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EXPERIMENTAL RESULTS
Overview: A non-flow vessel or cell included a pair of platinum
electrodes 1 mm apart inserted into a well 1.56 mm in length and 8.32 mm
in depth. The non-flow vessel was connected to a direct current source
capable of creating an electric field at a constant voltage and constant
amperage. Into this well was laced a suspension of the human immunodeficiency
virus type 1 (HIV-1) at a concentration of 1,000,000 infectious particles
per ml. An aliquot of approximately 10 ul of the virus suspension was placed
into the well. Thereafter, the viral suspension was exposed to direct currents
ranging from 0 microamps (uA) for up to 12 minutes, to 100 microamps for
up to 6 minutes. Intermediate currents of 25, 50 and 75 microamps were
used to expose similar viral aliquots. After exposure of the viral suspension
to electric currents, the contents of the non-flow vessel were removed
and placed into sterile microtubes. 5 ul of each sample were removed and
diluted with 95 ul tissue culture medium supplemented with 10% fetal calf
serum (FCS. unborn calf blood)
In Experiment 1, the resuspended and treated viral stocks were incubated
with a human T lymphoblastoid cell line named CEM-SS. This cell line, upon
exposure to HIV-1, forms syncytia (giant cells). It is well documented
that the viral titer (amount) used is directly correlated with the number
of syncytia formed. Therefore, evaluation of infectivity of HIV-1 can be
used with this assay. In contrast, Experiment No. 2 used a differnet human
T lymphoblastoid cell line named H9. This cell line, in contrast to CEM-SS
cells, produces, upon exposure to HIV-1, many viral particles. The amount
of virus produced is proportional to the amount of virus to which the cells
are exposed. Therefore, quantitation of viral particles, or more commonly
associated viral protein (in this case reverse transcriptase), can be used
as an index of viral infection. In both assays, the CEM syncytia forming
assay and the H9 viral protein assay, similar type results were obtained.
That is, with the CEM cells, although syncytium formation and quantitation
is preferrable, one can quantitate the HIV-1 associated protein (reverse
transcriptase) activity and conversely with the H9 cells, although reverse
transcriptase quantitation is preferred, one can quantitate giant cell
(syncytia) formation. Both of these assays are widely used as reproducible
measures of viral infection and can be used to determine if alterations
in viral infectivity as a product of this electrical treatment can be detected.
Experiment #1
Approximately 100,000 CEM-SS cells per sample were incubated with a
treated or untreated (control) viral aliquot for up to 4 days. The cells
were placed into microtiter plate wells and monitored for formation of
syncytia every 24 hours by microscopic observation. In a standardized fashion,
as it has been reported in the literature and is currently being conducted
in many laboratories, the number of syncytia at 3 and 4 days was determined.
Table 2 summarizes the results from a representative experiment using this
assay. As can be noted, the number of syncytia formed was inversely proportional
to the amount of electric current. That is, additionally, with increased
current (100 vs 50 uA) there was a reduction in the number of syncytia
formed. These results and those of additional experiments using the CEM-SS
cell line indicate a consistent finding that electrical treatment of the
RF strain of HIV-1 attentuates the virus potential for inducing syncytium
formation in this cell line.
Experiment #2
A separate and independent assay to determine the ability of electric
current to alter HIV-1 infectivity using H9 cells was employed. The basic
strategy was similar to that used for the CEM cells with the exception
that the initial suspension of treated and controlled (non-treated) viral
stock was incubated with 100,000 H9 cells for 2 hours at 37 degees Celsius.
Thereafter, the cell virus suspensions were further diluted to 5 ml in
standard tissue culture medium. The cell-viral suspensions were then incubated
for up to 14 days at 37 degrees Celsius with 5% carbon dioxide. At 3 day
intervals (beginning at day 2), aliquots of cell suspension were removed
from each sample. The aliquots were centrifuged at 1,000 rpm for 5 minutes
in order to pellet the cells. After centrifugation, the supernatant and
cell pellets were seperated. The supernatant was cyropreserved for subsequent
reverse transcriptase assay and the cell pellets were resuspended in fixatives
and maintained in a tissue bank for additional studies employing in situ
hybridization and immunocytochemistry to detect qualitatively and semi-qualitatively
viral infection by HIV-1. At the end of each experiment, the supernatant
samples from each of the tests and time points were examined using standard
reverse transcriptase assay. The results of the representative experiment
are shown in Table 3. The results of this experiment indicate the ability
of HIV-1 to infect H9 cells is attenuated by the magnitude of the electrical
currents to which the virus is exposed. Additionally, at lower current
magnitude, but with prolonged exposure time, attenuation of viral infectivity
is achieved. That is, analogous to the results observed using syncytium
formation and the CEM-SS cell line, either increased current or increased
duration of exposure time was inversely proportional to the amount of reverse
transcriptase produced by the cell line.
In conclusion, these experiments which have been repeated several times, and those using the CEM-SS cell line, indicate at a statistically significant level that direct electrical current at biocompatible amperages for discrete exposure time intervals can attenuate the ability of HIV-1 to infect normally healthy cells which are susceptible to the HIV-1 AIDS virus.
TABLE 2
Syncytium Formation
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Dilution of virus (Number of Syncytia)
-------- --------------------------------
1:20 TNTC TNTC 28 66 15
1:40 TNTC 175 22 42 7
1:80 TNTC 90 20 25 4
1:160 180 44 9 9 2
1:320 115 28 4 6 0
1:640 70 10 0 2 0
1:1280 40 7 0 0 0
1:2560 28 4 0 0 0
1:5120 15 2 0 0 0
1:10,240 10 1 0 0 0
1:20,480 4 0 0 0 0
------ ----- ----- ----- ------
0uA 25uA 50uA 75uA 100uA
---------------------------------------------
(TNTC=too numerous to count)
TABLE 3
Reverse Transcriptase Activity
(count per million x .001)
-------------------------------------
Days of Incubation
------------------
uAmps/Time(min.) 2 days 4 days
---------------- ------- ------
0/6 0 13.8
0/12 0 11.7
50/3 0 9.1
50/6 0 9.1
50/12 0 4.8
100/3 0 5.7
100/6 0 3.6
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