An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism.

GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.

Efficacy and safety of crizotinib in ALK-positive systemic anaplastic large-cell lymphoma in children, adolescents, and adult patients: results of the French AcSé-crizotinib trial.

BACKGROUND: The French phase II AcSé-crizotinib trial aimed to evaluate the safety and efficacy of crizotinib in patients with ALK, ROS1, and MET-driven malignancies, including ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL). METHODS: ALK+ ALCL patients 12 months or older with measurable disease and no standard care options available received crizotinib twice daily at 165 mg/m2 in children and adolescents and 250 mg in adults. The primary end-point was the response rate at 8 weeks. RESULTS: Twenty-eight patients were enroled between February 2014 and March 2018. Three patients who were not treated were excluded from the analysis. The median age was 19 years. The median previous line of chemotherapy was two. In the 24 patients with an evaluable response, the response rate at 8 weeks was 67% (95% CI: 47-82%). All patients discontinued crizotinib after a median treatment duration of 3.7 months: eight for progression, two for adverse events (AEs) related to prior treatments, and 15 by choice, including six for allogeneic stem-cell transplantation. The median follow-up was 45 months. Nine patients experienced an event: eight relapses (seven after crizotinib discontinuation and one after dose reduction), and one died in complete remission. The median duration of response was 43.3 months (95% CI: 8.3-not reached). The 3-year progression-free and overall survival rates were 40% (95% CI: 23-59%) and 63% (95% CI: 43-79%). Grade 3 or 4 treatment-related AEs occurred in 32% of patients. CONCLUSION: Crizotinib shows efficacy and an acceptable safety profile in ALK+ ALCL relapsed/refractory patients. However, a large proportion of patients experience a relapse after crizotinib discontinuation. Future studies will assess if prolonged ALK inhibitor exposure has curative potential without consolidation.

Topical delivery of mitogen-activated protein kinase inhibitor binimetinib prevents the development of cutaneous neurofibromas in neurofibromatosis type 1 mutant mice.

Cutaneous neurofibromas (cNFs) are a hallmark of patients with the neurofibromatosis type 1 (NF1) genetic disorder. These benign nerve sheath tumors, which can amount to thousands, develop from puberty onward, often cause pain and are considered by patients to be the primary burden of the disease. Mutations of NF1, encoding a negative regulator of the RAS signaling pathway, in the Schwann cell (SCs) lineage are considered to be at the origin of cNFs. The mechanisms governing cNFs development are poorly understood, and therapeutics to reduce cNFs are missing, mainly due to the lack of appropriate animal models. To address this, we designed the Nf1-KO mouse model that develops cNFs. Using this model, we found that cNFs development is a singular event and goes through 3 successive stages: initiation, progression, and stabilization characterized by changes in the proliferative and MAPK activities of tumor SCs. We found that skin trauma accelerated the development of cNFs and further used this model to explore the efficacy of the MEK inhibitor binimetinib to cure these tumors. We showed that while topically delivered binimetinib has a selective and minor effect on mature cNFs, the same drug prevents their development over long periods.

Improving Emergency Department Patient-Physician Conversation Through an Artificial Intelligence Symptom-Taking Tool: Mixed Methods Pilot Observational Study.

BACKGROUND: Establishing rapport and empathy between patients and their health care provider is important but challenging in the context of a busy and crowded emergency department (ED). OBJECTIVE: We explore the hypotheses that rapport building, documentation, and time efficiency might be improved in the ED by providing patients a digital tool that uses Bayesian reasoning-based techniques to gather relevant symptoms and history for handover to clinicians. METHODS: A 2-phase pilot evaluation was carried out in the ED of a German tertiary referral and major trauma hospital that treats an average of 120 patients daily. Phase 1 observations guided iterative improvement of the digital tool, which was then further evaluated in phase 2. All patients who were willing and able to provide consent were invited to participate, excluding those with severe injury or illness requiring immediate treatment, with traumatic injury, incapable of completing a health assessment, and aged <18 years. Over an 18-day period with 1699 patients presenting to the ED, 815 (47.96%) were eligible based on triage level. With available recruitment staff, 135 were approached, of whom 81 (60%) were included in the study. In a mixed methods evaluation, patients entered information into the tool, accessed by clinicians through a dashboard. All users completed evaluation Likert-scale questionnaires rating the tool's performance. The feasibility of a larger trial was evaluated through rates of recruitment and questionnaire completion. RESULTS: Respondents strongly endorsed the tool for facilitating conversation (61/81, 75% of patients, 57/78, 73% of physician ratings, and 10/10, 100% of nurse ratings). Most nurses judged the tool as potentially time saving, whereas most physicians only agreed for a subset of medical specialties (eg, surgery). Patients reported high usability and understood the tool's questions. The tool was recommended by most patients (63/81, 78%), in 53% (41/77) of physician ratings, and in 76% (61/80) of nurse ratings. Questionnaire completion rates were 100% (81/81) by patients and 96% (78/81 enrolled patients) by physicians. CONCLUSIONS: This pilot confirmed that a larger study in the setting would be feasible. The tool has clear potential to improve patient-health care provider interaction and could also contribute to ED efficiency savings. Future research and development will extend the range of patients for whom the history-taking tool has clinical utility. TRIAL REGISTRATION: German Clinical Trials Register DRKS00024115; https://drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00024115.

Cutaneous neurofibromas: patients' medical burden, current management and therapeutic expectations: results from an online European patient community survey.

BACKGROUND: Neurofibromatosis type 1 is an inherited condition with variable phenotypic expression and a high medical and social burden. The objectives of this patient survey were to better understand the real-world experiences of patients living with cutaneous neurofibromas (cNF), to perceive their satisfaction and feelings about cNF current management (only laser and surgery are currently available), and to highlight their expectations of new therapeutic modalities. RESULTS: One hundred seventy patients from 4 European countries took part in the study, 65% (n = 110) were women and mean age was 39 years old. 96% (n = 164) of respondents have cNF on visible parts of the body and the survey confirmed that total number of cNF and visibility increase with age. Patients reported that cNF mainly impacts everyday mood, general daily life and social life. The visibility of cNF had a higher impact than their number. 92% (n = 156) of patients have a regular and multidisciplinary medical follow-up. The dermatologist is one of the most consulted healthcare professionals. 76% (n = 130) of respondents have treated their cNF: 65% (n = 111) had surgery and 38% (n = 64) had multiple laser sessions. Frequency of operations and regrowth of cNF were the two most unsatisfactory aspects with both treatments for patients. Indeed, after removal, new cNF appear in more than 75% (n = 128) of cases. As a future treatment, patients expected a topical (30%, n = 51) or oral medication (29%, n = 50). Around 2 out of 3 patients would agree to take it at least once a day or more for life but they would like a well-tolerated treatment. According to patients, the most important effectiveness criteria of a new treatment are to block cNF growth and reduce their number. 70% (n = 119) of patients would consider a future treatment moderately effective to very effective if it could clear 30% of cNF. CONCLUSIONS: This first cNF European patient community survey confirmed that the visible stigma and unaesthetic aspect of cNF have an important impact on patients' quality of life. The survey highlighted that patients were not entirely satisfied with the actual surgery and laser treatments and revealed their clear and realistic expectations for future treatment of cNF.

Risk behaviour and patient preferences for an improved non-melanoma skin cancer prevention modality for organ-transplanted patients: a European, multi-country, online patient community study.

Immunosuppressants used in organ transplant patients increase the risk of non-melanoma skin cancer. This study aimed to evaluate patient behaviours towards skin cancer prevention methods and to understand characteristics of a future prevention strategy based on patients' perspective. Carenity, a global online patient community, enabled the recruitment of 200 adult patients with solid organ transplants from four European countries: France, Italy, Spain and Germany. Most patients were well informed about the risk of skin cancer, but only 27% (53/200) monitored their skin. Most patients exposed themselves to intense sun exposure once a month or more. Nevertheless, more than half of patients were motivated to use additional prevention strategies and limit their sun exposure. The most appropriate prevention strategy was reported to be the use of a cosmetically attractive, water-resistant, paraben/fragrance-free cream. A one-size-fits-all approach is not an appropriate prevention strategy and an adapted approach based on patients' preferences may significantly contribute to better compliance and adherence.

Intraoperatively malpositioned stent as a complication of common bile duct injury during laparoscopic cholecystectomy.

Injuries occurring during laparoscopic bile duct exploration in the course of laparoscopic cholecystectomy may represent threatening complications and lead to inappropriate management. We present a case of patient with biliary colic who underwent laparoscopic cholecystectomy. During the procedure, a common bile duct injury occurred, compelling conversion to open approach, and the patient was treated using a manually inserted biliary stent. She was referred with severe right upper quadrant pain six weeks after the surgery. Investigation with endoscopic retrograde cholangiopancreatography showed a malpositioned biliary stent with completely extra-biliary trajectory. This is thought to be the first description of a malpositioned common bile duct stent through the common biliary duct as a complication of the commonly performed surgical procedure of bile duct exploration.

Intraneural ganglion cysts of the lower limb. / Intraneurale Ganglionzysten der unteren Extremität.

BACKGROUND: Intraneural ganglion cysts are rare. They affect the peripheral nerves. According to the most widely accepted theory (articular/synovial theory), the cysts are formed from a capsular defect of an adjacent joint, so that synovial fluid spreads along the epineurium of a nerve branch. This leads to diverse neurological symptoms. We will illustrate this disease based on three of our own cases. METHODS: Patients were examined between 2011 and 2018 using lower limb MRI. MRI scans were also performed for the follow-up examinations. CASE STUDIES AND DISCUSSION: The patients had many symptoms. We were able to accurately detect the intraneural ganglion cysts on MRI and provide the treating surgeons with the basis for the operation to be performed. The success of surgical therapy depends on the resection of the nerve endings supplying the joint as the only way to treat the origin of the disease and prevent recurrence. Based on our case studies, we can support the commonly favored articular/synovial theory. KEY POINTS: · Intraneural ganglion cysts can cause diverse neurological symptoms depending on their location.. · The pathogenesis is reasonably explained by the articular/synovial theory, which states that cysts are the result of a capsular defect of a joint.. · MRI is the method of choice for diagnosing intraneural ganglion cysts. However, ultrasound is also important.. · Surgery is the only curative treatment with treatment success being dependent on ligature of the nerve endings supplying the articular branch.. CITATION FORMAT: · Fricke T, Schmitt AD, Jansen O. Intraneural ganglion cysts of the lower limb. Fortschr Röntgenstr 2019; 191: 732 - 738.

Raman characterization of human skin aging.

BACKGROUND: Skin aging is a complex biological process mixing intrinsic and extrinsic factors, such as sun exposure. At the molecular level, skin aging affects in particular the extracellular matrix proteins. MATERIALS AND METHODS: Using Raman imaging, which is a nondestructive approach appropriate for studying biological samples, we analyzed how aging modifies the matrix proteins of the papillary and reticular dermis. Biopsies from the buttock and dorsal forearm of volunteers younger than 30 and older than 60 were analyzed in order to identify chronological and photoaging processes. Analyses were performed on skin section, and Raman spectra were acquired separately on the different dermal layers. RESULTS: We observed differences in dermal matrix structure and hydration state with skin aging. Chronological aging alters in particular the collagen of the papillary dermis, while photoaging causes a decrease in collagen stability by altering proline and hydroxyproline residues in the reticular dermis. Moreover, chronological aging alters glycosaminoglycan content in both dermal compartments. CONCLUSION: Alterations of the papillary and reticular dermal matrix structures during photo- and chronological aging were clearly depicted by Raman spectroscopy.

Modulations in extracellular calcium lead to H+-ATPase-dependent acid secretion: a clarification of PPI failure.

The H+,K+-ATPase was identified as the primary proton secretory pathway in the gastric parietal cell and is the pharmacological target of agents suppressing acid secretion. Recently, we identified a second acid secretory protein expressed in the parietal cell, the vacuolar H+-ATPase (V-type ATPase). The aim of the present study was to further characterize H+-ATPase activation by modulations in extracellular calcium via the calcium sensing receptor (CaSR). Isolated gastric glands were loaded with the pH indicator dye BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester] to measure intracellular pH. Experiments were conducted in the absence of sodium and potassium to monitor H+-ATPase-specific transport activity. CaSR was activated with the calcimimetic R568 (400 nM) and/or by modulations in extracellular Ca2+. Elevation in calcium concentrations increased proton extrusion from the gastric parietal cell. Allosteric modification of the CaSR via R568 and calcium increased vacuolar H+-ATPase activity significantly (ΔpH/minlowCa2+(0.1mM) = 0.001 ± 0.001, ΔpH/minnormalCa2+(1.0mM) = 0.033 ± 0.004, ΔpH/minhighCa2+(5.0mM) = 0.051 ± 0.005). Carbachol significantly suppressed calcium-induced gastric acid secretion via the H+-ATPase under sodium- and potassium-free conditions. We conclude that the V-type H+-ATPase is tightly linked to CaSR activation. We observed that proton pump inhibitor (PPI) exposure does not modulate H+-ATPase activity. This elevated blood calcium activation of the H+-ATPase could provide an explanation for recurrent reflux symptoms while taking a PPI therapy. NEW & NOTEWORTHY This study emphasizes the role of the H+-ATPase in acid secretion. We further demonstrate the modification of this proton excretion pathway by extracellular calcium and the activation of the calcium sensing receptor CaSR. The novelty of this paper is based on the modulation of the H+-ATPase via both extracellular Ca (activation) and the classical secretagogues histamine and carbachol (inactivation). Both activation and inactivation of this proton pump are independent of PPI modulation.

Isolation and Characterization of Fast-Migrating Human Glioma Cells in the Progression of Malignant Gliomas.

Gliomas are the most common primary brain tumors. The most malignant form, the glioblastoma multiforme (GBM; WHO IV), is characterized by an invasive phenotype, which enables the tumor cells to infiltrate into adjacent brain tissue. When investigating GBM migration and invasion properties in vitro, in most cases GBM cell lines were analyzed. Comprehensive investigations focusing on progression-dependent characteristics of migration processes using fresh human glioma samples of different malignancy grades do not exist. Thus, we isolated fast-migrating tumor cells from fresh human glioma samples of different malignancy grades (astrocytomas WHO grade II, grade III, GBM, and GBM recurrences) and characterized them with regard to the transcription of genes involved in the migration and invasion, tumor progression, epithelial-to-mesenchymal transition, and stemness. In addition, we transferred our results to GBM cell lines and glioma stem-like cells and examined the influence of temozolomide on the expression of the above-mentioned genes in relation to migratory potential. Our results indicate that "evolutionary-like" expression alterations occur during glioma progression when comparing slow- and fast-migrating cells of fresh human gliomas. Furthermore, a close relation between migratory and stemness properties seems to be most likely. Variations in gene expression were also identified in GBM cell lines, not only when comparing fast- and slow-migrating cells but also regarding temozolomide-treated and untreated cells. Moreover, these differences coincided with the expression of stem cell markers and their migratory potential. Expression of migration-related genes in fast-migrating glioma cells is not only regulated in a progression-dependent manner, but these cells are also characterized by specific stem cell-like features.

Proteomic and transcriptomic investigation of acne vulgaris microcystic and papular lesions: Insights in the understanding of its pathophysiology.

BACKGROUND: The pathogenesis of acne vulgaris involves several phases including androgen-dependent hyper-seborrhea, colonization by Propionibacterium acnes, and inflammation. Recent investigations have shown that in fact P. acnes provokes the activation of the inflammasome present in macrophages and dendritic cells. This signaling pathway leads to excessive production of interleukin IL-1ß, a proinflammatory cytokine. Nevertheless, these well-studied phenomena in acne fail to elucidate the mechanisms responsible for the appearance of different lesions. METHODS: We investigate response pathways for specific acne lesions such as microcysts and papules using shot-gun proteomic followed by systemic biology and transcriptomic approaches. RESULTS: Results show that most of the proteins identified as differentially expressed between the normal and acne tissue biopsies associated with the immune system response were identified as highly or exclusively expressed in the papule biopsies. They were also expressed in microcysts, but in lower amounts compared to those in papules. These results are supported by the identification of CAMP factor protein produced by P. acnes in microcysts, indicating its enhanced proliferation in this type of lesion CONCLUSIONS: As CAMP factor protein was not detected in papule biopsies, we can see a clear delineation in the stages of progression of acne pathogenesis, which begins with a hyphenated inflammatory response in the papule stage, followed by imbalance of lipid production, which in turn triggers the enhanced proliferation of P. acnes. GENERAL SIGNIFICANCE: We demonstrate that expression inflammation varies across the two types of lesions, suggesting different pathways enhanced as a function of the progression of P. acnes.

"Inverse signaling" of the transmembrane chemokine CXCL16 contributes to proliferative and anti-apoptotic effects in cultured human meningioma cells.

BACKGROUND: Chemokines and their receptors play a decisive role in tumor progression and metastasis. We recently found a new signaling mechanism in malignant glioma cells mediated by transmembrane chemokines that we termed "inverse signaling". According to this hypothesis, soluble (s)-CXCL16 binds to the surface-expressed transmembrane (tm) -CXCL16, and induces signaling and different biological effects in the stimulated cells, so that the transmembrane ligand itself acts as a receptor for its soluble counterpart. Now, we hypothesized that "inverse signaling" via tm-CXCL16 might also take place in meningiomas, a completely different, benign tumor entity. METHODS: We used quantitative reverse-transcription polymerase chain reaction, immunocytochemistry and western blot to detect CXCL16 and CXCR6 in human meningioma cells isolated from 28 human meningiomas. Subsequently, we stimulated cultured human tm-CXCL16-positive, CXCR6-negative meningioma cells with recombinant s-CXCL16 and analyzed binding, signaling and biological effects using RNAi silencing to verify specificity. RESULTS: In fact, cultured human meningioma cells considerably express CXCL16, but substantially lack CXCR6, the only known CXCL16 receptor. These receptor-negative cells could bind s-CXCL16, and responded to s-CXCL16 application with activation of the intracellular kinases ERK1/2 und Akt. As a consequence, we observed increased proliferation and rescue of apoptosis of cultured meningioma cells. Since binding and signaling were abolished by siRNA silencing, we concluded that tm-CXCL16 specifically acts as a receptor for s-CXCL16 also in human meningioma cells. CONCLUSION: These findings underline our recent report on the mechanism of inverse signaling as a broad biological process also observable in more benign tumor cells and contributing to tumor progression.

Optimising treatment strategies in spinal ependymoma based on 20years of experience at a single centre.

Spinal ependymomas are rare tumours, with total resection favoured where possible. Several case series assessing the outcome following neurosurgical treatment for spinal ependymoma advocate the usage of adjuvant radiotherapy in cases of subtotal resection, or in unencapsulated tumours. We assessed the outcome of 61 consecutive cases of spinal ependymoma in a single centre over a 20year period using a variety of outcome measures. Sex distribution was equal, with a mean age at surgery of 43.6years (range 5-76years). Overall, most tumours occurred in the lumbosacral region (70.5%), with fewer in the thoracic (27.9%) and cervical regions (18.0%). Myxopapillary features were seen in 41.0% of tumours, and were more common when occurring in the lumbar region (51.2%). Gross total resection was achieved in 52.5%, subtotal resection in 37.7% and biopsy alone in 9.8% of patients and 31.1% received adjuvant radiotherapy. Two-thirds of patients achieved an excellent post-operative neurological outcome (Frankel grade E). Tumour recurrence was rare. Gross total resection and good preoperative neurological condition were most strongly predictive of good outcome. Post-operative radiotherapy did not seem to confer survival benefit in this case series, even in cases of incomplete resection, leading us to question its utility for all cases of spinal cord ependymoma.

Down regulation of Akirin-2 increases chemosensitivity in human glioblastomas more efficiently than Twist-1.

The Twist-1 transcription factor and its interacting protein Akirin-2 regulate apoptosis. We found that in glioblastomas, highly malignant brain tumors, Akirin-2 and Twist-1 were expressed in glial fibrillary acidic protein positive tumor regions as well as in tumor endothelial cells and infiltrating macrophages / microglia. Temozolomide (TMZ) induced the expression of both molecules, partly shifting their nuclear to cytosolic localization. The knock-down (kd) of Akirin-2 increased the activity of cleaved (c)Caspase-3/-7, the amounts of cCaspases-3, -7 and cPARP-1 and resulted in an increased number of apoptotic cells after TMZ exposure. Glioblastoma cells containing decreased amounts of Akirin-2 after kd contained increased amounts of cCaspase-3 as determined by the ImageStreamx Mark II technology. For Twist-1, similar results were obtained with the exception that the combination of TMZ treatment and Twist-1 kd failed to significantly reduce chemoresistance compared with controls. This could be attributed to a cell population containing only slightly increased cCaspase-3 together with decreased Twist-1 levels, which was clearly larger than the respective population observed under Akirin-2 kd. Our results showed that, compared with Twist-1, Akirin-2 is the more promising target for RNAi strategies antagonizing Twist-1/Akirin-2 facilitated glioblastoma cell survival.

Elastin Modification by 4-Hydroxynonenal in Hairless Mice Exposed to UV-A. Role in Photoaging and Actinic Elastosis.

Chronic exposure to ultraviolet (UV) radiation causes oxidative stress, which is involved in photoaging and actinic elastosis. UV and reactive oxygen species generate lipid peroxidation products, including the α, ß-unsaturated carbonyl compounds such as acrolein or 4-hydroxynonenal (4-HNE). These aldehydes can modify proteins of the extracellular matrix, but their role in the pathogenesis of photoaging is not clarified. The aim of this study was to investigate whether these aldehydes contribute to alter elastin metabolism and whether topical carbonyl scavengers delay UV-induced skin photoaging. Hairless mice (4-6-week old) daily exposed to UV-A (20 J cm(-2) per day, up to 600 J cm(-2)) exhibited the typical features of photoaging, associated with a significant increase in 4-HNE- and acrolein-adduct content, and elastotic material deposition. Immunofluorescence studies showed the accumulation of 4-HNE adducts on elastin in the dermis of UV-A-exposed mice. This was mimicked in vitro by incubating orcein-elastin with 4-HNE or acrolein, which altered its digestion by leukocyte-elastase, a feature possibly involved in the accumulation of elastotic material. A daily topical application of carnosine completely reversed the development of photoaging alterations and 4-HNE-adduct formation on elastin. These data emphasize the role of 4-HNE and acrolein in the mechanism of photoaging, and the preventive effect of carbonyl scavengers.

Variation between specialist uropatholgists in reporting extraprostatic extension after radical prostatectomy.

AIMS: Extraprostatic extension of prostate cancer in radical prostatectomy specimens significantly affects patient management. We evaluated the degree of interobserver variation between uropathologists at a tertiary referral teaching hospital in assessing the extraprostatic extension of prostate cancer in radical prostatectomy specimens. METHODS: Histopathological data from a consecutive series of 293 radical prostatectomy specimens (January 2007-December 2012) were reviewed. A subset of 50 consecutive radical prostatectomy cases originally staged as tumours confined to the prostate (pT2) or tumours extending into periprostatic tissue (pT3a) during this period were reviewed by four specialist uropathologists. RESULTS: Five consultant histopathologists reported these specimens with significant differences in the reported stage (p=0.0164) between pathologists. Double-blind review by 4 uropathologists of 50 consecutive radical prostatectomy cases showed a lack of consensus in 16/50 (32%) cases (κ score 0.58, moderate agreement). A consensus meeting was held, but consensus could still not be reached in 9/16 cases. CONCLUSIONS: Our findings highlight variability in the reporting of pT stage in radical prostatectomy specimens even by specialist uropathologists. Assessment of extraprostatic extension has important implications for patient management and there is a need for more precise guidance.

Chemokine expression profile of freshly isolated human glioblastoma-associated macrophages/microglia.

Several studies have substantiated the hypothesis that tumor progression is not only driven by the tumor cells themselves but also by their interaction with intrinsic and surrounding stromal cells. Tumor-associated macrophages and microglial cells (TAMs) represent one major stromal cell component of glioblastomas. Additionally, in many gliomas, chemokines are highly expressed and some chemokines were already linked to settlement of TAMs in tumors. However, although chemoattraction mechanisms mediated by chemokines and their receptors are well documented, information on their expression and role in TAMs, particularly in patients, is limited. Therefore, we investigated the transcription of the chemokine-receptor combinations CXCL12-CXCR4-CXCR7, CXCL16-CXCR6 and CX3CL1-CX3CR1 in freshly isolated TAMs from 20 human glioblastomas in relation to in vitro polarized M1- and M2-macrophages. We demonstrated that TAMs express both M1- and M2-markers. Compared to in vitro polarized macrophages, the M1-marker interleukin (IL)-6 was similarly expressed, whereas IL-1ß and tumor necrosis factor (TNF)-α were found at lower levels. The M2-marker IL-10 was comparably expressed, while CD163 and transforming growth factor (TGF)-ß were detected with one tenth lower intensities in TAMs. All investigated chemokines/receptors were transcribed at moderate to high levels in TAMs as well as in vitro polarized macrophages. However, CX3CR1 was markedly higher and CXCR7 was somewhat higher expressed in TAMs, whereas M2-macrophages were characterized by the highest CXCL12 and a moderate CX3CL1 expression. Collectively, TAMs share properties of M1- and M2-macrophages and show a considerably higher expression of the chemokine receptors CXCR7 and CX3CR1.

4-Hydroxynonenal impairs transforming growth factor-ß1-induced elastin synthesis via epidermal growth factor receptor activation in human and murine fibroblasts.

Elastin is a long-lived protein and a key component of connective tissues. The tissular elastin content decreases during chronological aging, and the mechanisms underlying its slow repair are not known. Lipid oxidation products that accumulate in aged tissues may generate protein dysfunction. We hypothesized that 4-hydroxynonenal (4-HNE), a highly reactive α,ß-aldehydic product generated from polyunsaturated fatty acid peroxidation, could contribute to inhibiting elastin repair by antagonizing the elastogenic signaling of transforming growth factor-ß1 (TGF-ß1) in skin fibroblasts. We report that a low 4-HNE concentration (2µmol/L) inhibits the upregulation of tropoelastin expression stimulated by TGF-ß1 in human and murine fibroblasts. The study of signaling pathways potentially involved in the regulation of elastin expression showed that 4-HNE did not block the phosphorylation of Smad3, an early step of TGF-ß1 signaling, but inhibited the nuclear translocation of Smad2. Concomitantly, 4-HNE modified and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR) and subsequently ERK1/2 activation, leading to the phosphorylation/stabilization of the Smad transcriptional corepressor TGIF, which antagonizes TGF-ß1 signaling. Inhibitors of EGFR (AG1478) and MEK/ERK (PD98059), and EGFR-specific siRNAs, reversed the inhibitory effect of 4-HNE on TGF-ß1-induced nuclear translocation of Smad2 and tropoelastin synthesis. In vivo studies on aortas from aged C57BL/6 mice showed that EGFR is modified by 4-HNE, in correlation with an increased 4-HNE-adduct accumulation and decreased elastin content. Altogether, these data suggest that 4-HNE inhibits the elastogenic activity of TGF-ß1, by modifying and activating the EGFR/ERK/TGIF pathway, which may contribute to altering elastin repair in chronological aging and oxidative stress-associated aging processes.