Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR

Background

Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context.

Methods

We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests.

Results

HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001).

Conclusions

Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC.     

Visual Function Assessment in Simulated Real-Life Situations in HIV-Infected Subjects

Visual function abnormalities are common in people living with HIV disease (PLWH) without retinitis, even after improvement in immune status. Abnormalities such as reduced contrast sensitivity, altered color vision, peripheral visual field loss, and electrophysiological changes are related to a combination of retinal dysfunctions, involving inner and outer retinal structures. The standard protocol for testing vision performance in clinical practice is the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. However, this method poorly correlates with activities of daily living that require patients to assess visual stimuli in multiple light/contrast conditions, and with limited time. We utilized a novel interactive computer program (Central Vision Analyzer) to analyze vision performance in PLWH under a variety of light/contrast conditions that simulate stressful and real-world environments. The program tests vision in a time-dependent way that we believe better correlates with daily living activities than the non-timed ETDRS chart. We also aimed to correlate visual scores with retinal neuro-fiber layer thickness on optical coherence tomography. Here we show that visual acuity is more affected in PLWH in comparison to HIV-seronegative controls in varying contrast and luminance, especially if the nadir CD4+ T-cell count was lower than 100 cells/mm³. Visual impairment reflects the loss of retinal nerve fiber layer thickness especially of the temporal-inferior sector. In PLWH the ETDRS chart test led to better visual acuity compared to the Central Vision Analyzer equivalent test, likely because patients had indefinite time to guess the letters. This study confirms and strengthens the finding that visual function is affected in PLWH even in absence of retinitis, since we found that the HIV serostatus is the best predictor of visual loss. The Central Vision Analyzer may be useful in the diagnosis of subclinical HIV-associated visual loss in multiple light/contrast conditions, and may offer better understanding of this entity called “neuroretinal disorder”.     

Comparison of Protein-Extraction Methods for Gills of the Shore Crab,Carcinus maenas (L.), and Application to 2DE

As it is well-established that protein extraction constitutes a crucial step for two-dimensional electrophoresis (2DE), this work was done as a prerequisite to further the study of alterations in the proteome in gills of the shore crab Carcinus maenas under contrasted environmental conditions. Because of the presence of a chitin layer, shore crab gills have an unusual structure. Consequently, they are considered as a hard tissue and represent a challenge for optimal protein extraction. In this study, we compared three published extraction procedures for subsequent applications to 2DE: the first one uses homogenization process, the second one included an additional TCA-acetone precipitation step, and finally, the third one associated grinding in liquid nitrogen (N₂) and TCA-acetone precipitation. Extracted proteins were then resolved using 1DE and 2DE. Although interesting patterns were obtained using 1DE with the three methods, only the one involving grinding in liquid N₂ and TCA-acetone precipitation led to proper resolution after 2DE, showing a good level of reproducibility at technical (85%) and biological (84%) levels. This last method is therefore proposed for analysis of gill proteomes in the shore crab.     

Isolation and characterization of dihydropteridine reductase from Pseudomonas species.

Dihydropteridine reductase isolated from the bacterium Pseudomonas species (ATCC 11299a) has been purified approximately 450-fold byammonium sulfate precipitation and diethylaminoethyl-cellulose chromatographic procedures. The preparation is at least 80% pure as judged by polyacrylamide gels. Its molecular weight was determined to be about 44,000. Both dihydropteridine reductase and phenylalanine hydroxylase activities were found to be higher in cells adapted to a medium containing L-phenylalanine or L-tyrosine as the sole carbon source than in those grown in L-asparagine. The substrate of the reductase is quinonoid dihydropteridine, and the product is tentatively identified as a tetrahydropteridine through its ability to serve as a cofactor for phenylalanine hydroxylase. The enzyme shows no marked specificity for the pteridine cofactor that occurs naturally in this organism, L-threo-neopterin. The pH optimum for the reductase is 7.2, and nicotinamide adenine dinucleotide, reduced form, is the preferred cosubstrate. Inhibition of the reduced and untreated enzyme by several sulfhydryl reagents was observed. A metal requirement for the reductase could not be demonstrated. Dihydropteridine reductase was found to be inhibited by aminopterin in a competitive manner with respect to the quinonoid dihydro form of 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine.     

Induction of xylanase by β-methylxyloside in Cryptococcus albidus

Abstract The compound β-methylxyloside (β-MX) was found to induce the production of extracellular xylanase (EC 3.2.1.8) by the yeast Cryptococcus albidus . The induction of xylanase by β-MX requires de novo protein synthesis and proceeds via de novo accumulation of translatable mRNA coding for xylanase as demonstrated by the addition of cycloheximide. In vitro translation of cellular messenger RNA followed by immunoprecipitation with xylanase-specific antibodies reveals that the stimulation leads to the appearance of xylanase-coding mRNA. In vivo labeling experiments show that the β-MX induces specifically the 48 kDa xylanase, and that the addition of xylose in the culture medium reverses the β-MX action by suppressing completely the production of xylanase by the cells.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.