Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain.

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X7 ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X7 receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y2 in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y2 expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y2 sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y2 mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X7-antagonists oATP and BBG. The BzATP-evoked P2Y2 receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y2siRNA, thus supporting evidence of increased P2Y2 activity. To further investigate the mechanisms by which P2X7 receptors mediated the P2Y2 mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y2 mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X7-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X7 receptors in astrocytes enhances P2Y2 mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways

Changes in matrix extracellular phosphoglycoprotein expression before and during in vitro osteogenic differentiation of human dental papilla mesenchymal cells.

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing β-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration

Mechanism of differential Zika and dengue virus neutralization by a public antibody lineage targeting the DIII lateral ridge

We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1

Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV

Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes

The main source of cholesterol in the central nervous system (CNS) is represented by glial cells, mainly astrocytes, which also synthesise and secrete apolipoproteins, in particular apolipoprotein E (ApoE), the major apolipoprotein in the brain, thus generating cholesterol-rich high density lipoproteins (HDLs). This cholesterol trafficking, even though still poorly known, is considered to play a key role in different aspects of neuronal plasticity and in the stabilisation of synaptic transmission. Moreover, cell cholesterol depletion has recently been linked to a reduction in amyloid beta formation. Here we demonstrate that guanosine, which we previously reported to exert several neuroprotective effects, was able to increase cholesterol efflux from astrocytes and C6 rat glioma cells in the absence of exogenously added acceptors. In this effect the phosphoinositide 3 kinase/extracellular signal-regulated kinase 1/2 (PI3K/ERK1/2) pathway seems to play a pivotal role. Guanosine was also able to increase the expression of ApoE in astrocytes, whereas it did not modify the levels of ATP-binding cassette protein A1 (ABCA1), considered the main cholesterol transporter in the CNS. Given the emerging role of cholesterol balance in neuronal repair, these effects provide evidence for a role of guanosine as a potential pharmacological tool in the modulation of cholesterol homeostasis in the brain

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms

Potently neutralizing and protective human antibodies against SARS-CoV-2

The COVID-19 pandemic is a major threat to global health1 for which there are limited medical countermeasures2,3. Moreover, we currently lack a thorough understanding of mechanisms of humoral immunity4. From a larger panel of human monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein5, we identified several that exhibited potent neutralizing activity and fully blocked the receptor-binding domain of S (SRBD) from interacting with human ACE2 (hACE2). Competition-binding, structural, and functional studies allowed clustering of the mAbs into classes recognizing distinct epitopes on the SRBD as well as distinct conformational states of the S trimer. Potent neutralizing mAbs recognizing non-overlapping sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two mouse models of SARS-CoV-2 infection, passive transfer of either COV2-2196 or COV2-2130 alone or a combination of both mAbs protected mice from weight loss and reduced viral burden and inflammation in the lung. In addition, passive transfer of each of two of the most potently ACE2 blocking mAbs (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutics