Biphasic and bilateral changes in striatal VGLUT1 and 2 protein expression in hemi-Parkinson rats

Parkinson's disease is characterized by disturbed glutamatergic neurotransmission in the striatum. Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1 and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na(+)/K(+)-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2 transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time intervals post unilateral 6-OHDA injection into the medial forebrain bundle versus controls. Three weeks post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2 expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-Parkinson rats whereas the striatal expression of VGLUT2 was comparable to control rats. No effect could be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic neurotransmission from the two types of striatal afferents.status: publishe

Using conventional HIV tests on oral fluid36499

There is need for more evaluations of non-invasive tests in order to broaden the reach of testing programs and to perform large scale epidemiological studies. In this study, three different human immunodeficiency virus (HIV) enzyme linked immunosorbent assays (ELISAs) and one line immunoassay were evaluated to detect HIV antibodies in oral fluid samples. Specimens were collected, after informed consent was obtained, with the Oracol (MMD, Worcester, England) device. A total IgG quantitation test was performed to demonstrate the quality of the sample. Assessment of a modified protocol of the Vironostika(R) HIV Ag/Ab, Enzygnost(R) Anti-HIV 1/2 Plus Genscreen HIV-1/2 Version 2 and a line immune confirmatory assay the INNO-LIA HIV I/II score was done, using oral fluid specimens of 325 HIV positive and negative individuals. For the ELISAs, the addition of an extra internal oral fluid control was evaluated as well as different cut-offs, time between sampling and testing and the effect of drinking water just before sampling. Finally, the confirmatory test and some testing algorithms and combination of tests were discussed. The results obtained from the oral fluid specimens were compared with the gold standard on paired serum specimens. Firstly, there was no significant difference observed between the use of the kit controls and the oral fluid controls. New protocols and calculation of cut-offs were defined for two of the three ELISAs. High sensitivities and specificities were obtained with all three ELISAs without any statistical difference between the three tests. Secondly, no statistically significant difference was observed when samples were stored for different time periods between sampling and testing, meaning that a period of seven days at room temperature before testing is still acceptable. Thirdly, drinking water before sample collection did not interfere with the testing, although lower optical densities were observed. None of the positive samples were missed. In addition, the line immunoassay INNO-LIA HIV I/II score test is a promising test for confirmation of reactive oral fluid specimen, but more samples need to be validated in order to adapt the interpretation rules specifically for oral fluid specimens. Different choices/algorithms adapted for the purpose of testing can be proposed. In conclusion, it can be said that the commercial ELISAs with adapted protocol and cut-off values are suitable tools for making HIV test performance accessible to people. With this non-invasive sampling method, more eligible individuals can and will be selected for further HIV test on blood</p

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes

Time-dependent changes in GLT-1 functioning in striatum of hemi-Parkinson rats

Striatal dopamine loss in Parkinson's disease is accompanied by a dysregulation of corticostriatal glutamatergic neurotransmission. Within this study, we investigated striatal expression and activity of the glial high-affinity Na+/K+-dependent glutamate transporters, GLT-1 and GLAST, in the 6-hydroxydopamine hemi-Parkinson rat model at different time points after unilateral 6-hydroxydopamine injection into the medial forebrain bundle. Using semi-quantitative Western blotting and an ex vivo D-[H-3]-aspartate uptake assay, we showed a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1. At 3 and 12 weeks post-lesion, striatal GLT-1 function was bilaterally upregulated whereas at 5 weeks there was no change. Even though our data do not allow a straightforward conclusion as for the role of glutamate transporters in the pathogenesis of the disease, they do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of a rat model for Parkinson's disease. (C) 2010 Elsevier Ltd. All rights reserved

Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays

HIV-1 Env pseudotyped viruses (PV) are an attractive tool for studying the antiviral activities of compounds interfering with virus entry into a target cell. To investigate whether results obtained in PV assays are relevant biologically, the antiviral activity of 6 reference compounds was compared on 5 virus isolates of different clades using three assays: (1) replicating virus in peripheral blood mononuclear cells (PBMCs), (2) PV in CD4 and CCR5- or CXCR4 co-receptor expressing Ghost cells, and (3) PV in PBMCs. A significant linear relationship was found between both single-cycle PV assays (P<0.0001, R2=0.75). Moreover, both assays showed enhanced sensitivity to the antiretrovirals tested (P=0.013 and 0.015, respectively) as compared to the PBMC assay with replication-competent virus. Most importantly, results from the latter assay could be predicted significantly from both PV assays, in which either Ghost target cells (P<0.0001, R2=0.61) or PBMCs (P<0.0001, R2=0.55) were used. The usefulness of the PV assay was demonstrated further by investigating the impact of the HIV-1 Env subtype on the antiviral activity of five new compounds derived from the entry inhibitor BMS806

Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model