PATHOGENETIC PROCESSES IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION 
Dorothy E. Lewis, Ph.D., Assistant Investigator 
The overall objective of the research being per- 
formed in Dr. Lewis's laboratory is to understand 
the biology of human immunodeficiency virus 
(HIV) infection. During the past year, several areas 
of investigation have been undertaken. 
I. In Vitro Transmission of HIV: Derivation of CDS"*" 
HIV-infected Cell Lines. 
Dr. Lewis originally demonstrated that CDS"*^ 
cells from most asymptomatic HIV-infected individ- 
uals are both phenotypically and functionally 
abnormal. In the past year, an in vitro system to 
study the effect of HIV on CD8^ cells has been 
explored. In this experimental system, HIV is trans- 
mitted via infected, irradiated cells to activated 
lymphocytes from the same individual. In 5-7 
days, most of the CD4^ cells are killed, concomi- 
tant with a burst in viral output. The cells remain- 
ing are predominantly CDS""^ cells, which have been 
maintained in interleukin-2 (IL-2)-containing me- 
dium for 4-5 months after initiation of cultures. 
The cells have been shown to be infected produc- 
tively with Hiy as detected by p24 antigen produc- 
tion, in situ hybridi2ation, and electron microscopy. 
The cells are T cells, as detected by anti-CD3 and 
T cell receptor (TCR) staining; most are DR^ and 
CDSS"*", but only a few are IL-2 receptor positive. 
CD57 is not detectable in these cell lines. Function- 
ally the cells are not as responsive to T cell signal- 
ing, and, in most cultures, T cell activation has a 
detrimental effect on cellular viability. Experiments 
are in progress to determine whether a CD4 mes- 
sage exists in these cell lines and whether transmis- 
sion of HIV requires CD4 or is mediated by some 
other molecule, such as lymphocyte function anti- 
gen 1 (LFA-1). 
II. Anti-HIV Effect of Plant Phospholipids. 
Dr. Lewis's laboratory also has examined the ef- 
fects of certain soybean-derived phospholipids on 
HIV production in vitro. The compounds have 
proven effective in reducing HIV viral levels in 
tumor cell lines and in in vitro-iniect^d human 
lymphocytes, as detected by p24 antigen ELISA. Un- 
saturation at the Sn 2 position of the fatty acid 
chain is required for activity. At high concentrations 
(100 |jlM), the compounds are toxic to cells in 
vitro; but at lower concentrations (25 \xM), the 
compounds reduce HIV production 50-80%, with 
minimal toxicity. Cellular proliferation is slowed at 
these concentrations, but there is a preferential ef- 
fect on HIV-infected cell lines. The compounds do 
not prevent syncytia formation and require several 
days to reach maximal effect. The compounds can 
be washed out, and viral production is still reduced 
up to four days after elimination of the compounds, 
suggesting a long-lasting effect. The compounds re- 
duce HIV message production, as detected by in 
situ hybridization. The compounds do not inhibit 
the p24 antigen ELISA determination per se. 
In preliminary in vivo animal toxicity tests, the 
compounds are not toxic in gram dosages. The 
compounds also have been shown to kill multire- 
sistant tumor cells preferentially in vitro, presum- 
ably via accumulation of toxic fatty acid products 
after phosphorylase A^ action. Experiments are in 
progress to determine the mode of action of these 
drugs on HIV production. 
III. In Situ Hybridization Detection Using Confocal 
Microscopy. 
Most investigators have suggested that very few 
transcriptionally active HIV-infected cells are pres- 
ent in vivo. This is based on in situ hybridization, 
using ^^S-labeled HIV RNA and conventional mi- 
croscopy as the detection system. This is difficult to 
comprehend, because of the massive CD4''^ cellular 
depletion that occurs and the immunodeficiency 
that is probably responsible for most of the pathol- 
ogy. There are models to explain the depletion, in- 
cluding syncytia formation or cytotoxic T cell or 
ADCC (antibody-dependent cellular cytotoxicity) 
killing of infected cells; however, none of these 
mechanisms have been shown to be significant in 
vivo. Dr. A. S. Fauci has recently presented data 
from four acquired immune deficiency syndrome 
(AIDS) patients suggesting that 1 per 100 or 1 per 
1,000 CD4+ cells contain HIV DNA, as detected by 
polymerase chain reaction amplification. Dr. Lewis 
and her colleagues have coupled conventional ^^S- 
based RNA in situ hybridization with an alternative 
detection system, the confocal laser digital imaging 
microscope, which enhances in situ detection 
~100-fold. The ability to focus the laser light en- 
hances contrast so that the background, which is in 
the same plane as the cells, disappears and the re- 
flectance image, which is above the plane of the 
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