Oligodendroglial maturation is dependent on intracellular protein shuttling.

Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.

Influence of quinacrine and chloroquine on the in vitro 3'-azido-3'-deoxythymidine antiretroviral effect.

BACKGROUND: Antimalarials quinacrine (Qc) and chloroquine (Cq) intercalate DNA, potentiate the activity of other drugs and have lysosomotropic, anti-inflammatory and antiviral activities that could increase the effect of the 3'-azido-3'-deoxythymidine (AZT) antiretroviral agent. The aim of the current study was to evaluate if Qc and Cq could improve the in vitro effect of the antiretroviral AZT agent. FINDINGS: Inhibition of viral replication in human immunodeficiency virus (HIV)SF33-infected peripheral blood mononuclear cells treated with Qc or Cq, alone or combined with a low dose of AZT was measured. Viral replication increased with Qc and decreased with high doses of Cq. The increase of replication caused by Qc was reversed by AZT. Neither Qc nor Cq significantly changed the antiviral activity of AZT. CONCLUSION: Cq does not potentiate the effect of AZT, but it is effective by itself at high doses. The rise of HIV replication by Qc could be deleterious in HIV endemic regions, where it is used as antimalarial. The mechanisms associated to this phenomenon must be identified.

Histone methyltransferase enhancer of zeste homolog 2 regulates Schwann cell differentiation.

Epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. However, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging. Here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (EZH2) gene, on Schwann cell differentiation. In sciatic nerves, EZH2 expression was found in Schwann cells and to peak perinatally. Suppression of EZH2 expression in cultured primary rat Schwann cells reduced the length of cell processes. These morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of Schwann cell maturation. In addition, we show that EZH2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. Chromatin immunoprecipitation analysis revealed binding of EZH2 at the p57kip2 promoter and reduction of histone H3K27 trimethylation upon gene suppression. EZH2 suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of EZH2. Furthermore, we describe Hes5 as transcriptional repressor of myelin genes in Schwann cells, which was induced upon EZH2 suppression and downregulated in p57kip2-suppressed Schwann cells. Therefore, we have identified a molecular link between histone methylation and control of Schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed.

Vinpocetine inhibits oligodendroglial precursor cell differentiation.

BACKGROUND: In multiple sclerosis during periods of remission a limited degree of myelin repair can be observed mediated by oligodendroglial precursor cells. Phosphodiesterase inhibitors act as anti-inflammatory agents and might hold promise for future multiple sclerosis treatment. AIMS: To investigate whether phosphodiesterase inhibitors could also influence myelin repair. METHODS: We stimulated primary oligodendroglial precursor cells with cilostazol, rolipram and vinpocetine and assessed their effects on repair related cellular processes. RESULTS: We found that vinpocetine exerted a strong negative effect on myelin expression while cilostazol and rolipram did not show such effects. In addition, vinpocetine decreased morphological complexities suggesting an overall negative impact on oligodendroglial cell maturation. We provide evidence that this is not mediated via a blockade of phosphodiesterase-1 but rather by inhibition of IĸB kinase. CONCLUSION: These findings suggest that vinpocetine via IĸB inhibition exerts a strong negative impact on oligodendroglial cell maturation and may therefore provide the rationale to restrict its application during periods of remission in multiple sclerosis patients. This is of particular interest since vinpocetine is widely used as a health supplement thought to act as a cognitive and memory enhancer for healthy people and patients with neurological or muscle diseases.

APOBEC3G mRNA expression in exposed seronegative and early stage HIV infected individuals decreases with removal of exposure and with disease progression.

BACKGROUND: APOBEC3G is an antiretroviral factor that acts by inducing G to A mutations. In this study, we examined the expression of APOBEC3G in uninfected HIV-1 exposed individuals at the time of their partner's diagnosis and one year later. We then compared this expression with that of infected individuals at different disease stages. APOBEC3G mRNA was measured in PBMCs from three groups: healthy controls with no known risk factor to HIV infection (n = 26), exposed uninfected individuals who had unprotected sex with their HIV+ partners for at least 3 months (n = 37), and HIV infected patients at various disease stages (n = 45), including 8 patients with low HIV viral loads < 10,000 copies/mL (LVL) for at least 3 years. Additionally, we obtained sequences from the env, gag, pol, nef, vif and the LTR of the patients' virus. RESULTS: Exposed uninfected individuals expressed higher APOBEC3G than healthy controls (3.86 vs. 1.69 relative expression units), and their expression significantly decreased after a year from the HIV diagnosis and subsequent treatment of their partners. Infected individuals showed a positive correlation (Rho = 0.57, p = 0.00006) of APOBEC3G expression with CD4+ T cell count, and a negative correlation with HIV viremia (Rho = -0.54, p = 0.00004). The percentage of G to A mutations had a positive correlation (Rho = 0.43, p = 0.0226) with APOBEC3G expression, and it was higher in LVL individuals than in the other patients (IQR 8.27 to 9.64 vs. 7.06 to 8.1, p = 0.0084). Out of 8 LVLs, 3 had hypermutations, and 4 had premature stop codons only in viral vif. CONCLUSION: The results suggest that exposure to HIV may trigger APOBEC3G expression in PBMCs, in the absence of infection. Additionally, cessation of exposure or advanced disease is associated with decreased APOBEC3G expression.

Sildenafil Inhibits Myelin Expression and Myelination of Oligodendroglial Precursor Cells.

Phosphodiesterases (PDEs) have previously been implicated in oligodendrocyte maturation and myelination of central nervous system axons. Sildenafil citrate is a phosphodiesterase inhibitor known to block PDE5, which also reduces inflammation in the experimental autoimmune encephalomyelitis demyelinating model. To find out whether this inhibitor might exert beneficial effects on central nervous system myelin repair activities, we investigated to what degree sildenafil modulates differentiation and maturation of cultured primary rat oligodendroglial precursor cells (OPCs). To this end, gene and protein expression of 2',3'-cyclic-nucleotide 3'-phosphodiesterase, myelin basic protein, and myelin oligodendrocyte glycoprotein, as well as of negative regulators of myelin expression (Hes1, Hes5, Id2, Id4, Rock2, and p57Kip2) were measured in OPCs treated with sildenafil. Moreover, the subcellular distribution of the p57kip2 protein was determined after sildenafil treatment, as this revealed to be an early predictor of the oligodendroglial differentiation capacity. In vitro myelination assays were done to measure the myelination capacity of oligodendrocytes treated with sildenafil. We found that sildenafil significantly diminished myelin gene expression and protein expression. Moreover, sildenafil also increased the expression of Id2 and Id4 negative transcriptional regulators, and the degree of OPCs with cytoplasmic p57kip2 protein localization was reduced, providing evidence that the PDE blocker impaired the differentiation capacity. Finally, sildenafil also interfered with the establishment of internodes as revealed by in vitro myelination assays. We therefore conclude that blocking PDE5 activities exerts a negative impact on intrinsic oligodendroglial differentiation processes.

Complete Genome Sequences, before and after Mammalian Cell Culture, of Zika Virus Isolated from the Serum of a Symptomatic Male Patient from Oaxaca, Mexico.

Zika virus (ZIKV) is an emerging arthropod-borne flavivirus associated with severe congenital malformations and neurological complications. Although the ZIKV genome is well characterized, there is limited information regarding changes after cell isolation and culture adaptation. We isolated, and passaged in Vero cells, ZIKV from the serum of a symptomatic male patient and compared the viral genomes before and after culture. Single nucleotide polymorphisms were characteristic among serum-circulating genomes, while such diversity decreased after cell culture.

CD8+ cell noncytotoxic anti-HIV response: restoration by HAART in the late stage of infection.

Highly active antiretroviral therapy (HAART) is currently the best HIV infection management strategy. However, its effects on the CD8+ T cell noncytotoxic anti-HIV response (CNAR) are not well known. We investigated if HAART has different effects on CNAR in patients at the intermediate and late stages of HIV infection. Untreated healthy HIV-infected subjects with a mean CD4+ T cell count of 606 cells/microl were examined as a reference group. Plasma viral load, CD4+ T cell count, and CNAR activity were measured at baseline and regular intervals for at least 48 weeks following initiation of HAART. Baseline CNAR activity in all subjects correlated inversely with viral load and directly with CD4 T+ cell counts. The level of CNAR in the latestage group was significantly lower than in the intermediate-stage and the healthy reference group (p < 0.01). Following initiation of HAART, substantial increases in CD4+ T cell counts and decreases in viral loads were observed in both groups, indicating treatment success. CNAR activity was found to be increased significantly during HAART, but only in the late-stage group (p < 0.01). This increase in CD8+ cell function was seen within 4 weeks of treatment initiation and resulted in levels of CNAR activity almost equal to those observed in the healthy reference subjects. Our findings suggest a beneficial effect on CNAR in those individuals with reduced activity, typically in late-stage infection.

Short communication: preferential concentration of hydroxychloroquine in adenoid tissue of HIV-infected subjects.

Hydroxychloroquine (HCQ) anti-HIV activity is well documented. To evaluate its distribution in lymphoid tissues, which are considered sanctuaries of HIV reservoirs and targets of early massive depletion of CD4(+) T cells, we assessed HCQ concentrations in adenoid tissue and plasma of HIV-infected subjects. A daily oral dose of 400 or 800 mg of HCQ was administered to eight HIV-infected subjects for 8 days. HCQ concentrations were measured in plasma and adenoid tissue by high-performance liquid chromatography. Mean concentrations of HCQ in adenoid tissue of subjects treated with 400 and 800 mg were 87,210 +/- 17,817 and 167,472 +/- 93,793 ng/g, respectively. In plasma, these values corresponded to 329 +/- 133 and 278 +/- 68 ng/g, respectively. HCQ concentrations were significantly higher in adenoid tissue than in plasma in both groups. The potential use of HCQ as adjuvant in the therapy of HIV deserves to be explored, as the drug accumulates in relevant tissues for HIV replication and immunopathogenesis.

Endoscopic assessment of adenoid size is an indicator of tissue virologic response to highly active antiretroviral therapy.

OBJECTIVE: To compare human immunodeficiency virus viral load (HIVVL) in plasma versus the adenoid HIVVL during highly active antiretroviral therapy (HAART). DESIGN: Adenoid biopsies were taken basally and after 3 and 6 months of treatment. Also, the adenoid diameter by simple endoscopy was measured, and its correlation with adenoid HIVVL was calculated. SETTING AND PATIENTS: A public tertiary care human immunodeficiency virus (HIV) hospital research centre. Twenty-seven antiretroviral-naive HIV-infected patients, with a mean age of 34.7 years, were included in the study. MAIN OUTCOME MEASURE: Correlation between adenoid diameter and plasma and tissue HIVVL. RESULTS: At 3 months, although plasma HIVVL reduced by almost 5 log to a level below 1 log, adenoid HIVVL only decreased 2.36 log, remaining well over 4 log. At 6 months, plasma HIVVL further decreased to 0.205 log, but adenoid HIVVL remained at 2.424 log. Adenoid diameter also decreased over time, with means at 8.52, 5.61, and 4 mm, respectively. It significantly correlated with plasma and adenoid viral load, but the correlation was higher with the biopsies. CONCLUSION: HIVVL in adenoid tissue is more resilient to HAART than plasma VL and may need more than 6 months to reach asymptomatic levels. Nevertheless, simple endoscopic measurement of the adenoid diameter is a good indicator of viral load decrease in this tissue.