Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

CD109 drives pro-tumorigenic cell properties in human non-small cell lung cancer through interaction with desmoglein-2.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein, expressed on epithelial and endothelial cells, CD4+ and CD8+ T-cells, and premature lymphocytes. CD109 interacts with different cell surface receptors and thereby modulates intracellular signaling pathways, which ultimately changes cellular functions. One well-studied example is the interaction of CD109 with the TGFß/TGFß-receptor complex at the cell surface. CD109 silences intracellular SMAD2/3 signaling and targets TGFß/TGFß-receptor to the endosomal/lysosomal compartment. In recent years, CD109 emerged as a tumor marker for different tumor entities and expression of CD109 could be linked to adverse outcome in patients. In this study, we show that silencing of CD109 in human non-small cell lung cancer (NSCLC) cells, returns these cells to an epithelial like growth phenotype. On the transcriptional level, we describe changes in cell-cell contact and epithelial-mesenchymal transition associated gene clusters. At the cell surface, we identify desmoglein-2 (DSG2) as a new interaction partner of CD109 and demonstrate CD109 dependent targeting of DSG2 to the apical cell surface, where it forms desmosomes between apical and basal cell poles. Both, CD109 and DSG2 are genetic risk factors, linked to reduced overall survival in lung adenocarcinoma patients (subtype of NSCLC). In this study, we show the expression of both proteins in the same tumor and suggest a new CD109-DSG2 axis in NSCLC patients that could present a targetable therapeutic option in the future.

Detecting Misfolded α-Synuclein in Blood Years before the Diagnosis of Parkinson's Disease.

BACKGROUND: Identifying individuals with Parkinson's disease (PD) already in the prodromal phase of the disease has become a priority objective for opening a window for early disease-modifying therapies. OBJECTIVE: The aim was to evaluate a blood-based α-synuclein seed amplification assay (α-syn SAA) as a novel biomarker for diagnosing PD in the prodromal phase. METHODS: In the TREND study (University of Tuebingen) biennial blood samples of n = 1201 individuals with/without increased risk for PD were taken prospectively over 4 to 10 years. We retrospectively analyzed blood samples of 12 participants later diagnosed with PD during the study to detect and amplify pathological α-syn conformers derived from neuronal extracellular vesicles using (1) immunoblot analyses with an antibody against these conformers and (2) an α-syn-SAA. Additionally, blood samples of n = 13 healthy individuals from the TREND cohort and n = 20 individuals with isolated rapid eye movement sleep behavior disorder (iRBD) from the University Hospital Cologne were analyzed. RESULTS: All individuals with PD showed positive immunoblots and a positive α-syn SAA at the time of diagnosis. Moreover, all PD patients showed a positive α-syn SAA 1 to 10 years before clinical diagnosis. In the iRBD cohort, 30% showed a positive α-syn SAA. All healthy controls had a negative SAA. CONCLUSIONS: We here demonstrate the possibility to detect and amplify pathological α-syn conformers in peripheral blood up to 10 years before the clinical diagnosis of PD in individuals with and without iRBD. The findings of this study indicate that this blood-based α-syn SAA assay has the potential to serve as a diagnostic biomarker for prodromal PD. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Resveratrol Alleviates the Early Challenges of Implant-Based Drug Delivery in a Human Glial Cell Model.

Brain diseases are oftentimes life-threatening and difficult to treat. The local administration of drug substances using brain implants can increase on-site concentrations and decrease systemic side effects. However, the biocompatibility of potential brain implant materials needs to be evaluated carefully as implants can trigger foreign body reactions, particularly by increasing the microglia and astrocyte reactivity. To date, these tests have been frequently conducted in very simple in vitro models, in particular not respecting the key players in glial cell reactions and the challenges of surgical implantation characterized by the disruption of oxygen and nutrient supply. Thus, we established an in vitro model in which we treated human glial cell lines with reduced oxygen and glucose levels. The model displayed cytokine and reactive oxygen species release from reactive microglia and an increase in a marker of reactive astrocytes, galectin-3. Moreover, the treatment caused changes in the cell survival and triggered the production of hypoxia-inducible factor 1α. In this comprehensive platform, we demonstrated the protective effect of the natural polyphenol resveratrol as a model substance, which might be included in brain implants to ease the undesired glial cell response. Overall, a glial-cell-based in vitro model of the initial challenges of local brain disease treatment may prove useful for investigating new therapy options.

Human APOE4 Protects High-Fat and High-Sucrose Diet Fed Targeted Replacement Mice against Fatty Liver Disease Compared to APOE3.

Recent genome- and exome-wide association studies suggest that the human APOE ε4 allele protects against non-alcoholic fatty liver disease (NAFLD), while ε3 promotes hepatic steatosis and steatohepatitis. The present study aimed at examining the APOE genotype-dependent development of fatty liver disease and its underlying mechanisms in a targeted replacement mouse model. Male mice expressing the human APOE3 or APOE4 protein isoforms on a C57BL/6J background and unmodified C57BL/6J mice were chronically fed a high-fat and high-sucrose diet to induce obesity. After 7 months, body weight gain was more pronounced in human APOE than endogenous APOE expressing mice with elevated plasma biomarkers suggesting aggravated metabolic dysfunction. APOE3 mice exhibited the highest liver weights and, compared to APOE4, massive hepatic steatosis. An untargeted quantitative proteome analysis of the liver identified a high number of proteins differentially abundant in APOE3 versus APOE4 mice. The majority of the higher abundant proteins in APOE3 mice could be grouped to inflammation and damage-associated response, and lipid storage, amongst others. Results of the targeted qRT-PCR and Western blot analyses contribute to the overall finding that APOE3 as opposed to APOE4 promotes hepatic steatosis, inflammatory- and damage-associated response signaling and fibrosis in the liver of obese mice. Our experimental data substantiate the observation of an increased NAFLD-risk associated with the human APOEε3 allele, while APOEε4 appears protective. The underlying mechanisms of the protection possibly involve a higher capacity of nonectopic lipid deposition in subcutaneous adipose tissue and lower hepatic pathogen recognition in the APOE4 mice.

Localization Pattern of Dispatched Homolog 2 (DISP2) in the Central and Enteric Nervous System.

Dispatched homolog (DISP) proteins have been implicated in the regulation of hedgehog signaling during embryologic development. Although DISP2 has recently been associated with neuronal development and control of cognitive functions, its localization pattern in the mammalian central and peripheral nervous system has not yet been investigated. In this study, the Disp2 expression profile was assessed in human tissues from publicly available transcriptomic datasets. The DISP2 localization pattern was further characterized in the human and rat central nervous system (CNS), as well as within the colonic enteric nervous system (ENS) using dual-label immunohistochemistry. Colocalization of DISP2 with neuronal and glial markers was additionally analyzed in murine primary ENS culture. At transcriptomic level, DISP2 expression was predominant in neuronal cell types of the CNS and ENS. DISP2 immunoreactivity was mainly located within PGP9.5-positive neurons rather than in S100-positive glial cells throughout the nervous system. Investigation of human and rat brain tissues, colonic specimens, and murine ENS primary cultures revealed that DISP2 was located in neuronal cell somata, as well as along neuronal processes both in the human and murine CNS and ENS. Our results indicate that DISP2 is prominently localized within neuronal cells of the CNS and ENS and support putative functions of DISP2 in these tissues.

Establishment of a Rodent Glioblastoma Partial Resection Model for Chemotherapy by Local Drug Carriers-Sharing Experience.

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application.

Resveratrol Mitigates Metabolism in Human Microglia Cells.

The recognition of the role of microglia cells in neurodegenerative diseases has steadily increased over the past few years. There is growing evidence that the uncontrolled and persisting activation of microglial cells is involved in the progression of diseases such as Alzheimer's or Parkinson's disease. The inflammatory activation of microglia cells is often accompanied by a switch in metabolism to higher glucose consumption and aerobic glycolysis. In this study, we investigate the changes induced by the natural antioxidant resveratrol in a human microglia cell line. Resveratrol is renowned for its neuroprotective properties, but little is known about its direct effect on human microglia cells. By analyzing a variety of inflammatory, neuroprotective, and metabolic aspects, resveratrol was observed to reduce inflammasome activity, increase the release of insulin-like growth factor 1, decrease glucose uptake, lower mitochondrial activity, and attenuate cellular metabolism in a 1H NMR-based analysis of whole-cell extracts. To this end, studies were mainly performed by analyzing the effect of exogenous stressors such as lipopolysaccharide or interferon gamma on the metabolic profile of microglial cells. Therefore, this study focuses on changes in metabolism without any exogenous stressors, demonstrating how resveratrol might provide protection from persisting neuroinflammation.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Breaking the circulus vitiosus of neuroinflammation: Resveratrol attenuates the human glial cell response to cytokines.

Neuroinflammation is both cause and effect of many neurodegenerative disorders. Activation of astrocytes and microglia leads to the release of cytokines and reactive oxygen species followed by blood-brain barrier leakage and neurotoxicity. Transient neuroinflammation is considered to be largely protective, however, chronic neuroinflammation contributes to the pathology of Alzheimer's disease, multiple sclerosis, traumatic brain injury, and many more. In this study, we focus on the aspect of cytokine-induced neuroinflammation in human microglia and astrocytes. Here we show by mRNA and protein analysis that cytokines, released not only by microglia but also by astrocytes, lead to a circuit of proinflammatory activation. Moreover, we present how the natural compound resveratrol can stop the circuit of proinflammatory activation and facilitate return to resting conditions. These results will contribute to distinguishing between the causes and the effects of neuroinflammation, a better understanding of underlying mechanisms, and potential treatment options.

Proteolysis of CD44 at the cell surface controls a downstream protease network.

The cell surface receptor cluster of differentiation 44 (CD44) is the main hyaluronan receptor of the human body. At the cell surface, it can be proteolytically processed by different proteases and was shown to interact with different matrix metalloproteinases. Upon proteolytic processing of CD44 and generation of a C-terminal fragment (CTF), an intracellular domain (ICD) is released after intramembranous cleavage by the γ-secretase complex. This intracellular domain then translocates to the nucleus and induces transcriptional activation of target genes. In the past CD44 was identified as a risk gene for different tumor entities and a switch in CD44 isoform expression towards isoform CD44s associates with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Here, we introduce meprin ß as a new sheddase of CD44 and use a CRISPR/Cas9 approach to deplete CD44 and its sheddases ADAM10 and MMP14 in HeLa cells. We here identify a regulatory loop at the transcriptional level between ADAM10, CD44, MMP14 and MMP2. We show that this interplay is not only present in our cell model, but also across different human tissues as deduced from GTEx (Gene Tissue Expression) data. Furthermore, we identify a close relation between CD44 and MMP14 that is also reflected in functional assays for cell proliferation, spheroid formation, migration and adhesion.

In Vitro Comparison of 2 Clinically Applied Biomaterials for Autologous Chondrocyte Implantation: Injectable Hydrogel Versus Collagen Scaffold.

OBJECTIVE: In autologous chondrocyte implantation (ACI), there is no consensus about used bioscaffolds. The aim of this study was to perform an in vitro comparative analysis of 2 clinically applied biomaterials for cartilage lesion treatment. DESIGN: Monolayer expanded human chondrocytes (n = 6) were embedded in a collagen scaffold (CS) and a hyaluronic acid-based hydrogel (HA). Cells were cultured in chondropermissive medium supplemented with and without interleukin-10 (IL-10) and bone morphogenetic protein-2 (BMP-2). Gene expression of chondrogenic markers (COL1A1, COL2A1, COL10A1, ACAN, SOX9) was detected via quantitative real-time-polymerase chain reaction (RT-qPCR). Biosynthesis of matrix compounds, cell viability, morphology as well as migration from surrounding native bovine cartilage into cell-free scaffolds were analyzed histologically. Adhesion of the material to adjacent cartilage was investigated by a custom-made push-out test. RESULTS: The shift of COL1/2 ratio toward COL2A1 was more pronounced in HA, and cells displayed a more spherical morphology compared with CS. BMP-2 and IL-10 significantly increased COL2A1, SOX9, and ACAN expression, which was paralleled by enhanced staining of glycosaminoglycans (GAGs) and type 2 collagen in histological sections of CS and HA. COL10A1 was not significantly expressed in HA and CS. Better interfacial integration and enhanced cell invasion was observed in CS. Push-out tests using CS showed higher bonding strength to native cartilage. CONCLUSION: HA-based hydrogel revealed a more chondrocyte-like phenotype but only allowed limited cell invasion, whereas CS were advantageous in terms of cellular invasion and interfacial adhesion. These differences may be clinically relevant when treating cartilaginous or osteochondral defects.

Comparative study of two re-embedding methods on the ultrastructure of corneal tissue.

PURPOSE: To expand the routine of pathological diagnostics of surgical keratoplasty specimens via transmission electron microscopy. The target was to identify the best re-embedding method for optimal structural preservation of formalin fixed paraffin embedded (FFPE) corneal tissue re-embedded into resin for ultrastructural analysis. BASIC PROCEDURES: Bovine FFPE corneal tissue was re-embedded into resin with either a rapid osmium-free four-hour-method or a four-day-routine-method known from nephropathology, compared with primary resin embedded bovine corneal tissue. The analysis involved the ultrastructure of cytoplasm, the intercellular interfaces of superficial epithelial cells, deepest basal epithelial cells and corneal endothelial cells, cell matrix interfaces, Bowman layer, corneal stroma, its microfibril bundles and Descemet membrane. MAIN FINDINGS: The main observation was the equally reduced preservation status of re-embedded FFPE corneal tissue independent of the used re-embedding method. This extends to the intercellular contacts of superficial epithelial cells and the apical tight junctions of corneal endothelial cells. Hemidesmosomal cell matrix contacts showed less demarcation in re-embedded specimens. Cell matrix interfaces of Bowman layer and Descemet membrane were more clearly bordered in primary resin embedded than re-embedded tissue. In contrast, gap junctions in re-embedded tissue were detected in deepest basal epithelial cells and corneal endothelial cells with comparable preservation to primary resin embedding. Bowman layer, corneal stromal extracellular matrix, its microfibril bundles and Descemet membrane showed equal ultrastructural preservation in all evaluated methods. PRINCIPAL CONCLUSION: Corneal tissue can be successfully analysed with transmission electron microscopy after a rapid osmium-free four hour re-embedding procedure from FFPE material. A comparable morphology with primary resin embedded material can be obtained for gap junctions of deepest basal epithelial cells and corneal endothelial cells, further for Bowman layer, corneal stromal extracellular matrix, its microfibril bundles and Descemet membrane.

Anti-Inflammatory Properties of the SGLT2 Inhibitor Empagliflozin in Activated Primary Microglia.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, are routinely used as antidiabetic drugs. Recent studies indicate that beside its beneficial effects on blood glucose level, empagliflozin may also exert vascular anti-inflammatory and neuroprotective properties. In the brain, microglia are crucial mediators of inflammation, and neuroinflammation plays a key role in neurodegenerative disorders. Dampening microglia-mediated inflammation may slow down disease progression. In this context, we investigated the immunomodulatory effect of empagliflozin on activated primary microglia. As a validated experimental model, rat primary microglial cells were activated into a pro-inflammatory state by stimulation with LPS. The influence of empagliflozin on the expression of pro-inflammatory mediators (NO, Nos2, IL6, TNF, IL1B) and on the anti-inflammatory mediator IL10 was assessed using quantitative PCR and ELISA. Further, we investigated changes in the activation of the ERK1/2 cascade by Western blot and NFkB translocation by immunostaining. We observed that empagliflozin reduces the expression of pro- and anti-inflammatory mediators in LPS-activated primary microglia. These effects might be mediated by NHE-1, rather than by SGLT2, and by the further inhibition of the ERK1/2 and NFkB pathways. Our results support putative anti-inflammatory effects of empagliflozin on microglia and suggest that SGLT2 inhibitors may exert beneficial effects in neurodegenerative disorders.

Detection of neuron-derived pathological α-synuclein in blood.

To date, no reliable clinically applicable biomarker has been established for Parkinson's disease. Our results indicate that a long anticipated blood test for Parkinson's disease may be realized. Following the isolation of neuron-derived extracellular vesicles of Parkinson's disease patients and non-Parkinson's disease individuals, immunoblot analyses were performed to detect extracellular vesicle-derived α-synuclein. Pathological α-synuclein forms derived from neuronal extracellular vesicles could be detected under native conditions and were significantly increased in all individuals with Parkinson's disease and clearly distinguished disease from the non-disease state. By performing an α-synuclein seeding assay these soluble conformers could be amplified and seeding of pathological protein folding was demonstrated. Amplified α-synuclein conformers exhibited ß-sheet-rich structures and a fibrillary appearance. Our study demonstrates that the detection of pathological α-synuclein conformers from neuron-derived extracellular vesicles from blood plasma samples has the potential to evolve into a blood-biomarker of Parkinson's disease that is still lacking so far. Moreover, the distribution of seeding-competent α-synuclein within blood exosomes sheds a new light of pathological disease mechanisms in neurodegenerative disorders.

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants.

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.

Inhibition of ADAM17 impairs endothelial cell necroptosis and blocks metastasis.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.

Influence of Simulated Deep Brain Stimulation on the Expression of Inflammatory Mediators by Human Central Nervous System Cells In Vitro.

Deep brain stimulation (DBS) seems to modulate inflammatory processes. Whether this modulation leads to an induction or suppression of inflammatory mediators is still controversially discussed. Most studies of the influence of electrical stimulation on inflammation were conducted in rodent models with direct current stimulation and/or long impulses, both of which differ from the pattern in DBS. This makes comparisons with the clinical condition difficult. We established an in-vitro model that simulated clinical stimulation patterns to investigate the influence of electrical stimulation on proliferation and survival of human astroglial cells, microglia, and differentiated neurons. We also examined its influence on the expression of the inflammatory mediators C-X-C motif chemokine (CXCL)12, CXCL16, CC-chemokin-ligand-2 (CCL)2, CCL20, and interleukin (IL)-1ß and IL-6 by these cells using quantitative polymerase chain reaction. In addition, protein expression was assessed by immunofluorescence double staining. In our model, electrical stimulation did not affect proliferation or survival of the examined cell lines. There was a significant upregulation of CXCL12 in the astrocyte cell line SVGA, and of IL-1ß in differentiated SH-SY5Y neuronal cells at both messenger RNA and protein levels. Our model allowed a valid examination of chemokines and cytokines associated with inflammation in human brain cells. With it, we detected the induction of inflammatory mediators by electrical stimulation in astrocytes and neurons.

Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy.

BACKGROUND: Selective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration. METHODS: For PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation. RESULTS: TSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells. CONCLUSIONS: SRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System.

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.