction in zymosan-induced paw inflammation. Since increased cannabinoid receptor type 1 signaling might influence AMP-activated kinase activity, we analyzed effects of anandamide on AMP-activated kinase in cell culture and observed a significant activation of AMP-activated kinase. Thus, endocannabionoid-induced AMP-activated kinase activation might be involved in antinociceptive effects after caloric restriction.
Conclusion: Our data suggest that caloric restriction has an impact on inflammatory nociception which might involve AMP-activated kinase activation and an increased activity of the endogenous endocannabinoid system by caloric restriction-induced cannabinoid receptor type 1 upregulation.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Introduction: Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse.
Methods: A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours.
Results: The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk.
Conclusion: This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.
Background: Imbalances in female career promotion are a key factor of gender disparities at the workplace. They may lead to stress and stress-related diseases including burnout, depression or cardiovascular diseases. Since this problem cannot be generalized and varies between different fields, new approaches are needed to assess and describe the magnitude of the problem in single fields of work.
Methods: To construct a new index, operating figures of female and male medical students were collected for Germany in a period over 15 years and their progression throughout their studies towards specialization and academic chair positions. By the use of different female to male ratios (f:m), we constructed an index that describes the extend by which women can ascent in their academic career by using the field of academic medicine as an example.
Results: A medical student f:m ratio of 1.54 (52,366 female vs. 34,010 male) was found for Germany in 2013. In 1998, this f:m ratio was 0.999. In the same year (2013), the OB/GYN hospital specialists’ f:m ratio was 1.566 (3347 female vs. 2137 male physicians) and 0.577 (516 female vs 894 male physicians) for ENT hospital specialists, respectively. The f:m ratios concerning chairs of OB/GYN and ENT were 0.105 and 0.1, respectively. Then an index was generated that incorporated these operating figures with the student f:m ratio as denominator and the chair f:m ratio as numerator while the hospital specialist f:m ratio served as a corrector in the numerator in order to adjust to the attraction of a given field to female physicians. As a result, the index was 0.044 for OB/GYN and 0.113 for ENT instead of ideally ~1 in a completely gender harmonized situation.
Conclusion: In summary, a new index to describe female career advancement was established for academic medicine. By the use of this index, different academic and medical fields can now be compared to each other and future benchmarks could be proposed. Also, country differences may be examined using the proposed index and the success of specific funding programs.
Anticoagulation with warfarin and rivaroxaban ameliorates experimental autoimmune encephalomyelitis
(2017)
Background: In multiple sclerosis, coagulation factors have been shown to modulate inflammation. In this translational study, we investigated whether long-term anticoagulation with warfarin or rivaroxaban has beneficial effects on the course of autoimmune experimental encephalomyelitis (EAE).
Methods: Female SJL/J mice treated with anticoagulants namely warfarin or rivaroxaban were immunized with PLP139–151. Stable anticoagulation was maintained throughout the entire experiment. Mice without anticoagulation treated with the vehicle only were used as controls. The neurological deficit was recorded during the course of EAE, and histopathological analyses of inflammatory lesions were performed.
Results: In preventive settings, both treatment with warfarin and rivaroxaban reduced the maximum EAE score as compared to the control group and led to a reduction of inflammatory lesions in the spinal cord. In contrast, therapeutic treatment with warfarin had no beneficial effects on the clinical course of EAE. Signs of intraparenchymal hemorrhage at the site of the inflammatory lesions were not observed.
Conclusion: We developed long-term anticoagulation models that allowed exploring the course of EAE under warfarin and rivaroxaban treatment. We found a mild preventive effect of both warfarin and rivaroxaban on neurological deficits and local inflammation, indicating a modulation of the disease induction by anticoagulation.
Malignant brain tumors, including gliomas, brain metastases and anaplastic meningiomas, are associated with poor prognosis, and represent an unmet medical need. ASA404 (DMXAA), a vascular disrupting agent, has demonstrated promising results in several preclinical tumor models and early phase clinical trials. However, two phase III trials in non-small cell lung cancer reported insufficient results. The aim of the present study was to determine the effects of ASA404 on brain tumors. The effects of ASA404 were evaluated in vitro and in vivo using subcutaneous, and orthotopical models for malignant glioma (U-87, LN-229, U-251, LN-308 and Tu-2449), brain metastasis (HT-29) and malignant meningioma (IOMM-Lee). The acute effects of ASA404 on tumor tissue were analyzed using conventional and immunohistochemical staining techniques [hematoxylin and eosin, MIB-1 antibody/proliferation maker protein Ki-67, cleaved caspase-8, stimulator of interferon genes (STING), ionized calcium-binding adapter molecule 1]. Furthermore, the sizes of subcutaneous tumors were measured and the symptom-free survival rates of animals with intracranial tumors receiving ASA404 treatment were analyzed. ASA404 demonstrated low toxicity in vitro, but exhibited strong effects on subcutaneous tumors 24 h following a single dose of ASA404 (25 mg/kg). ASA404 induced necrosis, hemorrhages and inhibited the proliferation, and growth of tumors in the subcutaneous glioma models. However, ASA404 failed to demonstrate comparable effects in any of the intracranial tumor models examined and did not result in a prolongation of survival. Expression of STING, the molecular target of ASA404, and infiltration of macrophages, the cells mediating ASA404 activity, did not differ between subcutaneous and intracranial tumors. In conclusion, ASA404 demonstrates clear efficacy in subcutaneous tumor models, but has no relevant activity in orthotopic brain tumor models. The expression of STING and infiltration with macrophages were not determined to be involved in the differential activity observed among tumor models. It is possible that the low penetration of ASA-404 into the brain prevents concentrations sufficient enough reaching the tumor in order to exhibit acute effects in vivo.
Reactive oxygen species are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases are important reactive oxygen species targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of protein-tyrosine phosphatase oxidation in signal transduction, the cell biological details of reactive oxygen species-catalyzed protein-tyrosine phosphatase inactivation have remained largely unclear, due to our inability to visualize protein-tyrosine phosphatase oxidation in cells. By combining proximity ligation assay with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor evokes transient oxidation on or close to RAB5+/ early endosome antigen 1− endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with platelet-derived growth factor receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that platelet-derived growth factor-dependent “redoxosomes,” contribute to proper signal transduction.
Oncologic therapy is currently undergoing significant changes. A number of innovative targeted medications currently in clinical development have raised high expectations. With that in mind, discussions about terms such as "clinical benefit" and "clinical relevance" are highly topical. This also applies to further developments in the field of adjuvant systemic therapies for early-stage breast cancer. As the treatment aim is curative, assessment of the clinical benefit of adjuvant therapies must be largely based on efficacy outcomes. The focus must be on improving disease-free survival rates and lowering the risk of recurrence. Because of the current low mortality rates, statements about overall survival rates are only possible after very long observation periods. Consequently, new drugs in adjuvant therapies should be considered as offering a clinical benefit, if they reduce the risk of recurrence below current low levels of risk. The evidence for established adjuvant therapy standards in early-stage breast cancer can be used as objective criteria for comparison. This review article considers the requirements for clinical benefit of new adjuvant therapies for early breast cancer, based on examples from adjuvant endocrine therapy, adjuvant polychemotherapy and adjuvant anti-HER2 therapy.
Zika virus (ZIKV) infection, a public health emergency of international concern, has recently been confirmed in Indonesia. However, to date, there has been no study to assess how prepared healthcare workers in Indonesia are to confront this emerging infectious disease. The aim of this study was to assess the attitudes of medical doctors in Indonesia towards ZIKV infection and its associated explanatory variables. A cross-sectional self-administered online survey was conducted from 3 May to 3 June 2016 in Aceh province, Indonesia. A pre-tested questionnaire was used to collect data on doctors’ attitudes towards ZIKV infection and a range of explanatory variables (basic demographic data, professional characteristics, workplace characteristics and facilities, and medical experience related to ZIKV infection). Associations between attitude and explanatory variables were assessed using multiple-step logistic regression. We received 631 responses, 424 (67.19%) of which were included in the final analysis. Approximately 64% (271) of doctors had a poor attitude towards ZIKV infection. Experience considering ZIKV infection as a differential diagnosis and attendance at a national conference was associated with a good attitude, with odds ratios (OR) of 3.93 (95% confidence interval [CI]: 1.15–13.49) and 1.69 (95% CI: 1.03–2.76), respectively. Unexpectedly, doctors who had attended an international conference and those working at places that had molecular diagnostic (polymerase chain reaction based testing) facilities had lower odds of having a good attitude (OR: 0.35 [95% CI: 0.15–0.84] and 0.42 [95% CI: 0.19–0.95], respectively). In conclusion, the attitude towards ZIKV infection is relatively poor among doctors in Aceh. Therefore, strategies for enhancing their capacity to respond to ZIKV infection are needed. The survey concept and tools were well accepted by the participants of this study, suggesting that this rapid assessment could be rolled out across the Indonesian archipelago and elsewhere to identify and regionally differentiate unmet needs of disease and outbreak preparedness.
In the later stages of addiction, automatized processes play a prominent role in guiding drug-seeking and drug-taking behavior. However, little is known about the neural correlates of automatized drug-taking skills and drug-related action knowledge in humans. We employed functional magnetic resonance imaging (fMRI) while smokers and non-smokers performed an orientation affordance task, where compatibility between the hand used for a behavioral response and the spatial orientation of a priming stimulus leads to shorter reaction times resulting from activation of the corresponding motor representations. While non-smokers exhibited this behavioral effect only for control objects, smokers showed the affordance effect for both control and smoking-related objects. Furthermore, smokers exhibited reduced fMRI activation for smoking-related as compared to control objects for compatible stimulus-response pairings in a sensorimotor brain network consisting of the right primary motor cortex, supplementary motor area, middle occipital gyrus, left fusiform gyrus and bilateral cingulate gyrus. In the incompatible condition, we found higher fMRI activation in smokers for smoking-related as compared to control objects in the right primary motor cortex, cingulate gyrus, and left fusiform gyrus. This suggests that the activation and performance of deeply embedded, automatized drug-taking schemata employ less brain resources. This might reduce the threshold for relapsing in individuals trying to abstain from smoking. In contrast, the interruption or modification of already triggered automatized action representations require increased neural resources.
Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins
(2017)
Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFβ-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFβ. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. TRAF3 autophagy is driven by RAS and results in activation of the NF-κB family member RELB. We show that RELB represses TGFβ target promoters independently of DNA binding at NF-κB recognition sequences, instead binding with SMAD family member(s) at SMAD-response elements. Thus, autophagy antagonises TGFβ gene expression. Finally, autophagy-deficient A549 cells regain tumorigenicity upon SMAD4 knockdown. Thus, at least in this setting, a physiologic function for autophagic regulation of gene expression is tumour growth.
Autophagy in cancer therapy
(2017)
Autophagy represents a catabolic program involved in the degradation of cellular components via lysosomes. It serves to mitigate cellular stress and to provide metabolic precursors especially upon starvation. Thereby, autophagy can support the survival of cancer cells. In addition, there is now convincing evidence showing that under certain conditions autophagy can also foster cell death. This dual function of autophagy is also relevant upon anticancer treatment, as many chemotherapeutic agents engage autophagy. A better understanding of the molecular mechanisms that are critical for mediating autophagic cell death in cancer cells will be instrumental to selectively interfere with this cellular program in order to increase the cancer cell’s response to cytotoxic drugs. This review illustrates how anticancer drug-induced autophagy is involved in mediating cell death.
Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient’s own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF) was compared to standard platelet-rich plasma (PRP) with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g) for 3 min) were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), collagen1 (COL1) and fibronectin (FN). The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA) levels of PDGF, TGF-β, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.
Benzene is a ubiquitous, volatile pollutant present at high concentrations in toxins (e.g. tobacco smoke) known to increase cardiovascular disease (CVD) risk. Despite its prevalence, the cardiovascular effects of benzene have rarely been studied. Hence, we examined whether exposure to benzene is associated with increased CVD risk. The effects of benzene exposure in mice were assessed by direct inhalation, while the effects of benzene exposure in humans was assessed in 210 individuals with mild to high CVD risk by measuring urinary levels of the benzene metabolite trans,trans-muconic acid (t,t-MA). Generalized linear models were used to assess the association between benzene exposure and CVD risk. Mice inhaling volatile benzene had significantly reduced levels of circulating angiogenic cells (Flk-1+/Sca-1+) as well as an increased levels of plasma low-density lipoprotein (LDL) compared with control mice breathing filtered air. In the human cohort, urinary levels of t,t-MA were inversely associated several populations of circulating angiogenic cells (CD31+/34+/45+, CD31+/34+/45+/AC133–, CD34+/45+/AC133+). Although t,t-MA was not associated with plasma markers of inflammation or thrombosis, t,t-MA levels were higher in smokers and in individuals with dyslipidemia. In smokers, t,t-MA levels were positively associated with urinary metabolites of nicotine (cotinine) and acrolein (3-hydroxymercapturic acid). Levels of t,t-MA were also associated with CVD risk as assessed using the Framingham Risk Score and this association was independent of smoking. Thus, benzene exposure is associated with increased CVD risk and deficits in circulating angiogenic cells in both smokers and non-smokers.
Bestimmung des klinischen Nutzens systemischer adjuvanter Therapien beim frühen Mammakarzinom
(2017)
Die onkologische Therapie befindet sich im Umbruch. Hohe Erwartungen sind mit einer Reihe innovativer zielgerichteter Medikamente verknüpft, die sich derzeit in der klinischen Entwicklung befinden. Vor diesem Hintergrund erfahren Diskussionen um die Begriffe klinischer Nutzen oder klinische Relevanz neue Aktualität. Dies gilt auch für die Weiterentwicklungen der adjuvanten systemischen Therapie des frühen Mammakarzinoms. In Anbetracht der kurativen Zielsetzung erfolgt die Beurteilung des klinischen Nutzens einer adjuvanten Therapie maßgeblich anhand von Wirksamkeitsendpunkten. Der Fokus liegt hierbei auf Verbesserungen des krankheitsfreien Überlebens und des Rezidivrisikos. Eine Aussage zum Gesamtüberleben ist aufgrund der heute erreichten niedrigen Mortalitätsraten erst nach sehr langen Beobachtungszeiten möglich. Folgerichtig sollte neuen Medikamenten für die adjuvante Therapie ein klinischer Nutzen zugesprochen werden, wenn sie eine weitere Reduktion des Rezidivrisikos über den heutigen hohen Standard hinaus ermöglichen. Die Evidenz für etablierte adjuvante Therapiestandards beim frühen Mammakarzinom kann als objektiver Maßstab zum Vergleich herangezogen werden. Am Beispiel der adjuvanten endokrinen Therapie, der adjuvanten Polychemotherapie und der adjuvanten Anti-HER2-Therapie werden in diesem Übersichtsartikel die Anforderungen für den klinischen Nutzen neuer adjuvanter Therapien beim frühen Mammakarzinom abgeleitet.
Bevacizumab for patients with recurrent gliomas presenting with a gliomatosis cerebri growth pattern
(2017)
Bevacizumab has been shown to improve progression-free survival and neurologic function, but failed to improve overall survival in newly diagnosed glioblastoma and at first recurrence. Nonetheless, bevacizumab is widely used in patients with recurrent glioma. However, its use in patients with gliomas showing a gliomatosis cerebri growth pattern is contentious. Due to the marked diffuse and infiltrative growth with less angiogenic tumor growth, it may appear questionable whether bevacizumab can have a therapeutic effect in those patients. However, the development of nodular, necrotic, and/or contrast-enhancing lesions in patients with a gliomatosis cerebri growth pattern is not uncommon and may indicate focal neo-angiogenesis. Therefore, control of growth of these lesions as well as control of edema and reduction of steroid use may be regarded as rationales for the use of bevacizumab in these patients. In this retrospective patient series, we report on 17 patients with primary brain tumors displaying a gliomatosis cerebri growth pattern (including seven glioblastomas, two anaplastic astrocytomas, one anaplastic oligodendroglioma, and seven diffuse astrocytomas). Patients have been treated with bevacizumab alone or in combination with lomustine or irinotecan. Seventeen matched patients treated with bevacizumab for gliomas with a classical growth pattern served as a control cohort. Response rate, progression-free survival, and overall survival were similar in both groups. Based on these results, anti-angiogenic therapy with bevacizumab should also be considered in patients suffering from gliomas with a mainly infiltrative phenotype.
In patients with glioblastoma, antiangiogenic therapy with bevacizumab (BEV) has been shown to improve progression-free survival (PFS), but not overall survival (OS). Especially in patients with an unusual infiltrative phenotype as seen in multifocal glioblastoma, the use of BEV therapy is still more controversial. Therefore, we prepared a retrospective case series with 16 patients suffering from a multifocal glioblastoma treated with BEV. We compared these patients to a matched control cohort of 16 patients suffering from glioblastoma with a single lesion treated with BEV. The objective of this study was to evaluate whether the course of disease differs in glioblastoma patients with a multifocal disease pattern compared to those with a single lesion only. Patients were treated with BEV monotherapy or BEV in combination with irinotecan or lomustine (CCNU). Response rates and PFS were similar in both groups. There was a trend for an unfavorable OS in the patient group with multifocal glioblastoma, which was expected due to the generally worse prognosis of multifocal glioblastoma. We investigated whether BEV therapy affects the invasive growth pattern as measured by the appearance of new lesions on magnetic resonance imaging (MRI). Under BEV therapy, there was a trend for a lower frequency of new lesions both in multifocal and solitary glioblastoma. Based on these results, BEV therapy at relapse appears to be justified to no lesser extent in multifocal glioblastoma than in solitary glioblastoma.
Background: The aim of this meta-analysis was to evaluate efficacy and safety of first-line chemotherapy with or without a monoclonal antibody in elderly patients ( ≥ 70 years) with metastatic colorectal cancer (mCRC), since they are frequently underrepresented in clinical trials.
Results: Individual data from 10 studies were included. From a total of 3271 patients, 604 patients (18%) were ≥ 70 years (median 73 years, range 70–88). Of these, 335 patients were treated with a bevacizumab-based first-line regimen and 265 were treated with chemotherapy only. The median PFS was 8.2 vs. 6.5 months and the median OS was 16.7 vs. 13.0 months in patients treated with and without bevacizumab, respectively. The safety profile of bevacizumab in combination with first-line chemotherapy did not differ from published clinical trials.
Materials and Methods: PubMed and Cochrane Library searches were performed on 29 April 2013 and studies published to this date were included. Authors were contacted to request progression-free survival (PFS), overall survival (OS) data, patient data on treatment regimens, age, sex and potential signs of toxicity in patients ≥ 70 years of age.
Conclusions: This meta-analysis suggests that the addition of bevacizumab to standard first-line chemotherapy improves clinical outcome in elderly patients with mCRC and is well tolerated.
The sphingolipid sphingosine-1-phosphate (S1P) emerges as an important regulator of immunity, mainly by signaling through a family of five specific G protein-coupled receptors (S1PR1–5). While S1P signaling generally has the potential to affect not only trafficking but also differentiation, activation, and survival of a diverse range of immune cells, the specific outcome depends on the S1P receptor repertoire expressed on a given cell. Among the S1PRs, S1PR4 is specifically abundant in immune cells, suggesting a major role of the S1P/S1PR4 axis in immunity. Recent studies indeed highlight its role in activation of immune cells, differentiation, and, potentially, trafficking. In this review, we summarize the emerging data that support a major role of S1PR4 in modulating immunity in humans and mice and discuss therapeutic implications.
In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.
Galunisertib, a Transforming growth factor-βRI (TGF-βRI) kinase inhibitor, blocks TGF-β-mediated tumor growth in glioblastoma. In a three-arm study of galunisertib (300 mg/day) monotherapy (intermittent dosing; each cycle =14 days on/14 days off), lomustine monotherapy, and galunisertib plus lomustine therapy, baseline tumor tissue was evaluated to identify markers associated with tumor stage (e.g., histopathology, Ki67, glial fibrillary acidic protein) and TGF-β-related signaling (e.g., pSMAD2). Other pharmacodynamic assessments included chemokine, cytokine, and T cell subsets alterations. 158 patients were randomized to galunisertib plus lomustine (n = 79), galunisertib (n = 39) and placebo+lomustine (n = 40). In 127 of these patients, tissue was adequate for central pathology review and biomarker work. Isocitrate dehydrogenase (IDH1) negative glioblastoma patients with baseline pSMAD2+ in cytoplasm had median overall survival (OS) 9.5 months vs. 6.9 months for patients with no tumor pSMAD2 expression (p = 0.4574). Eight patients were IDH1 R132H+ and had a median OS of 10.4 months compared to 6.9 months for patients with negative IDH1 R132H (p = 0.5452). IDH1 status was associated with numerically higher plasma macrophage-derived chemokine (MDC/CCL22), higher whole blood FOXP3, and reduced tumor CD3+ T cell counts. Compared to the baseline, treatment with galunisertib monotherapy preserved CD4+ T cell counts, eosinophils, lymphocytes, and the CD4/CD8 ratio. The T-regulatory cell compartment was associated with better OS with MDC/CCL22 as a prominent prognostic marker.
We describe 70 cases of monocled cobra (Naja kaouthia) bite admitted to Chittagong Medical College Hospital, Bangladesh. The biting snakes were identified by examining the dead snake and/or detecting N. kaouthia venom antigens in patients' serum. Bites were most common in the early morning and evening during the monsoon (May-July). Ligatures were routinely applied to the bitten limb before admission. Thirty-seven patients consulted traditional healers, most of whom made incisions around the bite site. Fifty-eight patients experienced severe neurotoxicity and most suffered swelling and pain of the bitten limb. The use of an Indian polyvalent antivenom in patients exhibiting severe neurotoxicity resulted in clinical improvement but most patients experienced moderate-to-severe adverse reactions. Antivenom did not influence local blistering and necrosis appearing in 19 patients; 12 required debridement. Edrophonium significantly improved the ability of patients to open the eyes, endurance of upward gaze, and peak expiratory flow rate suggesting that a longer-acting anticholinesterase drug (neostigmine) could be recommended for first aid. The study suggested that regionally appropriate antivenom should be raised against the venoms of the major envenoming species of Bangladesh and highlighted the need to improve the training of staff of local medical centers and to invest in the basic health infrastructure in rural communities.
The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.
Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3′-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.
The disruption of coupling between brain areas has been suggested as the mechanism underlying loss of consciousness in anesthesia. This hypothesis has been tested previously by measuring the information transfer between brain areas, and by taking reduced information transfer as a proxy for decoupling. Yet, information transfer is a function of the amount of information available in the information source—such that transfer decreases even for unchanged coupling when less source information is available. Therefore, we reconsidered past interpretations of reduced information transfer as a sign of decoupling, and asked whether impaired local information processing leads to a loss of information transfer. An important prediction of this alternative hypothesis is that changes in locally available information (signal entropy) should be at least as pronounced as changes in information transfer. We tested this prediction by recording local field potentials in two ferrets after administration of isoflurane in concentrations of 0.0%, 0.5%, and 1.0%. We found strong decreases in the source entropy under isoflurane in area V1 and the prefrontal cortex (PFC)—as predicted by our alternative hypothesis. The decrease in source entropy was stronger in PFC compared to V1. Information transfer between V1 and PFC was reduced bidirectionally, but with a stronger decrease from PFC to V1. This links the stronger decrease in information transfer to the stronger decrease in source entropy—suggesting reduced source entropy reduces information transfer. This conclusion fits the observation that the synaptic targets of isoflurane are located in local cortical circuits rather than on the synapses formed by interareal axonal projections. Thus, changes in information transfer under isoflurane seem to be a consequence of changes in local processing more than of decoupling between brain areas. We suggest that source entropy changes must be considered whenever interpreting changes in information transfer as decoupling.
Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology
(2017)
Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Callous-unemotional traits are characterized by a lack of empathy, a disregard for others' feelings and shallow or deficient affect, such as a lack of remorse or guilt. Neuroanatomical correlates of callous-unemotional traits have been demonstrated in clinical samples (i.e., adolescents with disruptive behavior disorders). However, it is unknown whether callous-unemotional traits are associated with neuroanatomical correlates within normative populations without clinical levels of aggression or antisocial behavior. Here we investigated the relationship between callous-unemotional traits and gray matter volume using voxel-based morphometry in a large sample of typically-developing boys and girls (N = 189). Whole-brain multiple regression analyses controlling for site, total intracranial volume, and age were conducted in the whole sample and in boys and girls individually. Results revealed that sex and callous-unemotional traits interacted to predict gray matter volume when considering the whole sample. This interaction was driven by a significant positive correlation between callous-unemotional traits and bilateral anterior insula volume in boys, but not girls. Insula gray matter volume explained 19% of the variance in callous-unemotional traits for boys. Our results demonstrate that callous-unemotional traits are related to variations in brain structure beyond psychiatric samples. This association was observed for boys only, underlining the importance of considering sex as a factor in future research designs. Future longitudinal studies should determine whether these findings hold over childhood and adolescence, and whether the neuroanatomical correlates of callous-unemotional traits are predictive of future psychiatric vulnerability.
Cannabinoid antagonist SLV326 induces convulsive seizures and changes in the interictal EEG in rats
(2017)
Cannabinoid CB1 antagonists have been investigated for possible treatment of e.g. obesity-related disorders. However, clinical application was halted due to their symptoms of anxiety and depression. In addition to these adverse effects, we have shown earlier that chronic treatment with the CB1 antagonist rimonabant may induce EEG-confirmed convulsive seizures. In a regulatory repeat-dose toxicity study violent episodes of “muscle spasms” were observed in Wistar rats, daily dosed with the CB1 receptor antagonist SLV326 during 5 months. The aim of the present follow-up study was to investigate whether these violent movements were of an epileptic origin. In selected SLV326-treated and control animals, EEG and behavior were monitored for 24 hours. 25% of SLV326 treated animals showed 1 to 21 EEG-confirmed generalized convulsive seizures, whereas controls were seizure-free. The behavioral seizures were typical for a limbic origin. Moreover, interictal spikes were found in 38% of treated animals. The frequency spectrum of the interictal EEG of the treated rats showed a lower theta peak frequency, as well as lower gamma power compared to the controls. These frequency changes were state-dependent: they were only found during high locomotor activity. It is concluded that long term blockade of the endogenous cannabinoid system can provoke limbic seizures in otherwise healthy rats. Additionally, SLV326 alters the frequency spectrum of the EEG when rats are highly active, suggesting effects on complex behavior and cognition.
The objective of this study was to characterize blaOXA-23 harbouring Acinetobacter indicus-like strains from cattle including genomic and phylogenetic analyses, antimicrobial susceptibility testing and evaluation of pathogenicity in vitro and in vivo. Nasal and rectal swabs (n = 45) from cattle in Germany were screened for carbapenem-non-susceptible Acinetobacter spp. Thereby, two carbapenem resistant Acinetobacter spp. from the nasal cavities of two calves could be isolated. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified these isolates as A. indicus-like. A phylogenetic tree based on partial rpoB sequences indicated closest relation of the two bovine isolates to the A. indicus type strain A648T and human clinical A. indicus isolates, while whole genome comparison revealed considerable intraspecies diversity. High mimimum inhibitory concentrations were observed for carbapenems and other antibiotics including fluoroquinolones and gentamicin. Whole genome sequencing and PCR mapping revealed that both isolates harboured blaOXA-23 localized on the chromosome and surrounded by interrupted Tn2008 transposon structures. Since the pathogenic potential of A. indicus is unknown, pathogenicity was assessed employing the Galleria (G.) mellonella infection model and an in vitro cytotoxicity assay using A549 human lung epithelial cells. Pathogenicity in vivo (G. mellonella killing assay) and in vitro (cytotoxicity assay) of the two A. indicus-like isolates was lower compared to A. baumannii ATCC 17978 and similar to A. lwoffii ATCC 15309. The reduced pathogenicity of A. indicus compared to A. baumannii correlated with the absence of important virulence genes encoding like phospholipase C1+C2, acinetobactin outer membrane protein BauA, RND-type efflux system proteins AdeRS and AdeAB or the trimeric autotransporter adhesin Ata. The emergence of carbapenem-resistant A. indicus-like strains from cattle carrying blaOXA-23 on transposable elements and revealing genetic relatedness to isolates from human clinical sources requires further investigations regarding the pathogenic potential, genomic characteristics, zoonotic risk and putative additional sources of this new Acinetobacter species.
Aim: To evaluate the long-term outcome of catheter ablation of atrial fibrillation (AF) facilitated by preprocedural three-dimensional (3-D) transesophageal echocardiography.
Methods: In 50 patients, 3D transesophageal echocardiography (3D TEE) was performed immediately prior to an ablation procedure (paroxysmal AF: 30 patients, persistent AF: 20 patients). The images were available throughout the ablation procedure. Two different ablation strategies were used. In most of the patients with paroxysmal AF, the cryoablation technique was used (Arctic Front Balloon, CryoCath Technologies/Medtronic; group A2). In the other patients, a circumferential pulmonary vein ablation was performed using the CARTO system [Biosense Webster; group A1 (paroxysmal AF), group B (persistent AF)]. Success rates and complication rates were analysed at 4-year follow-up.
Results: A 3D TEE could be performed successfully in all patients prior to the ablation procedure and all four pulmonary vein ostia could be evaluated in 84% of patients. The image quality was excellent in the majority of patients and several variations of the pulmonary vein anatomy could be visualized precisely (e.g., common pulmonary vein ostia, accessory pulmonary veins, varying diameter of the left atrial appendage and its distance to the left superior pulmonary vein). All ablation procedures could be performed as planned and almost all pulmonary veins could be isolated successfully. At 48-mo follow-up, 68.0% of all patients were free from an arrhythmia recurrence (group A1: 72.7%, group A2: 73.7%, group B: 60.0%). There were no major complications.
Conclusion: 3D TEE provides an excellent overview over the left atrial anatomy prior to AF ablation procedures and these procedures are associated with a favourable long-term outcome.
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H2O2, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H2O2 treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H2O2 treatment, the expression of p53-dependent 5’-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H2O2-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.
There is a need for diagnostic biomarkers of epilepsy and status epilepticus to support clinical examination, electroencephalography and neuroimaging. Extracellular microRNAs may be potentially ideal biomarkers since some are expressed uniquely within specific brain regions and cell types. Cerebrospinal fluid offers a source of microRNA biomarkers with the advantage of being in close contact with the target tissue and sites of pathology. Here we profiled microRNA levels in cerebrospinal fluid from patients with temporal lobe epilepsy or status epilepticus, and compared findings to matched controls. Differential expression of 20 microRNAs was detected between patient groups and controls. A validation phase included an expanded cohort and samples from patients with other neurological diseases. This identified lower levels of miR-19b in temporal lobe epilepsy compared to controls, status epilepticus and other neurological diseases. Levels of miR-451a were higher in status epilepticus compared to other groups whereas miR-21-5p differed in status epilepticus compared to temporal lobe epilepsy but not to other neurological diseases. Targets of these microRNAs include proteins regulating neuronal death, tissue remodelling, gliosis and inflammation. The present study indicates cerebrospinal fluid contains microRNAs that can support differential diagnosis of temporal lobe epilepsy and status epilepticus from other neurological and non-neurological diseases.
Characterization of a novel KCNJ2 sequence variant detected in Andersen-Tawil syndrome patients
(2017)
Background: Mutations in the KCNJ2 gene encoding the ion channel Kir2.1 have been linked to the Andersen-Tawil syndrome (ATS). Molecular genetic screening performed in a family exhibiting clinical ATS phenotypes unmasked a novel sequence variant (c.434A > G, p.Y145C) in this gene. The aim of this study was to investigate the effect of this variant on Kir2.1 ion channel functionality.
Methods: Mutant as well as wild type GFP tagged Kir2.1 channels were expressed in HEK293 cells. In order to examine the effect of the new variant, electrophysiological measurements were performed using patch clamp technique. Cellular localization of the mutant in comparison to the wild type ion channel was analyzed by confocal laser scanning microscopy.
Results: The currents of cells expressing only mutant channels or a mixture of wild type and mutant were significantly reduced compared to those expressing wild type (WT) channels (p < 0.01). Whereas WT expressing cells exhibited at −120 mV an averaged current of −4.5 ± 1.9 nA, the mutant generates only a current of −0.17 ± 0.07 nA. A co-expression of mutant and WT channel generates only a partial rescue of the WT current. Confocal laser scanning microscopy indicated that the novel variant is not interfering with synthesis and/or protein trafficking.
Conclusions: The detected sequence variant causes loss-of-function of the Kir2.1 channel and explains the clinical phenotypes observed in Andersen-Tawil syndrome patients.
Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1−/−-MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1−/−-MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1−/−-MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1−/−-MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.
Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine
(2017)
Background: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer.
Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs.
Results: Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762.
Conclusions: AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
Dysfunction of the NO/sGC/cGMP signaling pathway has been implicated in the pathogenesis of pulmonary hypertension (PH). Therefore, agents stimulating cGMP synthesis via sGC are important therapeutic options for treatment of PH patients. An unwanted effect of this novel class of drugs is their systemic hypotensive effect. We tested the hypothesis that aerosolized intra-tracheal delivery of the sGC stimulator BAY41-8543 could diminish its systemic vasodilating effect.
Pharmacodynamics and -kinetics of BAY41-8543 after single intra-tracheal delivery was tested in healthy rats. Four weeks after a single injection of monocrotaline (MCT, 60 mg/kg s.c.), rats were randomized to a two-week treatment with either placebo, BAY 41-8543 (10 mg/kg per os (PO)) or intra-tracheal (IT) instillation (3 mg/kg or 1 mg/kg).
Circulating concentrations of the drug 10 mg/kg PO and 3 mg/kg IT were comparable. BAY 41-8543 was detected in the lung tissue and broncho-alveolar fluid after IT delivery at higher concentrations than after PO administration. Systemic arterial pressure transiently decreased after oral BAY 41-8543 and was unaffected by intratracheal instillation of the drug. PO 10 mg/kg and IT 3 mg/kg regimens partially reversed pulmonary hypertension and improved heart function in MCT-injected rats. Minor efficacy was noted in rats treated IT with 1 mg/kg. The degree of pulmonary vascular remodeling was largely reversed in all treatment groups.
Intratracheal administration of BAY 41-8543 reverses PAH and vascular structural remodeling in MCT-treated rats. Local lung delivery is not associated with systemic blood pressure lowering and represents thus a further development of PH treatment with sGC stimulators.
Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.
The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.
Many new strategies for the reconstruction of peripheral nerve injuries have been explored for their effectiveness in supporting nerve regeneration. However only a few of these materials were actually clinically evaluated and approved for human use. This open, mono-center, non-randomized clinical study summarizes the 12-month follow-up of patients receiving reconstruction of the sural nerve biopsy defect by the collagen-based nerve guide Neuromaix. Neuromaix was implanted as a micro-structured, two-component scaffold bridging 20–40 mm nerve defects after sural nerve biopsy in twenty patients (eighteen evaluated, two lost in follow-up). Safety of the material was evaluated by clinical examination of wound healing. Performance was assessed by sensory testing of modalities, pain assessment, and palpation for the Hoffmann–Tinel’s sign as well as demarcating the asensitive area at each follow-up visit. Every patient demonstrated uneventful wound healing during the complete 12-month time course of the study. Two patients reported complete return of sensation, whereas eleven out of eighteen patients reported a positive Hoffmann–Tinel’s sign at the lower leg with simultaneous reduction of the asensitive area by 12 months. Our data show that Neuromaix can be implanted safely in humans to bridge sural nerve gaps. No procedure-related, adverse events, or severe adverse events were reported. These first clinical data on Neuromaix provide promising perspectives for the bridging of larger nerve gaps in combined nerves, which should be investigated more through extensive, multi-center clinical trials in the near future.
Background. Colorectal cancers (CRC) shed DNA into blood circulation. There is growing evidence that the analysis of circulating tumor DNA can be effectively used for monitoring of disease, to track tumor heterogeneity and to evaluate response to treatment. Case Presentation. Here, we describe two cases of patients with advanced CRC. The first case is about a patient with no available tissue for analysis of RAS mutation status. Liquid biopsy revealed RAS-wild-type and the therapy with anti-EGFR (epidermal growth factor receptor) monoclonal antibody cetuximab could be initiated. In the second case, the mutational profile of a patient with initial wild-type RAS-status was continually tracked during the course of treatment. An acquired KRAS exon 3 mutation was detected. The number of KRAS mutated fragments decreased continuously after the discontinuation of the therapy with EGFR-specific antibodies. Conclusion. Liquid biopsy provides a rapid genotype result, which accurately reproduces the current mutation status of tumor tissue. Furthermore, liquid biopsy enables close monitoring of the onset of secondary resistance to anti-EGFR therapy.
Hematotoxicity is one of the major side effects of chemotherapy. The aim of this study was to examine the association between single nucleotide polymorphisms (SNPs) and hematotoxicity in breast cancer patients in a subset of patients of the SUCCESS prospective phase III chemotherapy study. All patients (n = 1678) received three cycles of 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) followed by three cycles of docetaxel or docetaxel/gemcitabine, depending on randomization. Germline DNA was genotyped for 246 SNPs selected from a previous genome-wide association study (GWAS) in a panel of lymphoblastoid cell lines, with gemcitabine toxicity as the phenotype. All SNPs were tested for their value in predicting grade 3 or 4 neutropenic or leukopenic events (NLEs). Their prognostic value in relation to overall survival and disease-free survival was also tested.
None of the SNPs was found to be predictive for NLEs during treatment with docetaxel/gemcitabine. Two SNPs in and close to the PIGB gene significantly improved the prediction of NLEs after FEC, in addition to the factors of age and body surface area. The top SNP (rs12050587) had an odds ratio of 1.38 per minor allele (95% confidence interval, 1.17 to 1.62). No associations were identified for predicting disease-free or overall survival.
Genetic variance in the PIGB gene may play a role in determining interindividual differences in relation to hematotoxicity after FEC chemotherapy.
Recent progress in the synaptic pathophysiology of brain diseases is reviewed. To emphasize the emergence of common motifs in synapse dysfunctions across neurodevelopmental, psychiatric and neurological disorders, conventional clinical boundaries are disregarded and a decidedly trans-diagnostic, potentially unifying view of altered synapse function is promoted. Based on the overlapping genetic architecture of brain disorders, which often converges on genes related to synaptic functions, disease-related changes in basic pre-synaptic and post-synaptic communication, neuromodulation-gated changes in Hebbian plasticity, dynamic interactions between Hebbian and homeostatic plasticity, and changes in synaptic maintenance by autophagy and glial-mediated phagocytosis are highlighted.
Background: Women’s participation in medicine and the need for gender equality in healthcare are increasingly recognised, yet little attention is paid to leadership and management positions in large publicly funded academic health centres. This study illustrates such a need, taking the case of four large European centres: Charité – Universitätsmedizin Berlin (Germany), Karolinska Institutet (Sweden), Medizinische Universität Wien (Austria), and Oxford Academic Health Science Centre (United Kingdom).
Case:The percentage of female medical students and doctors in all four countries is now well within the 40–60% gender balance zone. Women are less well represented among specialists and remain significantly under-represented among senior doctors and full professors. All four centres have made progress in closing the gender leadership gap on boards and other top-level decision-making bodies, but a gender leadership gap remains relevant. The level of achieved gender balance varies significantly between the centres and largely mirrors country-specific welfare state models, with more equal gender relations in Sweden than in the other countries. Notably, there are also similar trends across countries and centres: gender inequality is stronger within academic enterprises than within hospital enterprises and stronger in middle management than at the top level. These novel findings reveal fissures in the ‘glass ceiling’ effects at top-level management, while the barriers for women shift to middle-level management and remain strong in academic positions. The uneven shifts in the leadership gap are highly relevant and have policy implications.
Conclusion: Setting gender balance objectives exclusively for top-level decision-making bodies may not effectively promote a wider goal of gender equality. Academic health centres should pay greater attention to gender equality as an issue of organisational performance and good leadership at all levels of management, with particular attention to academic enterprises and newly created management structures. Developing comprehensive gender-sensitive health workforce monitoring systems and comparing progress across academic health centres in Europe could help to identify the gender leadership gap and utilise health human resources more effectively.
Clustering of cardiovascular risk factors and carotid intima-media thickness : the USE-IMT study
(2017)
Background: The relation of a single risk factor with atherosclerosis is established. Clinically we know of risk factor clustering within individuals. Yet, studies into the magnitude of the relation of risk factor clusters with atherosclerosis are limited. Here, we assessed that relation.
Methods: Individual participant data from 14 cohorts, involving 59,025 individuals were used in this cross-sectional analysis. We made 15 clusters of four risk factors (current smoking, overweight, elevated blood pressure, elevated total cholesterol). Multilevel age and sex adjusted linear regression models were applied to estimate mean differences in common carotid intima-media thickness (CIMT) between clusters using those without any of the four risk factors as reference group.
Results: Compared to the reference, those with 1, 2, 3 or 4 risk factors had a significantly higher common CIMT: mean difference of 0.026 mm, 0.052 mm, 0.074 mm and 0.114 mm, respectively. These findings were the same in men and in women, and across ethnic groups. Within each risk factor cluster (1, 2, 3 risk factors), groups with elevated blood pressure had the largest CIMT and those with elevated cholesterol the lowest CIMT, a pattern similar for men and women.
Conclusion: Clusters of risk factors relate to increased common CIMT in a graded manner, similar in men, women and across race-ethnic groups. Some clusters seemed more atherogenic than others. Our findings support the notion that cardiovascular prevention should focus on sets of risk factors rather than individual levels alone, but may prioritize within clusters.
The yeast bc1 complex (complex III) and cytochrome oxidase (complex IV) are mosaics of core subunits encoded by the mitochondrial genome and additional nuclear-encoded proteins imported from the cytosol. Both complexes build in the mitochondrial inner membrane various supramolecular assemblies. The formation of the individual complexes and their supercomplexes depends on the activity of dedicated assembly factors. We identified a so far uncharacterized mitochondrial protein (open reading frame YDR381C-A) as an important assembly factor for complex III, complex IV, and their supercomplexes. Therefore, we named this protein Cox interacting (Coi) 1. Deletion of COI1 results in decreased respiratory growth, reduced membrane potential, and hampered respiration, as well as slow fermentative growth at low temperature. In addition, coi1Δ cells harbour reduced steady-state levels of subunits of complexes III and IV as well as of the assembled complexes and supercomplexes. Interaction of Coi1 with respiratory chain subunits seems transient, as it appears to be a stoichiometric subunit neither of complex III nor of complex IV. Collectively, this work identifies a novel protein that plays a role in the assembly of the mitochondrial respiratory chain.