Abstract

his article has two purposes: to review techniques that have been employed for assessment of CNS HIV burden to and address new quantitative RNA assays used to assess HIV load in body fluids and tissue homogenates.

Introduction

Background

HIV-1 associated dementia complex (ADC) occurs in approximately one quarter of all terminally-ill AIDS patients. While the clinical manifestations of some CNS opportunistic diseases may mimic dementia, a causal relationship between HIV and ADC was first suggested in 1985 by Shaw, et al. Using Southern blot analysis and in situ hybridization (ISH), they detected HIV DNA and RNA in cerebral tissue from a small group of patients with ADC. In one patient, the abundance of viral DNA in the brain exceeded that found in spleen or lymph nodes (Shaw et al. 1985). In the same year, Levy, et al. and Ho, et al. successfully isolated HIV from the neural tissues and cerebrospinal fluid of patients with ADC by co-cultivation with peripheral blood mononuclear cells (Levy et al. 1985; Ho et al. 1985). Subsequent clinical-pathological studies have suggested that a chronic productive HIV-1 infection in the brain (i.e. HIV encephalitis) is the pathological substrate of ADC (Navia et al. 1986; Wiley and Achim 1994).

Gap in Knowledge

There is some ongoing controversy regarding the importance of CNS viral burden in mediating neurologic disease (Wiley and Achim 1994; Brew et al. 1995; Glass et al. 1995; Johnson et al. 1996). Previous studies on relationship between CNS viral burden and ADC have not yet reached agreement. Part of this controversy is attributed to the variety of detection techniques employed in different laboratories to assess HIV burden. Even using similar methods, different study groups often generate vastly different results. A reproducible quantitative assay that can be used in any laboratory is needed to permit ready cross-study comparisons. In addition, it is known that HIV infection is not uniformly distributed throughout the CNS, but is preferentially localized to certain anatomical regions (Kure et al. 1990; Brew et al. 1995). Therefore, the extent of CNS tissue sampling is also important, and may require a consensus guideline.

Techniques I

In situ hybridization

Data: ISH has been employed by several groups to detect HIV RNA in tissue sections. Shaw, et al. applied this technique on frozen cerebral sections and found positive signals in 4 of 15 brains from patients with ADC (Shaw et al. 1985). Numerous other groups have found varying numbers of HIV probe-labeled cells in autopsied brains. Initial attempts to quantify this signal employed counting cells and grains per cells. Recently, Haase, et al. applied isotopic ISH and quantitative image analysis to assess HIV RNA load in human lymphoid tissues (Haase et al. 1996). Similar technology has been applied to SIV infection in rhesus macaques where viral RNA loads in CNS tissue sections were determined by a 4-tiered scoring system derived from counting viral RNA-positive cells per tissue sectional area (Reinhart et al. 1997).

Conclusions: None of these semi-quantitative techniques have gained widespread acceptance or been compared between laboratories.

PCR-ISH and Reverse Transcriptase (RT) in situ PCR

Data: Studies using PCR-ISH technology from different laboratories have shown conflicting results. Nuovo, et al. showed that a number of HIV DNA-positive cells was increased by 10- to more than 100-fold, compared to the standard ISH method (Nuovo et al. 1994). In this study, significant proportions of neurons and astrocytes were shown to contain viral DNA and RNA, in addition to microglia/macrophages. Using a combination of PCR-ISH, RT-PCR-ISH, and fluorescent imunocytochemistry (ICC), Bagasra, et al. concluded that HIV also infected neurons, endothelial cells, astrocytes, and oligodendrocytes (Bagasra et al. 1996). In dorsal root ganglia, Brannagan, et al. detected HIV DNA and RNA in satellite cells and occasional neurons of HIV-infected patients with peripheral neuropathy (Brannagan III et al. 1997). In contrast to the studies mentioned above, Takahashi, et al. found no convincing evidence of HIV infection in neurons, oligodendrocytes, and endothelial cells by PCR-ISH, although HIV DNA was detected in fewer than 1% of astrocytes in sections with HIV encephalitic features (Takahashi et al. 1996).

Conclusions: Potentiality of PCR-ISH technology would allow HIV detection in tissue at the level of single copy of viral DNA and concomitantly to determine its cellular localization. However, clearly great variability exists between different laboratories using this technology. Orders of magnitude differences suggest it may be a long time, if ever, before a consensus on this technology is reached.

Immunocytochemistry

Data: Detection of HIV proteins on CNS tissue sections has been achieved by ICC with antibodies to gag proteins (p17 and p24) and to transmembrane envelope protein (gp41) (Wiley et al. 1986; Sinclair et al. 1994; Achim et al. 1994). Studies from Kure, et al. and Budka, et al. had shown that a monoclonal antibody to gp41 (Clone 41.1, Genetic Systems) was most sensitive on paraffin CNS sections, compared to antibodies to p17 and p24 (DuPont) (Kure et al. 1990; Budka 1990). Our own experience confirmed this observation.

Specifically, ICC with avidin-biotin-peroxidase complex (ABC) method for gp41 (Genetic Systems) yielded many more positive cells than that for p24 (DAKO) on serial paraffin sections. Multinucleated giant cells, microglial nodules, and variable numbers of microglia and perivascular macrophages labeled intensely with the anti-gp41 antibody. Strangely, to date, no ICC has successfully detected the other portion of the envelope protein gp120 in fixed tissue (Budka 1990). Recently, a new signal amplification method was introduced into ICC protocols. This method amplifies signal via binding of tyramide to proteins near the site of peroxidase-labeled antibodies or strepavidin (von Wasielewski et al. 1997). Strappe, et al. reported a significant increase in sensitivity of ICC for p24 (DuPont) with tyramide signal amplification (TSA, DuPont) on paraffin brain sections. With microwave irradiation of brain sections in citrate buffer, the TSA method yielded higher staining intensity and up to 2-fold increases of the number of p24-positive foci, when compared to the ABC method. However, this study did not compare the sensitivity of TSA method for p24 to that of the ABC method for gp41 (Strappe et al. 1997).

Quantification of HIV-stained cells had been attempted in several studies. Vazeux, et al. assessed overall CNS viral load with 4-tiered estimation of the number of HIV-stained cells per section, and determination of the proportion of positive tissue blocks to all blocks examined in each case (Vazeux et al. 1987). Our group devised a scoring system for semi-quantification of gp41-stained cells (Genetic Systems) (Masliah et al. 1992). Three CNS regions (i.e. cortical gray matter, cerebral white matter, and deep gray matter) were assessed on paraffin sections taken from the mid-frontal gyrus and basal ganglia. We had applied this scoring system to determine the overall brain viral burden, and correlated brain viral load with histologic evidence of neocortical damage, clinical diagnosis of ADC, length of survival after AIDS diagnosis (Masliah et al. 1992; Wiley and Achim 1994; Soontornniyomkij et al. 1997). Glass, et al. also quantified gp41 immunostaining on paraffin sections of the mid-frontal gyrus and basal ganglia with a scoring system quite different from that developed by our group (Glass et al. 1995). Brew, et al. sampled brains and spinal cords systematically and extensively, and determined the proportion of p24-positive tissue sections in each case (Brew et al. 1995).

Conclusions: While the immunocytochemical semi-quantification of CNS HIV burden has shed some light on the pathogenesis of HIV encephalitis, several drawbacks are inherent in this technology. Numerous fixation parameters limit successful ICC on tissue sections, leading to false-negative and false-positive staining. Additionally, semi-quantification of morphology-based assessment (like ICC) requires extensive professional interpretation and would thus be expected to be difficult to replicate between laboratories.

Techniques II"

ELISA-based antigen capture assay

Data: Our group applied an ELISA-based antigen capture assay (ACA) (Coulter) to quantify HIV p24 protein in brain tissue homogenates (Achim et al. 1994). This quantitative technique was less sensitive than the semi-quantitative ICC for gp41 in detecting HIV proteins in brain tissue, possibly because of non-specific blocking proteins and lipids.

Conclusions: In the future the sensitivity of ELISA-based ACA may be increased by improvements in detection method (e.g. chemiluminescence) or higher affinity antibodies.

PCR and RT-PCR

Data: An assortment of PCR-based methods and quantitative analyses have been applied to assess CNS HIV load in different laboratories. Most previous studies were carried out on tissue from only one cerebral region (Boni et al. 1993;Sinclair et al. 1994; Johnson et al. 1996; An et al. 1996; Lazarini et al. 1997). Our group employed PCR on tissue from the mid-frontal cortex, white matter, and basal ganglia, and quantified HIV DNA using an enzyme oligonucleotide assay (Cellular Products) (Achim et al. 1994). We found HIV DNA was most abundant in the basal ganglia. Fujimura, et al. examined tissue from several brain regions by a quantitative PCR method, and found variable levels of HIV DNA in different regions (Fujimura and Bockstahler 1995). The viral levels in the hippocampus were 3-fold higher than those in the basal ganglia.

Conclusions: As different PCR and quantitative techniques were applied, several study groups have generated conflicting results. Limited tissue sampling may also contribute to misleading interpretation.

Quantitative HIV RNA assays

Data: Recently HIV has been quantified in plasma using a variety of highly-sensitive RNA assays based on either signal amplification (e.g., branched-DNA assay, Quantiplex HIV RNA, ChironCorporation) or template amplification (e.g., RT-PCR assay, Amplicor HIV-1 Monitor, Roche MolecularSystems, and nucleic acid sequence-based assay, NASBA HIV-1 RNA QT, Organon Teknika) (Schuurman et al. 1996). The Quantiplex assay has also been applied to lymphoid tissues (Haase et al. 1996; Harris et al. 1997). Recently, our group has employed both Quantiplex and Amplicor to determine the regional distribution of brain viral load in AIDS autopsies (Wiley et al. submitted 1997). We found a good correlation between these two techniques in quantitative performance. In addition, the HIV RNA load generally correlated well with HIV gp41 protein load assessed by our immunocytochemical scoring system.

Conclusions: Of the truly quantitative techniques, HIV RNA assays of tissue homogenates hold the greatest promise for objective cross-study comparisons.

Conclusions"

A variety of techniques to quantify HIV in the CNS have led to vastly different results in different laboratories. It is important to identify a reproducible quantitative assay that can be applied in any laboratory. Of the truly quantitative techniques, HIV RNA assays of tissue homogenates hold the greatest promise for objective cross-study comparisons. In addition, the extent of CNS tissue sampling is another issue that needs to be resolved. A consensus guideline for tissue sampling may be necessary. A wide range of studies on different patient populations can be compared systematically when the same reproducible technique and tissue sampling guideline are applied.

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