Emerging Role of miR-21-5p in Neuron-Glia Dysregulation and Exosome Transfer Using Multiple Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with neuron-glia dysfunction and dysregulated miRNAs. We previously reported upregulated miR-124/miR-21 in AD neurons and their exosomes. However, their glial distribution, phenotypic alterations and exosomal spread are scarcely documented. Here, we show glial cell activation and miR-21 overexpression in mouse organotypic hippocampal slices transplanted with SH-SY5Y cells expressing the human APP695 Swedish mutation. The upregulation of miR-21 only in the CSF from a small series of mild cognitive impairment (MCI) AD patients, but not in non-AD MCI individuals, supports its discriminatory potential. Microglia, neurons, and astrocytes differentiated from the same induced pluripotent stem cells from PSEN1ΔE9 AD patients all showed miR-21 elevation. In AD neurons, miR-124/miR-21 overexpression was recapitulated in their exosomes. In AD microglia, the upregulation of iNOS and miR-21/miR-146a supports their activation. AD astrocytes manifested a restrained inflammatory profile, with high miR-21 but low miR-155 and depleted exosomal miRNAs. Their immunostimulation with C1q + IL-1α + TNF-α induced morphological alterations and increased S100B, inflammatory transcripts, sAPPß, cytokine release and exosomal miR-21. PPARα, a target of miR-21, was found to be repressed in all models, except in neurons, likely due to concomitant miR-125b elevation. The data from these AD models highlight miR-21 as a promising biomarker and a disease-modifying target to be further explored.

Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment.

BACKGROUND: Inhibiting programmed cell death protein 1 (PD-1) or PD-ligand 1 (PD-L1) has shown exciting clinical outcomes in diverse human cancers. So far, only monoclonal antibodies are approved as PD-1/PD-L1 inhibitors. While significant clinical outcomes are observed on patients who respond to these therapeutics, a large proportion of the patients do not benefit from the currently available immune checkpoint inhibitors, which strongly emphasize the importance of developing new immunotherapeutic agents. METHODS: In this study, we followed a transdisciplinary approach to discover novel small molecules that can modulate PD-1/PD-L1 interaction. To that end, we employed in silico analyses combined with in vitro, ex vivo, and in vivo experimental studies to assess the ability of novel compounds to modulate PD-1/PD-L1 interaction and enhance T-cell function. RESULTS: Accordingly, in this study we report the identification of novel small molecules, which like anti-PD-L1/PD-1 antibodies, can stimulate human adaptive immune responses. Unlike these biological compounds, our newly-identified small molecules enabled an extensive infiltration of T lymphocytes into three-dimensional solid tumor models, and the recruitment of cytotoxic T lymphocytes to the tumor microenvironment in vivo, unveiling a unique potential to transform cancer immunotherapy. CONCLUSIONS: We identified a new promising family of small-molecule candidates that regulate the PD-L1/PD-1 signaling pathway, promoting an extensive infiltration of effector CD8 T cells to the tumor microenvironment.

BASHY Dye Platform Enables the Fluorescence Bioimaging of Myelin Debris Phagocytosis by Microglia during Demyelination.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the presence of demyelinated regions with accumulated myelin lipid debris. Importantly, to allow effective remyelination, such debris must be cleared by microglia. Therefore, the study of microglial activity with sensitive tools is of great interest to better monitor the MS clinical course. Using a boronic acid-based (BASHY) fluorophore, specific for nonpolar lipid aggregates, we aimed to address BASHY's ability to label nonpolar myelin debris and image myelin clearance in the context of demyelination. Demyelinated ex vivo organotypic cultures (OCSCs) and primary microglia cells were immunostained to evaluate BASHY's co-localization with myelin debris and also to evaluate BASHY's specificity for phagocytosing cells. Additionally, mice induced with experimental autoimmune encephalomyelitis (EAE) were injected with BASHY and posteriorly analyzed to evaluate BASHY+ microglia within demyelinated lesions. Indeed, in our in vitro and ex vivo studies, we showed a significant increase in BASHY labeling in demyelinated OCSCs, mostly co-localized with Iba1-expressing amoeboid/phagocytic microglia. Most importantly, BASHY's presence was also found within demyelinated areas of EAE mice, essentially co-localizing with lesion-associated Iba1+ cells, evidencing BASHY's potential for the in vivo bioimaging of myelin clearance and myelin-carrying microglia in regions of active demyelination.

Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor.

Neuronal miRNA dysregulation may have a role in the pathophysiology of Alzheimer's disease (AD). miRNA(miR)-124 is largely abundant and a critical player in many neuronal functions. However, the lack of models reliably recapitulating AD pathophysiology hampers our understanding of miR-124's role in the disease. Using the classical human SH-SY5Y-APP695 Swedish neuroblastoma cells (SH-SWE) and the PSEN1 mutant iPSC-derived neurons (iNEU-PSEN), we observed a sustained upregulation of miR-124/miR-125b/miR-21, but only miR-124 was consistently shuttled into their exosomes. The miR-124 mimic reduced APP gene expression in both AD models. While miR-124 mimic in SH-SWE neurons led to neurite outgrowth, mitochondria activation and small Aß oligomer reduction, in iNEU-PSEN cells it diminished Tau phosphorylation, whereas miR-124 inhibitor decreased dendritic spine density. In exosomes, cellular transfection with the mimic predominantly downregulated miR-125b/miR-21/miR-146a/miR-155. The miR-124 inhibitor upregulated miR-146a in the two experimental cell models, while it led to distinct miRNA signatures in cells and exosomes. In sum, though miR-124 function may be dependent on the neuronal AD model, data indicate that keeping miR-124 level strictly controlled is crucial for proper neuronal function. Moreover, the iNEU-PSEN cellular model stands out as a useful tool for AD mechanistic studies and perhaps for the development of personalized therapeutic strategies.

Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS-Activated Acrylamides.

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N-hydroxysuccinimide (NHS) activated esters, are combined in NHS-activated acrylamides for efficient chemoselective amino-sulfhydryl stapling on native peptides and proteins. NHS-activated acrylamides allow for a fast functionalization of N-terminal cysteines (k2 =1.54±0.18×103  M-1 s-1 ) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross-linking of in-chain or C-terminal cysteines with nearby lysine residues. NHS-activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual-modifications. This strategy was successfully applied to the late-stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS-activated acrylamides is expected to promote amino-sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates.

Frailty-A promising concept to evaluate disease vulnerability.

With the emergence of diseases, people become frailer and are expected to be less tolerant of adverse outcomes. Frailty was first described to explain the variability in life expectancy in individuals of the same age. Nowadays, it is described as a syndrome and as a state. It is used to explain the heterogeneity of people not only in their responses to biological ageing but also in their responses to illness. In this review, we explore the role of frailty both in age-related diseases, including dementia, cancer and cardiovascular disease, and in non-age-related diseases, such as Human Immunodeficiency Virus. We describe how high levels of frailty in such disorders predict worse outcomes and play a direct role in disease progression and in prognostic prediction. Overall, the potential for frailty to predict adverse health outcomes among young people as well as in non-age-related diseases is an evolving topic. Understanding how frailty contributes to poor health and how it can be modified to prevent or delay disease progression will ultimately enhance quality of life in affected individuals.

Reduction of inflammation and colon injury by a Pennyroyal phenolic extract in experimental inflammatory bowel disease in mice.

PURPOSE: Little is known about the pharmacological effects of the phenolic compounds of Pennyroyal (Mentha pulegium). This Mediterranean aromatic plant, used as a gastronomic spice and as food preservative by the food industry has been studied mainly due to its essential oil antibacterial properties, composed primarily by monoterpenes. With this work, we aimed to evaluate the effects of a phenolic extract of pennyroyal in the impairment of inflammatory processes in Inflammatory Bowel Diseases (IBD) and in the potential inhibition of progression to colorectal cancer (CRC). METHODS: To that purpose, we evaluated the effect of pennyroyal extract administration in a model of TNBS-induced colitis in mice and further determined its effect on human colon carcinoma cell proliferation and invasion. RESULTS: The phenolic extract of pennyroyal exhibited antioxidant properties in in vitro assays and administration of the extract in a rat model of carrageenan-induced paw oedema led to significant anti-inflammatory effects. Further results evidenced a beneficial effect of the phenolic extract in the attenuation of experimental colitis and a potential antiproliferative effect on cultured colon cancer cells, effects not previously described, to our knowledge. A reduction in several markers of colon inflammation was observed following administration of the extract to colitis-induced mice, including functional and histological indicators. A successful inhibition of cancer cell invasion and proliferation was also observed in in vitro studies with HT-29 cells. Furthermore, the extract also led to a reduced expression of iNOS/COX-2 in the colon of colitis-induced mice, both being crucial mediators of intestinal inflammation. CONCLUSIONS: Taking into consideration the central role of inflammation in the pathophysiology of CRC and the recognised connection between inflammatory events and cancer, these results enlighten the relevance of the phenolic constituents of pennyroyal as important pharmacological sources in the investigation of new treatment options for patients with inflammatory bowel diseases.

Reduction of Inflammation and Colon Injury by a Spearmint Phenolic Extract in Experimental Bowel Disease in Mice.

Background: Inflammatory Bowel Diseases (IBD) encompasses both Crohn's Disease and Ulcerative Colitis, known to be connected to an enlarged risk for developing colorectal cancer (CRC). Spearmint (Mentha spicata L.) is a Mediterranean plant used as an aromatic agent, and studies have mainly focused on the essential oil suggesting an anti-inflammatory activity. This work aimed to perform a preliminary screening of the in vivo anti-inflammatory effects of a spearmint phenolic extract in an acute inflammation model, in a chronic inflammation model of colitis, and also study the effects in vitro on a colon cancer model. Methods: Spearmint extract was administered to rats of a paw oedema model (induced by carrageenan) and to mice from a TNBS-induced colitis model in parallel with studies using HT-29 CRC cells. Results: Administration of the extract led to reduced paw inflammation, reduction of colon injury and inflammation, with attenuation of histological markers, and reduction of iNOS expression. It repressed the in vitro movement of HT-29 cells in a wound healing assay. Conclusions: These findings suggest that spearmint extract exhibits acute and chronic anti-inflammatory activity and is able to inhibit migration of cancer cells, suggesting a potential role in the supplementary therapy of IBD patients.

Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases.

OBJECTIVE: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. METHODS: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several "hallmark of aging" pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six "hallmark of aging" pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. RESULTS: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. CONCLUSION: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFß (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Secretome from SH-SY5Y APPSwe cells trigger time-dependent CHME3 microglia activation phenotypes, ultimately leading to miR-21 exosome shuttling.

Exosome-mediated intercellular communication has been increasingly recognized as having a broad impact on Alzheimer's disease (AD) pathogenesis. Still, limited information exists regarding their "modus operandi", as it critically depends on exosomal cargo, environmental context and target cells. Therefore, a more thorough understanding of the role of exosomes from different cell types as mediators of neuroinflammation in AD context is a decisive step to open avenues for innovative and efficient therapies. In this study, we demonstrate that SH-SY5Y cells transfected with the Swedish mutant of APP695 (SHSwe) remarkably express increased inflammatory markers, combined with higher APP and Aß1-40 production, when compared to naïve SH-SY5Y (SH) cells. Although exerting an early clearance effect on extracellular APP and Aß accumulation when in co-culture with SHSwe cells, human CHME3 microglia gradually lose such property, and express both pro-inflammatory (iNOS, IL-1ß, TNF-α, MHC class II, IL-6) and pro-resolving genes (IL-10 and Arginase 1), while also evidence increased senescence-associated ß-galactosidase activity. Interestingly, upregulation of inflammatory-associated miRNA (miR)-155, miR-146a and miR-124 by SHSwe secretome shows to be time-dependent and to inversely correlate with their respective targets (SOCS-1, IRAK1 and C/EBP-α). We report that microglia also internalize exosomes released from SHSwe cells, which are enriched in miR-155, miR-146a, miR-124, miR-21 and miR-125b and recapitulate the cells of origin. Furthermore, we show that SHSwe-derived exosomes are capable of inducing acute and delayed microglial upregulation of TNF-α, HMGB1 and S100B pro-inflammatory markers, from which only S100B is found on their derived exosomes. Most importantly, our data reveal that miR-21 is a consistent biomarker that is found not only in SHSwe cells and in their released exosomes, but also in the recipient CHME3 microglia and derived exosomes. This work contributes to the increased understanding of neuron-microglia communication and exosome-mediated neuroinflammation in AD, while highlights miR-21 as a promising biomarker/target for therapeutic intervention.

Downregulated Glia Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

Dyospiros kaki phenolics inhibit colitis and colon cancer cell proliferation, but not gelatinase activities.

Polyphenols from persimmon (Diospyros kaki) have demonstrated radical-scavenging and antiinflammatory activities; however, little is known about the effects of persimmon phenolics on inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Therefore, we aimed in this work to characterize the antiinflammatory and antiproliferative effects of a persimmon phenolic extract (80% acetone in water), using an in vivo model of experimental colitis and a model of cancer cell invasion. Our results show, for the first time, a beneficial effect of a persimmon phenolic extract in the attenuation of experimental colitis and a potential antiproliferative effect on cultured colon cancer cells. Administration of persimmon phenolic extract to mice with TNBS-induced colitis led to a reduction in several functional and histological markers of colon inflammation, namely: attenuation of colon length decrease, reduction of the extent of visible injury (ulcer formation), decrease in diarrhea severity, reduced mortality rate, reduction of mucosal hemorrhage and reduction of general histological features of colon inflammation. In vitro studies also showed that persimmon phenolic extract successfully impaired cell proliferation and invasion in HT-29 cells. Further investigation showed a decreased expression of COX-2 and iNOS in the colonic tissue of colitis mice, two important mediators of intestinal inflammation, but there was no inhibition of the gelatinase MMP-9 and MMP-2 activities. Given the role of inflammatory processes in the progression of CRC and the important link between inflammation and cancer, our results highlight the potential of persimmon polyphenols as a pharmacological tool in the treatment of patients with IBD.

S100B as a Potential Biomarker and Therapeutic Target in Multiple Sclerosis.

Multiple sclerosis (MS) pathology is characterized by neuroinflammation and demyelination. Recently, the inflammatory molecule S100B was identified in cerebrospinal fluid (CSF) and serum of MS patients. Although seen as an astrogliosis marker, lower/physiological levels of S100B are involved in oligodendrocyte differentiation/maturation. Nevertheless, increased S100B levels released upon injury may induce glial reactivity and oligodendrocyte demise, exacerbating tissue damage during an MS episode or delaying the following remyelination. Here, we aimed to unravel the functional role of S100B in the pathogenesis of MS. Elevated S100B levels were detected in the CSF of relapsing-remitting MS patients at diagnosis. Active demyelinating MS lesions showed increased expression of S100B and its receptor, the receptor for advanced glycation end products (RAGE), in the lesion area, while chronic active lesions displayed increased S100B in demyelinated areas with lower expression of RAGE in the rim. Interestingly, reactive astrocytes were identified as the predominant cellular source of S100B, whereas RAGE was expressed by activated microglia/macrophages. Using an ex vivo demyelinating model, cerebral organotypic slice cultures treated with lysophosphatidylcholine (LPC), we observed a marked elevation of S100B upon demyelination, which co-localized mostly with astrocytes. Inhibition of S100B action using a directed antibody reduced LPC-induced demyelination, prevented astrocyte reactivity and abrogated the expression of inflammatory and inflammasome-related molecules. Overall, high S100B expression in MS patient samples suggests its usefulness as a diagnostic biomarker for MS, while the beneficial outcome of its inhibition in our demyelinating model indicates S100B as an emerging therapeutic target in MS.

Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation.

Rosmarinic acid is a polyphenolic compound and main constituent of Rosmarinus officinalis and has been shown to possess antioxidant and anti-inflammatory properties. We aimed to evaluate the anti-inflammatory properties of rosmarinic acid and of an extract of R. officinalis in local inflammation (carrageenin-induced paw oedema model in the rat), and further evaluate the protective effect of rosmarinic acid in rat models of systemic inflammation: liver ischaemia-reperfusion (I/R) and thermal injury models. In the local inflammation model, rosmarinic acid was administered at 10, 25 and 50 mg/kg (p.o.), and the extract was administered at 10 and 25 mg/kg (equivalent doses to rosmarinic acid groups) to male Wistar rats. Administration of rosmarinic acid and extract at the dose of 25 mg/kg reduced paw oedema at 6 hr by over 60%, exhibiting a dose-response effect, suggesting that rosmarinic was the main contributor to the anti-inflammatory effect. In the liver I/R model, rosmarinic acid was administered at 25 mg/kg (i.v.) 30 min. prior to the induction of ischaemia and led to the significant reduction in the serum concentration of transaminases (AST and ALT) and LDH. In the thermal injury model, rosmarinic acid was administered at 25 mg/kg (i.v.) 5 min. prior to the induction of injury and significantly reduced multi-organ dysfunction markers (liver, kidney, lung) by modulating NF-κB and metalloproteinase-9. For the first time, the anti-inflammatory potential of rosmarinic acid has been identified, as it causes a substantial reduction in inflammation, and we speculate that it might be useful in the pharmacological modulation of injuries associated to inflammation.

Inhibition of glycogen synthase kinase-3ß attenuates organ injury and dysfunction associated with liver ischemia-reperfusion and thermal injury in the rat.

Glycogen synthase kinase 3 (GSK-3) is a serine-threonine kinase discovered decades ago to have an important role in glycogen metabolism. Today, we know that this kinase is involved in the regulation of many cell functions, including insulin signaling, specification of cell fate during embryonic development, and the control of cell division and apoptosis. Insulin and TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) are inhibitors of GSK-3ß that have been shown to possess organ-protective effects in inflammatory-mediated organ injury models. We aimed to evaluate the cytoprotective effect of GSK-3ß inhibition on rat models of liver ischemia-reperfusion and thermal injury. In the liver ischemia-reperfusion model, TDZD-8 and insulin were administered at 5 mg/kg (i.v.) and 1.4 IU/kg (i.v.), respectively, 30 min before induction of ischemia and led to the significant reduction of the serum concentration of aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, and lactate dehydrogenase. Beneficial effects were found to be independent from blood glucose levels. In the thermal injury model, TDZD-8 was administered at 5 mg/kg (i.v.) 5 min before induction of injury and significantly reduced multiple organ dysfunction markers (liver, neuromuscular, and lung). In the lung, TDZD-8 reduced the histological signs of tissue injury, inflammatory markers (cytokines), and neutrophil chemotaxis/infiltration; reduced GSK-3ß, nuclear factor-κB, and Akt activation; reduced caspase-3 and metalloproteinase-9 activation. Our study provides a new insight on the beneficial effects of GSK-3ß inhibition on systemic inflammation and further elucidates the mechanism and pathway crosstalks by which TDZD-8 reduces the multiple organ injury elicited by thermal injury.

Microglia and inflammation: conspiracy, controversy or control?

Microglial cells contribute to normal function of the central nervous system (CNS). Besides playing a role in the innate immunity, they are also involved in neuronal plasticity and homeostasis of the CNS. While microglial cells get activated and undergo phenotypic changes in different disease contexts, they are far from being the "villains" in the CNS. Mounting evidence indicates that microglial dysfunction can exacerbate the pathogenesis of several diseases in the CNS. Several molecular mechanisms tightly regulate the production of inflammatory and toxic factors released by microglia. These mechanisms involve the interaction with other glial cells and neurons and the fine regulation of signaling and transcription activation pathways. The purpose of this review is to discuss microglia activation and to highlight the molecular pathways that can counteract the detrimental role of microglia in several neurologic diseases. Recent work presented in this review support that the understanding of microglial responses can pave the way to design new therapies for inflammatory diseases of the CNS.

Neuroprotective effects of erythropoietin pretreatment in a rodent model of transient middle cerebral artery occlusion.

UNLABELLED: OBJECT.: There is an unmet clinical need to develop neuroprotective agents for neurosurgical and endovascular procedures that require transient cerebral artery occlusion. The aim in this study was to explore the effects of a single dose of recombinant human erythropoietin (rhEPO) before middle cerebral artery (MCA) occlusion in a focal cerebral ischemia/reperfusion model. METHODS: Twenty-eight adult male Wistar rats were subjected to right MCA occlusion via the intraluminal thread technique for 60 minutes under continuous cortical perfusion monitoring by laser Doppler flowmetry. Rats were divided into 2 groups: control and treatment. In the treated group, rhEPO (1000 IU/kg intravenously) was administered 10 minutes before the onset of the MCA ischemia. At 24-hour reperfusion, animals were examined for neurological deficits, blood samples were collected, and animals were killed. The following parameters were evaluated: brain infarct volume, ipsilateral hemispheric edema, neuron-specific enolase plasma levels, parenchyma histological features (H & E staining), Fluoro-Jade-positive neurons, p-Akt and total Akt expression by Western blot analysis, and p-Akt-positive nuclei by immunohistochemical investigation. RESULTS: Infarct volume and Fluoro-Jade staining of degenerating neurons in the infarct area did not vary between groups. The severity of neurological deficit (p < 0.001), amount of brain edema (78% reduction in treatment group, p < 0.001), and neuron-specific enolase plasma levels (p < 0.001) were reduced in the treatment group. Perivascular edema was histologically less marked in the treatment group. No variations in the expression or localization of p-Akt were seen. CONCLUSIONS: Administration of rhEPO before the onset of 60-minute transient MCA ischemia protected the brain from this insult. It is unlikely that rhEPO pretreatment leads to direct neuronal antiapoptotic effects, as supported by the lack of Akt activation, and its benefits are most probably related to an indirect effect on brain edema as a consequence of blood-brain barrier preservation. Although research on EPO derivatives is increasing, rhEPO acts through distinct neuroprotective pathways and its clinical safety profile is well known. Clinically available rhEPO is a potential therapy for prevention of neuronal injury induced by transitory artery occlusion during neurovascular procedures.

Cross-talk between neurons and astrocytes in response to bilirubin: adverse secondary impacts.

Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation,and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis,cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-a secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND.

Rat cerebellar slice cultures exposed to bilirubin evidence reactive gliosis, excitotoxicity and impaired myelinogenesis that is prevented by AMPA and TNF-α inhibitors.

The cerebellum is one of the most affected brain regions in the course of bilirubin-induced neurological dysfunction. We recently demonstrated that unconjugated bilirubin (UCB) reduces oligodendrocyte progenitor cell (OPC) survival and impairs oligodendrocyte (OL) differentiation and myelination in co-cultures of dorsal root ganglia neurons and OL. Here, we used organotypic cerebellar slice cultures, which replicate many aspects of the in vivo system, to dissect myelination defects by UCB in the presence of neuroimmune-related glial cells. Our results demonstrate that treatment of cerebellar slices with UCB reduces the number of myelinated fibres and myelin basic protein mRNA expression. Interestingly, UCB addition to slices increased the percentage of OPC and decreased mature OL content, whereas it decreased Olig1 and increased Olig2 mRNA expression. These UCB effects were associated with enhanced gliosis, revealed by an increased burden of both microglia and astrocytes. Additionally, UCB treatment led to a marked increase of tumor necrosis factor (TNF)-α and glutamate release, in parallel with a decrease of interleukin (IL)-6. No changes were observed relatively to IL-1ß and S100B secretion. Curiously, both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and TNF-α antibody partially prevented the myelination defects that followed UCB exposure. These data point to a detrimental role of UCB in OL maturation and myelination together with astrocytosis, microgliosis, and both inflammatory and excitotoxic responses, which collectively may account for myelin deficits following moderate to severe neonatal jaundice.

Evidence of tricellulin expression by immune cells, particularly microglia.

Tight junctions (TJs) are elaborate structures located on the apical region of epithelial cells that limit paracellular permeability. Tricellulin is a recently discovered TJ protein, which is concentrated at the structurally specialized tricellular TJs but also present at bicellular contacts between epithelial cells, namely in the stomach. Interestingly, several TJ proteins have been found in other than epithelial cells, as astrocytes, and tricellulin mRNA expression was reported in mature dendritic cells. These findings prompted us to look for tricellulin expression in both epithelial and immune cells in the stomach, as well as in microglia, the brain resident immunocompetent cells. Immunohistochemical analysis of human stomach tissue sections revealed peroxidase staining at three-corner contact sites, as well as at the contact between two adjacent epithelial cells, thus evidencing the expression of tricellulin not only at tricellullar but at bicellular junctions as well. Such analysis, further revealed tricellulin immunostaining in cells of the monocyte/macrophage lineage, scattered throughout the lamina propria. Cultured rat microglia exhibited a notorious tricellulin staining, consistent with an extensive expression of the protein along the cell, which was not absolutely coincident with the lysosomal marker CD68. Detection of mRNA expression by real-time PCR provided supportive evidence for the expression of the TJ protein in microglia. These data demonstrate for the first time that microglia express a TJ protein. Moreover, the expression of tricellulin both in microglia and in the stomach immune cells point to a possible role of this new TJ protein in the immune system.