An Integrative Approach to the Current Treatment of HIV-Associated Neurocognitive Disorders and the Implementation of Leukemia Inhibitor Factor as a Mediator of Neurocognitive Preservation.

HIV-associated neurocognitive disorders (HANDs) continue to impact patients despite antiretroviral therapy. A combination of antiretroviral therapies can diminish the HIV viral load to near undetectable levels, but fails to preserve neurocognitive integrity. The cytokine leukemia inhibitory factor (LIF) has shown neuroprotective properties that could mitigate neurodegeneration in HANDs. The LIF promotes neurogenesis, neural cell differentiation, and survival. Combination antiretroviral therapy reduces severe forms of HANDs, but neurocognitive impairment persists; additionally, some antiretrovirals have additional adverse neurotoxic effects. The LIF counteracts neurotoxic viral proteins and limits neural cell damage in models of neuroinflammation. Adding the LIF as an adjuvant therapy to enhance neuroprotection merits further research for managing HANDs. The successful implementation of the LIF to current therapies would contribute to achieving a better quality of life for the affected population.

Adjuvant effect of dendritic cells activator Imiquimod in genetic immunization with HIV-1 p55 Gag.

Dendritic cells (DC) are important antigen-presenting cells that have abilities to induce and maintain T-cell immunity, or attenuate it during hyperimmunization. Additional activation of DCs may be useful for vaccination purposes. Imiquimod is known to be a specific agonist of the Toll-like receptors (TLR7), which are located mainly on DCs. To study the effect of DC stimulation on the effectiveness of an HIV-1 p55 gag DNA vaccine in a mice model, we employed 25, 50, and 100 nM of Imiquimod as an adjuvant. Subsequently, Western blot analysis was used to quantify p55 protein production after the immunization. To characterize T-cells immune response, both the frequency of IFN-γ -secreting cells and IFN-γ and IL-4 production were measured, via an ELIspot assay and ELISA, respectively. Low concentrations of Imiquimod were found to effectively stimulate Gag production and the magnitude of the T-cell immune response, whereas higher concentrations reduced vaccination effects. Our results show that the adjuvant effects of Imiquimod depend on concentration. The use of Imiquimod may be helpful to study DC to T cell communication, including possible induction of immunotolerance.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Kinin-B2 receptor mediated neuroprotection after NMDA excitotoxicity is reversed in the presence of kinin-B1 receptor agonists.

BACKGROUND: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. PRINCIPAL FINDINGS: Bradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. CONCLUSIONS: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.

Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics.

In the past years, many reports have described the existence of neural progenitor and stem cells in the adult central nervous system capable of generating new neurons, astrocytes, and oligodendrocytes. This discovery has overturned the central assumption in the neuroscience field, of no new neurons being originated in the brain after birth and provided the fundaments to understand the molecular basis of neural differentiation and to develop new therapies for neural tissue repair. Although the mechanisms underlying cell fate during neural development are not yet understood, the importance of intrinsic and extrinsic factors and of an appropriate microenvironment is well known. In this context, emerging evidence strongly suggests that glial cells play a key role in controlling multiple steps of neurogenesis. Those cells, of particular radial glia, are important for migration, cell specification, and integration of neurons into a functional neural network. This review aims to present an update in the neurogenesis area and highlight the modulation of neural stem cell differentiation by neurotransmitters, growth factors, and their receptors, with possible applications for cell therapy strategies of neurological disorders.

Kinin-B2 receptor expression and activity during differentiation of embryonic rat neurospheres.

Neural progenitor cells were isolated from rat fetal telencephalon and proliferate as neurospheres in the presence of EGF, FGF-2, and heparin. In the absence of these growth factors, neurospheres differentiate into neurons, astrocytes, and oligodendrocytes. Using an embryonal carcinoma cell line as in vitro differentiation model, we have already demonstrated the presence of an autocrine loop system between kinin-B2 receptor activity and secretion of its ligand bradykinin (BK) as prerequisites for final neuronal differentiation (Martins et al., J Biol Chem 2005; 280: 19576-19586). The aim of this study was to verify the activity of the kallikrein-kinin system (KKS) during neural progenitor cell differentiation. Immunofluorescence studies and flow cytometry analysis revealed increases in glial fibrillary acidic protein and beta-3 tubulin expression and decrease in the number of nestin-positive cells along neurospheres differentiation, indicating the transition of neural progenitor cells to astrocytes and neurons. Kinin-B2 receptor expression and activity, secretion of BK into the medium, and presence of high-molecular weight kininogen suggest the participation of the KKS in neurosphere differentiation. Functional kinin-B2 receptors and BK secretion indicate an autocrine loop during neurosphere differentiation to neurons, astrocytes, and oligodendrocytes, reflecting events occurring during early brain development.

Development of the anti-VEGF aptamer to a therapeutic agent for clinical ophthalmology.

Age-related macular degeneration (AMD) is the main cause of loss of sight in the world and is characterized by neovascularization of the macula. The factors producing choroidal vascularization involve various growth factors, including the vascular endothelial growth factor (VEGF(165)). In this context, the systematic evolution of ligands by exponential enrichment (SELEX) became a tool for developing new therapeutic agents for AMD treatment. The SELEX is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules (aptamers) are identified by their ability to bind their targets with high affinity and specificity. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands for a wide range of proteins of clinical importance. For instance, Pegaptanib sodium, a 28-nucleotide polyethylene glycol RNA aptamer that selectively binds to VEGF(165) and inhibits angiogenesis, was approved by the Food and Drug Administration for the treatment of wet AMD, thereby providing significant benefits to a great number of patients with minimal adverse effects.

DNA and RNA aptamers: from tools for basic research towards therapeutic applications.

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.