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Objective JIA is a chronic inflammatory disease of unknown origin. The regulation of inflammatory processes involves multiple cellular steps including mRNA transcription and translation. Different miRNAs control these processes tightly. We aimed to determine the roles of specific miRNAs within JIA pathogenesis. Methods We performed a global miRNA expression analysis in parallel in cells from the arthritic joint and peripheral blood of oligoarticular JIA patients and healthy controls. Quantitative RT-PCR analysis was used to verify expression of miRNA in T cells. Ex vivo experiments and flow cytometric analyses were used to analyse proliferation and redox metabolism. Results Global miRNA expression analysis demonstrated a different composition of miRNA expression at the site of inflammation compared with peripheral blood. Bioinformatic analysis of predicted miRNA target genes suggest a huge overrepresentation of genes involved in metabolic and oxidative stress pathways in the inflamed joint. Despite enhanced reactive oxygen species (ROS) levels within the local inflammatory milieu, JIA T cells are hyperproliferative and reveal an overexpression of miR-23a, which is an inhibitor of Peptidyl-prolyl isomerase F (PPIF), the regulator of mitochondrial ROS escape. Mitochondrial ROS escape is diminished in JIA T cells, resulting in their prolonged survival. Conclusion Our data suggest that miRNA-dependent mitochondrial ROS shuttling might be a mechanism that contributes to T cell regulation in JIA at the site of inflammation.

Background An important hallmark of Alzheimer’s disease (AD) is the increase of Aβ1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia cell function represents an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aβ1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model. Methods To address this question, APP/PS1 double-transgenic AD mice were treated for 20 weeks with either the pro-inflammatory FPR agonist fMLF, the FPR1/2 antagonist Boc2 or the anti-inflammatory FPR2 agonist Ac2-26. Spatial learning and memory were evaluated using a Morris water maze test. Immunohistological staining, gene expression studies, and flow cytometry analyses were performed to study neuronal loss, gliosis, and Aß-load in the hippocampus and cortex, respectively. Results FPR antagonism by Boc2-treatment significantly improved spatial memory performance, reduced neuronal pathology, induced the expression of homeostatic growth factors, and ameliorated microglia, but not astrocyte, reactivity. Furthermore, the elevated levels of amyloid plaques in the hippocampus were reduced by Boc2-treatment, presumably by an induction of amyloid degradation. Conclusions We suggest that the modulation of FPR signaling cascades might be considered as a promising therapeutic approach for alleviating the cognitive deficits associated with early AD. Additional studies are now needed to address the downstream effectors as well as the safety profile of Boc2.

The aim of this study is to detect the presence of blood spinal cord barrier (BSCB) disruption in patients with degenerative cervical myelopathy (DCM). In this prospective non-randomized controlled cohort study, 28 patients with DCM were prospectively included. All patients had indication for neurosurgical decompression. Furthermore, 38 controls with thoracic abdominal aortic aneurysm (TAAA) and indication for surgery were included. All patients underwent neurological examination. Regarding BSCB disruption and intrathecal immunoglobulin (Ig) concentrations, cerebrospinal fluid (CSF) and blood serum were examined for albumin, IgG, IgA and IgM. Quotients (Q) (CSF/serum) were standardized and calculated according to Reibers’ diagnostic criteria. Patients and controls distinguished significantly in their clinical status. AlbuminQ, as expression of BSCB disruption, was significantly increased in the DCM patients compared to the controls. Quotients of IgG and IgA differed significantly between the groups as an expression of intrathecal diffusion. In the subgroup analysis of patients with mild/moderate clinical status of myelopathy and patients with severe clinical status, the disruption of the BSCB was significantly increased with clinical severity. Likewise, IgAQ and IgGQ presented increased quotients related to the clinical severity of myelopathy. In this study, we detected an increased permeability and disruption of the BSCB in DCM patients. The severity of BSCB disruption and the diffusion of Ig are related to the clinical status in our patient cohort. Having documented this particular pathomechanism in patients with DCM, we suggest that this diagnostic tool cloud be an important addition to surgical decision making in the future. These slides can be retrieved under Electronic Supplementary Material.

Molecular neurobiology (2021). doi:10.1007/s12035-021-02543-2, Published by Humana Press, Totowa, NJ

Accumulating evidence indicates a pivotal role for chronic inflammatory processes in the pathogenesis of neurodegenerative and psychiatric disorders. G protein-coupled formyl peptide receptor 2 (FPR2) mediates pro-inflammatory or anti-/pro-resolving effects upon stimulation with biased agonists. We aimed to evaluate the effects of a new FPR2 ureidopropanamide agonist, compound MR-39, on neuroinflammatory processes in organotypic hippocampal cultures (OHCs) derived from control (WT) and knockout FPR2−/− mice (KO) exposed to bacterial endotoxin (lipopolysaccharide, LPS). Higher LPS-induced cytokine expression and basal release were observed in KO FPR2 cultures than in WT cultures, suggesting that a lack of FPR2 enhances the OHCs response to inflammatory stimuli. Pretreatment with MR-39 abolished some of the LPS-induced changes in the expression of genes related to the M1/M2 phenotypes (including Il-1β, Il-6, Arg1, Il-4, Cd74, Fizz and Cx3cr1) and TNF-α, IL-1β and IL-4 release in tissue derived from WT but not KO mice. Receptor specificity was confirmed by adding the FPR2 antagonist WRW4, which abolished the abovementioned effects of MR-39. Further biochemical data showed an increase in the phospho-p65/total p65 ratio after LPS stimulation in hippocampal tissues from both WT and KO mice, and MR-39 only reversed this effect on WT OHCs. LPS also increased TRAF6 levels, which are critical for the TLR4-mediated NF-κB pro-inflammatory responses. MR-39 attenuated the LPS-evoked increase in the levels of the NLRP3 and caspase-1 proteins in WT but not KO hippocampal cultures. Since NLRP3 may be involved in the pyroptosis, a lytic type of programmed cell death in which the main role is played by Gasdermin D (GSDMD), we examined the effects of LPS and/or MR-39 on the GSDMD protein level. LPS only increased GSDMD production in the WT tissues, and this effect was ameliorated by MR-39. Collectively, this study indicates that the new FPR2 agonist efficiently abrogates LPS-induced neuroinflammation in an ex vivo model, as evidenced by a decrease in pro-inflammatory cytokine expression and release as well as the downregulation of NLRP3 inflammasome-related pathways.

BackgroundBacterial meningitis is, despite progress in research and the development of new treatment strategies, still a cause of severe neurological disability. The brain is protected from penetrating pathogens by the blood-brain barrier and the innate immune system. The invading pathogens are recognized by pattern recognition receptors including the G-protein coupled formyl peptide receptors (FPRs), which are expressed by immune cells of the central nervous system. FPRs show a broad spectrum of ligands including pro- and anti-inflammatory ones. Here, we investigated the effects of the AnnexinA1 mimetic peptide Ac2-26 in a mouse model of pneumococcal meningitis.MethodsWildtype (WT), Fpr1 and Fpr2-deficient mice were intrathecally infected with Streptococcus pneumoniae D39 (type 2). Subsequently, the different mice groups were treated by intraperitoneal injections of Ac2-26 (1 mg/kg body weight) 2, 8 and 24 hour after the infection. The extent of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time RT-PCR 30 h after infection.ResultsAc2-26 treated mice showed less severe neutrophil infiltration, paralleled by a reduced induction of pro-inflammatory glia cell responses. While meningitis was ameliorated in Ac2-26-treated Fpr1-deficient mice, this protective effect was not observed in Fpr2-deficient mice.ConclusionsEven with appropriate antimicrobial therapy, mortality during bacterial meningitis is high and so attention has recently focused on adjunctive therapies. Our results suggest that Ac2-26 might be a novel adjunctive therapy for Streptococcus pneumoniae-induced meningitis.* The two last authors contributed equally to this study.

Additional file 1: Fig. S1. Physical condition of animals is not affected neither in APP/PS1 mice nor by FPR ligands. To analyze the animals physical condition we used the Morris water maze. The average speed [m/s] of the flagged trials were recorded for each animal. There were no differences regarding the condition between the groups (two-way ANOVA with Turkey’s post hoc test). The data represent mean + SEM, n>11. Fig. S2. Illustration of Morris water maze training trials To investigate long-term memory we used the Morris water maze test. To analyze the learning process we used the latency time [s] of the training trials (1-12). Learning curves are separated for a better overview in A) WT group and B) in APP/PS1 mice group. The findings demonstrated that the animals improved their performance over one day (trial 1-6). Data represent mean + SEM; n > 11. Fig. S3. Quantification of amyloid-β 1-42 phagocytosis of microglial cells by flow cytometry. Cells were isolated from adult mice brains 3h after intraperitoneal injection of methoxy-04. Exemplary graphs of each group shows the evaluation of the phagocytosis rate (Q2). Fig. S4. FPR modulation does not affect astrocytes in APP/PS1 mice A) GFAP positive cells/mm² in the hippocampus where increased from WT control to APP/PS1 control mice.B) Also in the cortex we could see the same increased amount of GFAP positive cells in APP/PS1 control mice compared to WT control (n>15) C) Exemplary anti-GFAP staining’s of WT, APP/PS1 and APP/PS1+Boc2 mice in the cortex. D) Relative expression of Gfap mRNA in the hippocampus showed no differences but E) in the cortex we detected an increased Gfap mRNA expression in APP/PS1 control mice (n>6, ). Scale bar c 50 μm. Shown are the mean values of each group with SEM. Two-way ANOVA with turkey test *p

Background/aim In prostate cancer (PC), the formation of new blood vessels is stimulated by hypoxic conditions, androgens, and a number of molecular factors including microRNAs. MicroRNA-1 (miR-1) has been characterized in some tumor entities as anti-angiogenic, but this has not yet been investigated in PC. Materials and methods PC cells stably overexpressing miR-1 (LNCaP-miR-1) were incubated on an in vivo hen's egg test-chorioallantoic membrane (HET-CAM) model and compared to maternal LNCaP cells. Cell growth, blood vessel organisation, and total blood vessel area were analysed. Results Both matrigel-embedded LNCaP and LNCaP-miR-1 cells formed compact tumor-like cell aggregates on the CAM of the HET-CAM model. Although not quantifiable, bleeding of the CAM and remodelling of the blood vessel network in the CAM indicated an influence of miR-1 on the vascular system. The statistically significant decrease in the total surface area of blood vessels in the visible CAM section to 79.4% of control cells demonstrated the antiangiogenic properties of miR-1 for the first time. Conclusion MiR-1 had a tumor-suppressive and anti-angiogenic effect in an in vivo PC model. In the clinic, miR-1-mediated anti-angiogenesis would result in reduced tumor supply and increased hypoxic stress inside the tumor. Thus, miR-1 restoration by nucleic acid-based miR-1 mimetics would represent a promising option for future PC therapy.

Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. The factors triggering gliosis and demyelination are currently not well characterized. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. Especially during progressive disease, aberrant glia activation rather than the invasion of peripheral immune cells is accountable for progressive neuronal injury. The innate immune response can be induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs). In this study, we used the cuprizone model in mice to investigate the expression of TLR2 during the course of cuprizone-induced demyelination. In addition, we used TLR2-deficient mice to analyze the functional role of TLR2 activation during cuprizone-induced demyelination and reactive gliosis. We show a significantly increased expression of TLR2 in the corpus callosum and hippocampus of cuprizone-intoxicated mice. The absence of receptor signaling in TLR2-deficient mice resulted in less severe reactive astrogliosis in the corpus callosum and cortex. In addition, microglia activation was ameliorated in the corpus callosum of TLR2-deficient mice, but augmented in the cortex compared to wild-type littermates. Extent of demyelination and loss of mature oligodendrocytes was comparable in both genotypes. These results suggest that the TLR2 orchestrates glia activation during gray and white matter demyelination in the presence of an intact blood-brain barrier. Future studies now have to address the underlying mechanisms of the region-specific TLR2-mediated glia activation.

Introduction There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid β (Aβ)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD. Methods The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aβ burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze. Results Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aβ deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice. Conclusion In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs., Highlights • Methysticin activates the Nrf2/ARE system in the hippocampus of mice. • Methysticin protects AD mice against oxidative stress and associated neuroinflammation due to Nrf2 activation. • Methysticin improves long-term memory impairment in this mouse model of AD.

Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. Unclear are the factors triggering gliosis and demyelination. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. The innate immune response is induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the G-protein coupled with formyl peptide receptors (FPRs). Glial cells, the immune cells of the central nervous system, also express the PRRs. In this study, we used the cuprizone mice model to investigate the expression of the FPR1 in the course of cuprizone-induced demyelination In addition, we used FPR1-deficient mice to analyze glial cell activation through immunohistochemistry and real-time RT-PCR in cuprizone model. Our results revealed a significantly increased expression of FPR1 in the cortex of cuprizone-treated mice. FPR1-deficient mice showed a slight but significant decrease of demyelination in the corpus callosum compared to the wild-type mice. Furthermore, FPR1 deficiency resulted in reduced glial cell activation and mRNA expression of microglia/macrophages markers, as well as pro- and anti-inflammatory cytokines in the cortex, compared to wild-type mice after cuprizone-induced demyelination. Combined together, these results suggest that the FPR1 is an important part of the innate immune response in the course of cuprizone-induced demyelination.

Antimicrobial peptides (AMP) are an important part of the innate immune defense in the central nervous system (CNS). The expression of the AMP psoriasin (S100A7) is up-regulated during bacterial meningitis. However, the exact mechanisms induced by psoriasin to modulate glial cell activity are not yet fully understood. Our hypothesis is that psoriasin induced pro and anti-inflammatory signaling pathways as well as regenerative factors to contribute in total to a balanced immune response. Therefore, we used psoriasin stimulated glial cells and analyzed the translocation of the pro-inflammatory transcription factor nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NFκB) in murine glial cells and the expression of pro- and anti-inflammatory mediators by real time RT-PCR, ELISA technique and Western blotting. Furthermore, the relationship between psoriasin and the anti-oxidative stress transcription factor nuclear factor erythroid 2 related factor 2 (Nrf2) was investigated. Stimulation with psoriasin enhanced NFκB translocation and increased the expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) but also neurotrophin expression. Evidence for functional interactions between psoriasin and Nrf2 were detected in form of increased antioxidant response element (ARE) activity and induction of Nrf2/ARE-dependent heme oxygenase 1 (HO-1) expression in psoriasin-treated microglia and astrocytes. The results illustrate the ability of psoriasin to induce immunological functions in glia cells where psoriasin exerts divergent effects on the innate immune response. This article is protected by copyright. All rights reserved.

Background: An important hallmark of Alzheimer's disease (AD) is the increase of Aβ1-42 burden and its accumulation to senile plaques, leading the reactive gliosis and neurodegeneration. The modulation of glia cell function represent an attractive therapeutic strategy, but is currently limited by an incomplete understanding of its relevance for AD. The chemotactic G-protein coupled formyl peptide receptor (FPR), which is known to modulate Aβ1-42 uptake and signal transduction, might be one candidate molecule regulating glia function in AD. Here, we investigate whether the modulation of FPR exerts beneficial effects in an AD preclinical model. Methods: To address this question, APP/PS1 double-transgenic AD mice were treated for 20-weeks with either the pro-inflammatory FPR agonist fMLF, the FPR1/2 antagonist Boc2 or the anti-inflammatory FPR2 agonist Ac2-26. Spatial learning and memory were evaluated using a Morris water maze test. Immunohistological staining, gene expression studies and flow cytometry analyses were performed to study neuronal loss, gliosis and Aß-load in the hippocampus and cortex, respectively. Results: FPR antagonism by Boc2-treatment significantly improved spatial memory performance, reduced neuronal pathology, induced the expression of homeostatic growth factors, and ameliorated microglia, but not astrocyte, reactivity. Furthermore, the elevated levels of amyloid plaques in the hippocampus were reduced by Boc2-treatment, presumably by an induction of amyloid degradation.Conclusions: We suggest that the modulation of FPR signaling cascades might be considered as a promising therapeutic approach for alleviating the cognitive deficits associated with early AD. Additional studies are now needed to address the downstream effectors as well as the safety profile of Boc2.

Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hubel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.

Bacterial meningitis is - despite therapeutical progress during the last decades - still characterized by high mortality and severe permanent neurogical sequelae. The brain is protected from penetrating pathogens by both the blood–brain barrier and the innate immune system. Invading pathogens are recognized by so-called pattern recognition receptors including the Toll-like receptors (TLR) which are expressed by glial immune cells in the central nervous system. Among these, TLR2 is responsible for the detection of Gram-positive bacteria such as the meningitis-causing pathogen Streptococcus pneumoniae. Here, we used TLR2-deficient mice to investigate the effects on mortality, bacterial growth and inflammation in a mouse model of pneumococcal meningitis. Our results revealed a significantly increased mortality rate and higher bacterial burden in TLR2-deficient mice with pneumococcal meningitis. Furthermore, infected TLR2-deficient mice suffered from a significantly increased pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and Chemokine (C-C motif) ligand 2 (CCL2) or CCL3 chemokine expression and decreased expression of anti-inflammatory cytokines and antimicrobial peptides. In contrast, glial cell activation assessed by glial cell marker expression was comparable to wildtype mice. Taken together, the results suggest that TLR2 is essential for an efficient immune response against Streptococcus pneumoniae meningitis since lack of the receptor led to a worse outcome by higher mortality due to increased bacterial burden, weakened innate immune response and reduced expression of antimicrobial peptides.

Short non-coding microRNAs have been identified to orchestrate crucial mechanisms in cancer progression and treatment resistance. MicroRNAs are involved in posttranscriptional modulation of gene expression and therefore represent promising targets for anticancer therapy. As mircoRNA-1 (miR-1) exerted to be predominantly downregulated in the majority of examined tumors, miR-1 is classified to be a tumor suppressor with high potential to diminish tumor development and therapy resistance. Here we review the complex functionality of miR-1 in tumor biology.

Introduction: Inhibition of androgen synthesis by abiraterone acetate (AA) entails enhanced overall survival rates and clinical benefit for patients with locally advanced and metastasized prostate cancer (PC). The expression of heat shock protein 27 (HSP27) is generally associated with cytoprotection and was demonstrated to mediate chemoresistance under cytostatic therapy, for instance, docetaxel treatment. In this study, we investigated the impact of AA treatment on HSP27 expression and PC cell growth. Materials and Methods: HSP27 expression levels in docetaxel and AA-treated PC cell lines LNCaP and PC-3 were determined by SDS PAGE and Western blot analysis. Proliferation assays were performed using a CASY Cell Counter and Analyzer Model TT (Roche Applied Science). Results: Despite significantly increased HSP27 expression in PC cells incubated with docetaxel, Western blot analysis implicated a significant reduction of the cytoprotective HSP27 in AA-treated PC cells. Notably, HSP27 stably overexpressed in PC-3-HSP27 cells did not appear as an HSP27-mediated proliferation benefit in the presence of AA as shown in docetaxel incubation studies. Conclusion: In contrast to repeatedly demonstrated HSP27-driven chemoresistance related to chemotherapeutics, our results may constitute a broader molecular mode of action of AA chemotherapy. AA efficacy may exert an HSP27 suppressive role that goes beyond the primarily assumed inhibition of androgen biosynthesis.

Enzalutamide's accepted mode of action is by targeting the androgen receptor's (AR) activity. In clinical practice, enzalutamide demonstrates a good benefit-risk profile for the treatment of advanced prostate cancer (PC), even after poor response to standard antihormonal treatment. However, since both, well-established antiandrogens and enzalutamide, target AR functionality, we hypothesized that additional unknown mechanisms might be responsible for enzalutamide's superior anticancer activity. In the current study, PC cells were incubated with enzalutamide and enzalutamide-dependent modulation of apoptotic mechanisms were assessed via Western blot analysis, TDT-mediated dUTP-biotin nick end-labeling assay, and nuclear morphology assay. Alterations of heat shock protein (HSP), AR, and estrogen receptor (ER) expression were examined by Western blot analysis. Enzalutamide attenuated the proliferation of PC cells in a time- and dose-dependent manner. In the presence of enzalutamide, apoptosis occurred which was shown by increased BAX expression, decreased Bcl-2 expression, nuclear pyknosis, and genomic DNA fragmentation. Moreover, enzalutamide inhibited the expression of HSPs primarily involved in steroid receptor stabilization and suppressed AR and ERβ1 expression. This study demonstrates for the first time that enzalutamide treatment of PC cells triggers varying molecular mechanisms resulting in antiproliferative effects of the drug. In addition to the well-characterized antagonistic inhibition of AR functionality, we have shown that enzalutamide also affects the intracellular synthesis of steroid receptor-associated HSPs, thereby diminishing the expression of AR and ERβ1 proteins and inducing apoptotic pathways. According to an indirect attenuation of HSP-associated factors such as steroid receptors, endometrial carcinoma, uterine leiomyosarcoma, and mamma carcinoma cells also demonstrated inhibited cell growth in the presence of enzalutamide. Our data, therefore, suggest that enzalutamide's high efficacy is at least partially independent of AR and p53 protein expression, which are frequently lost in advanced PC.

Pneumococcal meningitis, caused by Streptococcus pneumoniae, is the most common type of bacterial meningitis. The clinical management of this disease has been challenged by the emergence of multidrug-resistant Streptococcus pneumoniae, requiring the urgent development of new therapeutic alternatives. Over the course of bacterial meningitis, pathogen invasion is accompanied by a massive recruitment of peripheral immune cells, especially neutrophil granulocytes, which are recruited under the coordination of several cytokines and chemokines. Here, we used chemokine (C-C motif) ligand 3 (Ccl3)-deficient mice to investigate the functional role of CCL3 in a mouse model of pneumococcal meningitis. Following intrathecal infection with Streptococcus pneumoniae Ccl3-deficient mice presented a significantly shorter survival and higher bacterial load than wildtype mice, paralleled by an ameliorated infiltration of neutrophil granulocytes into the CNS. Blood sample analysis revealed that infected Ccl3-deficient mice showed a significant decrease in erythrocytes, hemoglobin and hematocrit as well as in the number of banded neutrophils. Moreover, infected Ccl3-deficient mice showed an altered cytokine expression profile. Glial cell activation remained unchanged in both genotypes. In summary, this study demonstrates that CCL3 is beneficial in Streptococcus pneumoniae-induced meningitis. Pharmacological modulation of the CCL3 pathways might, therefore, represent a future therapeutic option to manage Streptococcus pneumoniae meningitis.

Background Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50–150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. Methods Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. Results Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. Conclusions The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth. Prostate © 2015 Wiley Periodicals, Inc.

Background: Antimicrobial peptides (AMP) are an important defense mechanism of the innate immune system and can modulate the course of various diseases. However, their significance during liver pathogenesis is currently not well defined. Methods: Patients with liver diseases were analyzed for LL-37/CRAMP, human beta-defensin-2 (hBD2), and complement 5a (C5a) serum levels. Mice deficient in CRAMP (Cathelicidin-related Antimicrobial Peptide), the mouse homolog for human LL-37, were fed with a methionine- and choline-deficient diet (MCD) and underwent bile-duct ligation (BDL). Results: First, serum samples from patients with chronic liver diseases were investigated. Therefore, significantly enhanced levels for LL-37, hBD2, and complement C5a were detected, all of which comprise antimicrobial properties. Next, CRAMP-knockout (CRAMP-KO) mice were investigated, to better define a functional role of LL-37/CRAMP in animal models of liver diseases. MCD feeding and bile-duct ligation of CRAMP-KO mice resulted in an enhanced degree of liver injury during the early treatment phase. MCD feeding in CRAMP-KO mice led to stronger intrahepatic fat accumulation and significantly enhanced matrix remodeling, whereas BDL caused more extensive liver necrosis. At the late 28 days time point, MCD-fed CRAMP-KO mice displayed a higher intrahepatic fat load. Long-term changes in bile-duct-ligated mice included higher collagen content as a sign of enhanced fibrosis progression if CRAMP was absent. Conclusion: The study shows a clear correlation of antimicrobial peptide serum levels in patients with chronic liver diseases. Furthermore, we were able to demonstrate protective functions of LL-37/CRAMP in two independent mouse models of chronic liver injury.

Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits.

Background/aim Due to its poor prognosis, it is increasingly necessary to understand the biology of renal cell cancer (RCC). Therefore, we investigated the role of microRNAs miR-1 and miR-21 in the growth of RCC cells compared to that of non-malignant renal cells. Materials and methods Four malignant cell lines (Caki-1, 786-O, RCC4, A498) were examined regarding their cell growth, microRNA and telomerase expression, and were compared to non-malignant RC-124 renal cells. Results Inconsistencies appeared in the panel of RCC cells regarding antiproliferative and proliferative properties of miR-1 and miR-21, respectively. Notably, and most likely due to immortaliziation, non-malignant RC-124 cells exhibited telomerase expression and activity. Conclusion miR-1 and miR-21 functionality in cancer progression, particularly in tumor growth, may be more dependent on the individual cellular context and may reflect RCC heterogeneity. Thus, both microRNAs, in combination with other stratifying biomarkers, may be useful in terms of RCC diagnosis, prognosis, or treatment response.

Analytical cellular pathology 2017, 7190546 (2017). doi:10.1155/2017/7190546, Published by Hindawi Publishing Corporation, New York

Additional Material and Methods. Fluorescence microscopy of TUNEL and Ki67: Primary mice astrocytes or microglia were seeded on cover glasses. After stimulation for 24 h, the cells werr fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.1% Triton X in 0.1% sodium citrate for 3 min at 4°C. Then, the slices were incubated at 37°C for 1 h with TUNEL reaction mixture according the manufacturer’s protocol (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). After washing with PBS and blocking with 1.5 BSA in PBS for 10 min, the slices were incubated at 4°C about the night with Ki67 antibody (rabbit polyclonal; ab15580, abcam, UK). Finally, the slices were incubated with anti-rabbit Cy3 (AP132C, Millipore, Darmstadt, Germany) for 1 h at room temperature. Nuclear counter-staining was performed with Diamidino-2-phenylindole dihydrochloride DAPI (Sigma 9542, Munich, Germany). Cells were digitally photographed using a Keyence digital microscope (BZ-9000, Neu Isenburg, Germany). Ki67+ positive cells were counted for each treatment, where five 63×fields were evaluated. The proliferation index was determined by the number of positive cells expressing Ki67 divided by the total number of cells in each field. (DOCX 16 kb)

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

International journal of molecular sciences 15(9), 15456-15474 (2014). doi:10.3390/ijms150915456, Published by Molecular Diversity Preservation International, Basel

Introduction Oxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2–ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). Methods Platelets and PRGF were obtained from healthy human donors. HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. Results The activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. Conclusions These results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2–ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.

Prolonged agonist exposure of many G-protein coupled receptors induces a rapid receptor phosphorylation and uncoupling from G-proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Numerous studies show the involvement of phospholipid-specific phosphodiesterase phospholipase D (PLD) in the receptor endocytosis and recycling of many G-protein coupled receptors e.g., opioid, formyl or dopamine receptors. The PLD hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), to phosphatidic acid (PA) and choline and is assumed to play an important function in cell regulation and receptor trafficking. Protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families regulate the two mammalian PLD isoforms 1 and 2. Mammalian and yeast PLD are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. The PA product is an intracellular lipid messenger. PLD and PA activities are implicated in a wide range of physiological processes and diseases including inflammation, diabetes, oncogenesis or neurodegeneration. This review discusses the characterization, structure, and regulation of PLD in the context of membrane located G-protein coupled receptor function.

The high variability of primary cells propagated in vitro led us to study the expression patterns of 11 most commonly accepted and widely used biomarkers specific for prostate cancer (PC) cells in primary cell models.Primary PC cells from five PC patients were partially subjected to RNA preparation immediately and remaining cells were propagated up to 84 days followed by RNA preparation. Subsequently, biomarker mRNA quantification was performed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and biomarker transcript concentrations before and after cultivation of primary PC cells were compared.Evaluation of androgen receptor, prostate-specific antigen, acid phosphatase, prostate-specific membrane antigen, fatty acid synthase, cytokeratin types 5/8/19, E-cadherin, epithelial cell adhesion molecule and fibroblast-specific protein 1 demonstrated temporal changes, as well as individual differences in expression, during primary PC cell propagation.Experimental design, as well as data evaluation, may need to take under consideration the high variability of biomarker expression in primary PC cells.

Mediators of inflammation 2016, 7678542 (2016). doi:10.1155/2016/7678542, Published by Hindawi Publishing Corp., Sylvania, Ohio

The prognosis of liver failure is often determined by infectious and cholestatic complications. As HGF/c-Met and interleukin (IL)-6/gp130 control hepatic cytoprotective pathways, we here investigated their cooperative role during the onset of cholestatic liver injury. Conditional hepatocyte-specific ((Δhepa)) c-Met, gp130 and c-Met/gp130 knockout mice (Cre-loxP system) were subjected to bile duct ligation (BDL) and lipopolysaccharide (LPS) stimulation. gp130(Δhepa) and c-Met/gp130(Δhepa) mice displayed increased lethality associated with severe bacteraemia early after BDL, whereas c-Met(Δhepa) and wild-type mice showed normal survival. Analysis of the innate immune response and the regulation of hepatic antibacterial pathways showed that the LPS-triggered hepatocellular response via the Toll-like receptor-4 pathway was regulated differentially by HGF/c-Met and IL-6/gp130. Activation of p38MAPK, c-Jun N-terminal kinase and signalling transducer and activator of transcription-3 was impaired in gp130(Δ) and c-Met(Δhepa) livers. In addition, the acute-phase response (APR) was reduced in c-Met(Δhepa) livers, whereas gp130(Δhepa) displayed a completely abolished APR. In contrast, TNF-α-dependent NF-κB activation was enhanced in gp130(Δhepa) and c-Met(Δhepa) mice and it was associated with a higher rate of apoptosis and inflammation. Moreover, expression of the neutrophil produced and secreted cathelin-related antimicrobial peptide and of genes related to the inflammasome complex correlated with the strength of the bacterial infection and with TNF-α expression. In conclusion, Gp130 and c-Met are involved in the hepatic antibacterial and innate immune response, control the APR and thus prevent sepsis and liver injury during cholestatic conditions.

Die Knochen-Knorpel-Transplantation unter Verwendung der Hohlfrasen des Osteochondral-Autograft-Transfer-System (OATS) hat sich bei umschriebenen aseptischen Osteonekrosen am Knie- und Sprunggelenk bewahrt. Wir beschreiben erstmals den Einsatz des OATS in der Behandlung einer partiellen Osteonekrose eines Mittelhandknochenkopfes; die Technik und der erfolgreiche Behandlungsverlauf werden dargestellt.

Experimental animal studies have demonstrated that oxidative stress plays an essential role during ischemic stroke. In addition to oxidizing macromolecules leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Nuclear factor erythroid 2-related factor 2 (Nrf2) represents one of the major regulators implicated in the endogenous defense system against oxidative stress. We have studied the expression and activation status of Nrf2 under stroke-like conditions using the temporary middle cerebral artery occlusion rat model. Inactive Nrf2 is proteasomal degraded within minutes but stabilized during activation. We analyzed Nrf2 activation and the resulting accumulation in post-ischemic rat brain cells using double immunofluorescence staining with antibodies directed against Nrf2 and cell type-specific markers. The core infarct region showed no obvious positive staining signal for Nrf2 24 h after the initiation of artery occlusion. However, Nrf2 immunoreactivity was detectable in the ipsilateral penumbra where microglia, astrocytes, and neurons contained Nrf2. Interestingly, Nrf2 was also significantly upregulated in neurons but not in other cell types of the unaffected contralateral site. These results provide strong evidence that Nrf2 is involved in acute stroke-dependent neurodegeneration in the penumbra but not core region and indicate the presence of a systemic Nrf2 activator independent from oxidative stress.

Background Thrombocyte concentrate i.e. platelet-rich plasma (PRP) has become a popular adjunct for many surgical procedures. It is believed to improve bone and soft tissue healing. Recently antimicrobial effects of the autologous preparation were reported by several groups. In this study we investigated the antimicrobial effect of PRP against gram-negative microbes which frequently cause severe complications in orthopaedic trauma surgery. Methods Platelet-rich plasma was produced from liquid preserved thrombocyte concentrates. ELISA, Western blot and immunohistochemistry were preformed to investigate the release and content of platelet concentrates. A radial diffusion assay was used to detect antimicrobial effects of PRP. Results We detected the human beta defensin-3 in bactericidal concentrations in platelet preparations by ELISA, Western blot and immunohistochemistry. In antimicrobial testing we demonstrated effective inhibition of Escherichia coli (ATCC 11303), Bacterium megaterium (ATCC 14581), Klebsiella pneumoniae (ATCC 13883), Enterococcus faecalis (ATCC 29212) and Proteus mirabilis (ATCC21100). Conclusion With this study we demonstrate antimicrobial action of a popular adjunct for orthopaedic and trauma surgery against gram-positive and gram-negative bacteria. We have identified a possible mechanism of action via the secretion of HBD-3 as a first line defence in contaminated wounds and in elective application of PRP. This finding supports a broader spectrum of clinical indications for an autologous platelet preparation.

Summary Aims Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. Methods and results We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n = 8) and 3rd (n = 8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. Conclusion The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell–cell and cell–matrix interactions between the placental syncytium and the maternal interface.

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.

Clinical observations have suggested a relationship between osteoarthritis and a changed sex-hormone metabolism, especially in menopausal women. This study analyzes the effect of 17β-estradiol on expression of matrix metalloproteinases-1, -3, -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) in articular chondrocytes. An imbalance of matrix metalloproteinases (MMPs) specialized on degradation of articular cartilage matrix over the respective inhibitors of these enzymes (TIMPs) that leads to matrix destruction was postulated in the pathogenesis of osteoarthritis. Primary human articular chondrocytes from patients of both genders were cultured in alginate beads at 5% O(2) to which 10(-11)M-10(-5)M 17β-estradiol had been added and analyzed by means of immunohistochemistry, immunocytochemistry and real-time RT-PCR. Since articular chondrocytes in vivo are adapted to a low oxygen tension, culture was performed at 5% O(2). Immunohistochemical staining in articular cartilage tissue from patients and immunocytochemical staining in articular chondrocytes cultured in alginate beads was positive for type II collagen, estrogen receptor α, MMP-1, and -13. It was negative for type I collagen, MMP-3, TIMP-1 and -2. Using real-time RT-PCR, it was demonstrated that physiological and supraphysiological doses of 17β-estradiol suppress mRNA levels of MMP-3 and -13 significantly in articular chondrocytes of female patients. A significant suppressing effect was also seen in MMP-1 mRNA after a high dose of 10(-5)M 17β-estradiol. Furthermore, high doses of this hormone led to tendentially lower TIMP-1 levels whereas the TIMP-2 mRNA level was not influenced. In male patients, only incubations with high doses (10(-5)M) of 17β-estradiol were followed by a tendency to suppressed MMP-1 and TIMP-1 levels while TIMP-2 mRNA level was decreased significantly. There was no effect on MMP-13 expression of cells from male patients. Taken together, application of 17β-estradiol in physiological doses will improve the imbalance between the amounts of MMPs and TIMPs in articular chondrocytes from female patients. Downregulation of TIMP-2 by 17β-estradiol in male patients would not be articular cartilage protective.