TLR2 controls random motility, while TLR7 regulates chemotaxis of microglial cells via distinct pathways

Microglial cells are the pathologic sensor of the brain, and any pathologic event triggers microglial activation, which involves migration of these cells to a lesion site. Employing different migration assays, we show that ligands for toll-like receptor (TLR) 2 stimulate random motility, while TLR7 ligands are chemoattractants. The subtype specificity of the TLR ligands was verified by using different TLR-deficient (TLRKO) mouse lines. PI3K and Rac inhibition impairs both TLR2- and TLR7-stimulated microglial migration. In contrast, Akt phosphorylation is only required for the TLR2-, but not for the TLR7-stimulated pathway. Interestingly, P2Y12 receptor signaling is involved in the TLR2 activation-induced microglial migration but not TLR7. Furthermore, TLR7 mRNA expression is down-regulated by TLR2 and TLR7 activation. We conclude that TLRs control the migratory behavior of microglia in a distinct manner

let-7 microRNAs regulate microglial function and suppress glioma growth through Toll-like receptor 7

Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma

Neurofibromatosis 1 - mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain

Activation of Toll-like receptor 5 in microglia modulates their function and triggers neuronal injury

Microglia are the primary immune-competent cells of the central nervous system (CNS) and sense both pathogen- and host-derived factors through several receptor systems including the Toll-like receptor (TLR) family. Although TLR5 has previously been implicated in different CNS disorders including neurodegenerative diseases, its mode of action in the brain remained largely unexplored. We sought to determine the expression and functional consequences of TLR5 activation in the CNS. Quantitative real-time PCR and immunocytochemical analysis revealed that microglia is the major CNS cell type that constitutively expresses TLR5. Using Tlr5(-/-) mice and inhibitory TLR5 antibody we found that activation of TLR5 in microglial cells by its agonist flagellin, a principal protein component of bacterial flagella, triggers their release of distinct inflammatory molecules, regulates chemotaxis, and increases their phagocytic activity. Furthermore, while TLR5 activation does not affect tumor growth in an ex vivo GL261 glioma mouse model, it triggers microglial accumulation and neuronal apoptosis in the cerebral cortex in vivo. TLR5-mediated microglial function involves the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, as specific inhibitors of this signaling pathway abolish microglial activation. Taken together, our findings establish TLR5 as a modulator of microglial function and indicate its contribution to inflammatory and injurious processes in the CNS

Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

A RETROSPECTIVE STUDY ON INTRAHEPATIC CHOLESTASIS OF PREGNANCY: MARKERS OF PREMATURE DELIVERY

Background: Intrahepatic Cholestasis of Pregnancy (ICP) carries with it a higher frequency of premature delivery (20 to 60%), a marker ofperinatal morbility/mortality. Therefore, it is important to identify markers of premature delivery. Aim: To retrospectively evaluate, in a large case study, the presence of markers predictive of premature delivery. Materials and Methods: Clinical records of all patients diagnosed with ICP in Policlinic S.Orsola-Malpighi (Bologna, Italy) from January 2001 until December 2007 have been collected. For all patients the following informations were available: subjective pruritus evaluation, standard biochemical parameters, serum bile acids (BA) determination, abdomen ultrasonography and APGAR index. From diagnosis until a week after delivery, biochemistry and fetal health have been controlled weekly. Student’s t Test, Mann-Whitney, Pearson’s correlation test and a stepwiselogistic regression analysis were performed as appropriate. Results: 169 clinical records have been gathered. Patients with premature delivery, compared to those who delivered closer to term, presented a significantly earlier appearance of pruritus (r = 0.4858) and higher levels of chenodeoxycholic acid (r = −0.2835). No significant differences were observed for APGAR index and rate of fetal complications according to treatment and time of delivery. Two fetal deaths occurred (fetal infection by Parvovirus B19 and haemolysis for ABO incompatibility) before week 34 with serum BA below 20 mM/l. According to the multivariate analysis, the only variable associated with preterm delivery is the early onset of cholestasis, that exposes to a risk twice than normal (OR = 2.044; 95% CI 1.40−2.98; p = 0.0002). Conclusions: The strongest predictor of premature delivery in our population is the time of onset of pruritus with an OR of 2.044

High dose lamivudine in HBV-related cirrhotic patients with unsatisfactory response after adefovir add-on

Background: Before tenofovir approval for chronic hepatitis B therapy, the clinical management of patients with suboptimal response or virological breakthrough during combination treatment with lamivudine and adefovir dipivoxil was a difficult clinical challenge. Aims In order to improve virologic response and reduce the risk of decompensation, we evaluate the efficacy of a high dose of lamivudine on chronic HBV patients who have previously presented an unsatisfactory response during treatment with lamivudine 100mg/day and adefovir 10mg/day. Methods: Six patients with HBV-related liver cirrhosis were prospectively enrolled. All were HBeAg-negative and presented a suboptimal response or virological breakthrough after \u2018\u2018adefovir add-on\u2019\u2019 because of development of clinical breakthrough during Lamivudine treatment. Lamivudine dose was increased to 200 or 300mg, depending on viral load. After 12 months of follow-up, virological and biochemical response were evaluated. Results: After 12 months of high-dose lamivudine, all patients (6/6, 100%) achieved a significant decrease of serum HBV DNA (mean reduction 2,62 \ub1 1,15 Log10 UI/ml, P = 0.03) and normalized ALT. In three patients (3/6, 50%), HBV DNA became undetectable within 6 months. No patient developed liver decompensation and no significant changes occurred in serum creatinine, serum and urinary electrolytes. No adverse events were registered. Conclusions: In our experience, rescue strategy with highdose lamivudine inhibited viral replication leading to undetectability of serum HBVDNA. This rescue treatment presented a good safety profile, without adverse events during the study period. Customized increase of nucleos(t)ide analogues dose in difficult-to-treat patients may be a proficient approach in challenging clinical setting

A RETROSPECTIVE STUDY ON INTRAHEPATIC CHOLESTASIS OF PREGNANCY: MARKERS OF PREMATURE DELIVERY

Background: Intrahepatic Cholestasis of Pregnancy (ICP) carries with it a higher frequency of premature delivery (20 to 60%), a marker ofperinatal morbility/mortality. Therefore, it is important to identify markers of premature delivery. Aim: To retrospectively evaluate, in a large case study, the presence of markers predictive of premature delivery. Materials and Methods: Clinical records of all patients diagnosed with ICP in Policlinic S.Orsola-Malpighi (Bologna, Italy) from January 2001 until December 2007 have been collected. For all patients the following informations were available: subjective pruritus evaluation, standard biochemical parameters, serum bile acids (BA) determination, abdomen ultrasonography and APGAR index. From diagnosis until a week after delivery, biochemistry and fetal health have been controlled weekly. Student\u2019s t Test, Mann-Whitney, Pearson\u2019s correlation test and a stepwiselogistic regression analysis were performed as appropriate. Results: 169 clinical records have been gathered. Patients with premature delivery, compared to those who delivered closer to term, presented a significantly earlier appearance of pruritus (r = 0.4858) and higher levels of chenodeoxycholic acid (r = −0.2835). No significant differences were observed for APGAR index and rate of fetal complications according to treatment and time of delivery. Two fetal deaths occurred (fetal infection by Parvovirus B19 and haemolysis for ABO incompatibility) before week 34 with serum BA below 20 mM/l. According to the multivariate analysis, the only variable associated with preterm delivery is the early onset of cholestasis, that exposes to a risk twice than normal (OR = 2.044; 95% CI 1.40−2.98; p = 0.0002). Conclusions: The strongest predictor of premature delivery in our population is the time of onset of pruritus with an OR of 2.044