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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 53-58
Detection and Titration of Human Herpesvirus-8-Specific
Antibodies in Sera From Blood Donors, Acquired Immunodeficiency
Syndrome Patients, and Kaposi's Sarcoma Patients Using a Whole
Virus Enzyme-Linked Immunosorbent Assay
By
Louise G. Chatlynne,
William Lapps,
Michael Handy,
Yao Q. Huang,
Rizwan Masood,
Ann S. Hamilton,
Jonathan W. Said,
H.P. Koeffler,
Mark H. Kaplan,
Alvin Friedman-Kien,
Parkash S. Gill,
James E. Whitman, and
Dharam V. Ablashi
From Advanced Biotechnologies Inc, Columbia, MD; New York University
Medical Center, New York, NY; University of Southern California School
of Medicine, Los Angeles, CA; Cedars-Sinai Medical Center, UCLA School
of Medicine, Los Angeles, CA; North Shore University Hospital,
Manhasset, NY; and Georgetown University School of Medicine,
Washington, DC.
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ABSTRACT |
A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay
(ELISA) with a whole virus lysate as antigen was developed and used to
measure the seroprevalence rate and levels of IgG antibodies to HHV-8
in sera/plasma of various patient groups and blood donors. The virus
antigen was prepared from the KS-1 cell line, which produces lytic
virus, and therefore contains a broad array of viral proteins.
Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of
72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an
average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency
virus (HIV) patients were positive with an average titer of 4,000. A
study on a very limited number of serial serum samples from patients
before and after diagnosis with KS showed highly elevated antibody
titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1+ homosexual patients
contain IgG antibodies to HHV-8 suggesting that this population is at
high risk for developing KS. Antibody results correlated well with the
confirmatory immunofluorescent assays (IFA) using KS-1 cells as the
substrate. This HHV-8 IgG antibody detection ELISA is sensitive and
specific and does not cross-react with Epstein-Barr virus
(EBV) or other human herpesviruses. The results of this
HHV-8 antibody survey suggest that this rapid ELISA assay can be used
to screen large numbers of sera to find those at risk for developing
KS.
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INTRODUCTION |
KAPOSI'S SARCOMA-associated herpesvirus
(KSHV) or human herpesvirus-8 (HHV-8) was first detected in KS
lesions by representational difference analysis by Chang et
al.1 The virus has also been found in body cavity-based
lymphomas (BCL or BCBL), also called primary effusion lymphoma
(PEL).2-4 The KS-1 cell line was established from the
pleural cavity-based lymphoma of a KSHV positive, human immunodeficiency virus (HIV)-1 and Epstein-Barr virus (EBV) negative patient.4 Intracellular viral particles have been detected in the KS-1 cell line,4 and sufficient quantities of
enveloped and nonenveloped virus (1.8 × 109
particles/L) can be pelleted from culture supernatants.5
Seroprevalence reports for HHV-8 antibodies in the general population
have been described as ranging from 0% to 25% based on
immunofluorescent assays (IFA) to latent and lytic
proteins,6-10 as well as single peptide or recombinant
antigen enzyme-linked immunosorbent assay (ELISA), and immunoblot
assays.10-13 This study describes the development and use
of an ELISA for the detection of HHV-8 IgG antibodies in clinical
samples using whole virus lysate as antigen containing most, if not
all, of the viral structural and nonstructural proteins. The ELISA is
free of cross-reactivity to other human herpesviruses.
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MATERIALS AND METHODS |
Sera/plasma panels.
All samples tested for this study were from adults. Blood donor sera
were from a well-characterized panel used for control reagents and
comparison studies at Advanced Biotechnologies Inc (Columbia,
MD). HIV-1 sera/plasma samples with or without diagnosis of KS were obtained from the North Shore University Hospital
(Manhasset, NY) and the University of Southern California School of
Medicine. The HIV-1/KS samples were received either coded or uncoded.
The uncoded group was presented along with dates of diagnosis of KS and
samples of KS patients' sera before KS diagnosis are identified in
this report as "pre-KS." Coded KS sera samples from classical KS
patients without HIV-1 coinfection were obtained from the New York
University Medical Center and the University of Southern California
School of Medicine. HIV-1/non-KS samples from North Shore University
Hospital were from a characterized group of sera from the late 1980s
and early 1990s that did not develop KS on follow-up; these samples are
referred to as retrospective. Follow-up on other HIV-1/non-KS patients
was not known when the samples were submitted. All sera/plasma panels
from these sources contained coded controls from patients without HIV-1
or KS. Assay results on coded samples were identified after testing and
these identifications are reported in the Results section and
Table 1.
Development of ELISA using HHV-8 viral lysate.
The KS-1 cell line was tested to insure freedom from adventitious
agents such as mycoplasma and other herpesviruses. Supernatant fluids
from KS-1 cultures were clarified by low speed (200xg) centrifugation before pelleting in a high-speed centrifuge (19,000 rpm
in a Beckman type 19 rotor, Beckman Instruments Inc, Palo Alto,
CA). Generally, virus pellets contained approximately 2 × 109 viral particles per liter when quantified by
electron microscopy using negatively stained preparations. The average
protein concentration of these virus pellet preparations was 1.58 mg/mL. The pellets were resuspended to a concentration 200× that
of the supernate. For purified virus preparations, the virus was
subjected to isopynic centrifugation on a 20% to 50% sucrose density
gradient, and the peak fractions collected and pooled after ELISA
detection using a reference HHV-8 antibody. The virus was lysed with
Triton X-100 and diluted 200× for direct pelleted virus and
300× for purified virus, to give 3 to 5 µg/mL preparation of
protein for coating, depending on the preparation. This viral
preparation was coated onto ELISA plates (Nunc polysorb #446140;
Roskilde, Denmark). Sera or plasma at various dilutions
were allowed to react for 30 minutes with the viral lysate. The unbound
serum was then rinsed away with a washing buffer containing Tween-20.
In the next step, a goat antihuman IgG horseradish peroxidase conjugate
was added. After a final rinsing, TMB (tetramethyl benzidine) was used
to develop the color. To assess the cross-reactivity of the HHV-8 virus
preparation from KS-1 cells with EBV, an EBV ELISA performed with same
serum using the same ELISA format except that direct pelleted EBV from
the B95-8 cell line (assayed to assure it was free of type C simian
virus) was used for coating the plates instead of the HHV-8.
Development of IFA to HHV-8 late antigens using KS-1 cells.
For the IFA IgG (Advanced Biotechnologies Inc, Cat. No. 15-330-000),
KS-1 cells were washed twice in phosphate-buffered saline (PBS), placed
on slides, air dried, and fixed for 15 minutes with cold acetone. Such
preparations generally expressed between 30% to 50% late antigen
positive cells by IFA, using HHV-8 positive polyclonal human sera from
a classical KS (HIV-1 negative) patient (Fig 1). The reactivity of this serum was
also confirmed by Western blot using purified HHV-8 antigen. Test sera
or plasma at various dilutions, starting with 1:20, were placed on the
fixed cells and incubated at 37°C for 0.5 hour. After the slides
were washed with PBS, a goat antihuman IgG fluorescein isothiocyanate
(FITC) conjugate containing Evans blue as a counter stain was placed on
the cells and the slides were again incubated at 37°C for 0.5 hour.
After washing in PBS, a coverslip was added with mounting solution and
the slides were observed with a fluorescence microscope. All sera or
plasma were tested at a starting dilution of 1:20. Figure 1A shows
staining of antigen positive cells by antibody positive serum and Fig
1B is a preparation stained with a serum from a healthy donor lacking
such antibodies.
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| Fig 1.
KS-1 cells (p39) were air-dried onto slides
and fixed with acetone. Plasmas were diluted 1:20 with PBS. In (A), the
cells were stained with a plasma from a classic KS patient; in (B), with plasma from a blood donor. FITC-conjugated goat antihuman IgG was
used as the secondary antibody. Cells were photographed at original
magnification × 400. Green staining cells indicate presence of HHV-8
antigens.
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Criteria for positive samples.
Based on our negative control sera and KS patient surveys, an ELISA
titer of 1:80 was considered positive. Blood donor, KS/HIV-1, and
classic KS samples with low ELISA titers were also tested by IFA.
Samples with ELISA titers greater than 1:80 were also positive by IFA;
but samples with ELISA titers lower than 1:80 were negative by IFA (at
1:20). The majority of the samples with ELISA titers of 1:80 were
positive by IFA (at 1:20), and we therefore have selected this dilution
as the cut-off value to evaluate the results of the ELISA. Also we
found that sera assayed at concentrations less than 1:80 by ELISA and
1:20 by IFA displayed some nonspecific reactivity especially with
Fc receptors. Therefore, samples with ELISA titers below 80 and IFA titers below 1:20 are reported as negative or zero.
Cross-reactivity with EBV.
EBV IgG antibody was detected by IFA using slides coated with
P3HR-1 cells expressing EBV-viral capsid antigen (VCA)
(Stellar Biosystems, Columbia, MD). A KS serum exhibiting antibody to
EBV-VCA (titer of  1:200) was adsorbed with live P3HR-1
cells (EBV producer cell line) overnight at 4°C to remove EBV
antibodies and to test for cross-reactions with HHV-8 by IFA;
significant loss in HHV-8 antibody titer would be indicative of a
cross-reaction of EBV antibody with HHV-8.
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RESULTS |
Antigen evaluation.
Table 2 shows that the serum antibody
titers of samples tested on ELISA plates coated with direct pelleted
virus varied very little from the titers obtained with plates coated
with sucrose purified virus. No antibody titer on the same serum varied
by more than one dilution, regardless of the preparation used for coating the plate as is shown in Table 1. Neither preparation showed
consistently higher titers than the other; therefore, we chose to use
direct pelleted viral lysate as the source of antigen for the ELISA.
Cross-reactions with other herpesviruses.
Human blood donor sera specifically tested and found to have high
antibody titers to other human herpesviruses (EBV, 10 sera; cytomegalovirus [CMV], 6 sera; herpes simplex virus-1
(HSV-1), 10 sera; HSV-2, 10 sera; HHV-6, 9 sera; and HHV-7, 1 serum)
and several monkey sera including squirrel monkeys, seropositive for herpesvirus saimiri (HVS) antibody, were assayed by HHV-8 ELISA and all
were found to give no signal on our HHV-8 IgG antibody ELISA, showing
the lack of antibody cross-reactivity with these other herpesviruses.
To confirm this finding, a serum from a KS/HIV patient with an HHV-8
antibody titer of 10,000, was tested for EBV antibodies by IFA and
found to have an EBV-VCA antibody titer of 200. Aliquots of this
serum were then treated with various dilutions of EBV antigen to absorb
out the antibodies. These aliquots were then assayed with an EBV
antibody ELISA and an HHV-8 antibody ELISA. The optical density (OD)
for the HHV-8 ELISA remained the same, regardless of how much EBV
antibody had been used for the absorption; and with the EBV ELISA the
OD decreased as increasing amounts of EBV antibody was absorbed out of
the serum.
In the reciprocal ELISA experiment, HHV-8 purified virus was added at
various dilutions to aliquots of the HHV-8 and EBV antibody positive
serum. In this experiment, the OD remained constant for the EBV ELISA,
but decreased for the HHV-8 ELISA as increasing amounts of HHV-8
antibodies were adsorbed out of the serum. This indicated that no
cross-reactivity occurred between antibodies to EBV and HHV-8 antigen
or between the HHV-8 antibodies and EBV antigen.
Similar results were obtained by IFA. The EBV absorbed serum showed no
signal for EBV-VCA at a 1:10 dilution; but on KS-1 cells, it was
strongly reactive up to a dilution of 1:200. The signal did not
weaken on the KS-1 cells after the EBV had been absorbed.
Furthermore, 91 normal donors were tested and only 11% were found to
give a positive signal on this ELISA. Because antibodies to EBV are
prevalent in approximately 85% of the normal population, one would
expect to find higher percentages of positive signal of this HHV-8 IgG
antibody ELISA if it had cross-reactivity for EBV.
Correlation of HHV-8 IgG antibody as detected by ELISA and IFA.
The titers obtained by IFA correlated very well (P < .0001, r = 0.915) with the antibody ELISA for the same samples, although the
titers by IFA were consistently lower (range, 1:20 to 1:5,120) than
those obtained with the ELISA, but they were proportional with the
ELISA. Because approximately 40% of the cells in the prepared IFA
slides were antigen positive, the remaining reddish colored negative
cells in the preparation could be used as an internal control for
nonspecific staining. Nonspecific staining tends to show up as a
general yellow-green haze, but as can be seen in Fig 1, the negative
cells only fluoresce red with the counter stain, Evan's blue. The
majority of the antibody positive samples exhibited intense diffuse
staining of the entire cell, which is typical for the expression of
late antigens. However, some sera from KS patients showed strong
reactivity at the cell margin, which is typical of antibodies to
envelope proteins. Some cells also showed intense nuclear staining,
which is associated with nonstructural proteins.
Seroprevalence.
The data in Table 1 show that 93% of 72 sera tested from classic KS
and 92% of the 62 sera or plasma of KS/HIV-1 patients had IgG antibody
to HHV-8. Eighty-four percent of 31 sera tested before patients
manifest symptoms of KS (pre-KS) were positive for HHV-8 antibodies.
Antibodies to the virus could be found in the plasma of 11% of 91 blood donors tested. The group labeled "HIV-1(non-KS)" contained
sera from patients who did not subsequently develop KS; in contrast,
the follow-up in the group labeled "HIV-1 (non-KS) homosexuals"
had not been done on the patients at the time the samples were sent to
us.
Figure 2 shows that the average and median
antibody titer of classic KS patients was higher (titer range, 0 to
128,000; average, 14,111; median, 5,120) than that of KS/HIV patients
(titer range, 0 to 64,000; average, 4,000; median, 400). In contrast,
the average titers of the positive blood donors were lower than those
of KS patients (titer range, 80 to 200; average, 118; median, 100) (Fig 2). The average titer of all blood donors tested was 14 and the median
was 0.
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| Fig 2.
Bar graph of Log10 average HHV-8 IgG antibody
titers: blood donors and KS patients. Group 1, positive blood donors;
group 2, KS/HIV-1 patients; group 3, classic KS patients.
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Longitudinal study on KS/HIV patients' sera.
Figure 3 shows the HHV-8 antibody
serotiters of four KS/HIV patients followed over time. Patient no. 1 had several serum samples before KS was diagnosed, but all samples had
a positive titer. This patient developed symptoms of KS 4 months after
the last sample and died a month later; while the CD4 count
of this patient dropped from 103 to 2/mm3 from the first
sample to the last. The second patient (no. 2) was also positive for
three samples before diagnosis and showed a similar pattern of falling
CD4 counts, from 218 to 47/mm3. Patient no. 3 samples were all drawn after the patient developed KS and these sera
had the highest HHV-8 antibody titers. The last patient (no. 4) was a
seroconverter. CD4 counts on patients 3 and 4 were not
available. These preliminary samples seem to indicate that the titer
increases slowly on sequential bleedings before KS was diagnosed, and
after diagnosis, the titer increases sharply. However, to use the
antibody titer to viral proteins to predict the development of KS, many
more patients will have to be evaluated to see if this pattern holds
true.
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| Fig 3.
Longitudinal follow-up of KS/HIV-1 patients for IgG
antibody to HHV-8. Patient no. 1 ( ) was diagnosed with KS a month
after the last serum sample and died a month after diagnosis. On
autopsy, KS was found in his internal organs. Patient no. 2 ( ) was
diagnosed with KS between his fourth and fifth samples. Patient no. 3 ( ) was diagnosed with KS before the first sample. Patient no. 4 ( ) was a seroconverter, his first sample had no HHV-8 IgG titer, but
all of his subsequent samples did; he was diagnosed with KS between his
third and fourth serum samples. Vertical line represents diagnosis of
KS. See text for more details.
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DISCUSSION |
Many reports have now appeared in the literature on the seroprevalence
of HHV-8,6-15 using a variety of methods, including ELISA
or immunoblot techniques using peptides of minor capsid protein, IFA to
latency-associated nuclear antigen (LANA), and lytic antibody IFA.
Generally, the methods using the peptides and the
LANA-IFA7,11 gave lower overall results than those using
the lytic antibody IFA, which includes a step with mouse monoclonal
antihuman IgG123 (American Type Culture
Collection HF6508).6 This is probably due to
the fact that the lytic antibody IFA method presents a wider array of
antigens with which the serum antibodies may react. The results
reported here using our whole virus ELISA fall roughly between the LANA
IFA7,11 and the lytic antibody IFA6 for both
blood donors and KS patients. Lennette et al,6 using the
BCBL-1 cell line as a substrate for IFA, reported on antibodies to both
lytic and latent proteins of HHV-8 and found that nearly 25% of the
general adult population has HHV-8 antibodies, which is higher than the
11% positive blood donors found by ELISA in our study. Their higher
value may be due to nonspecific cross-reactivity. We have also obtained
cross-reactivity in our samples if they were insufficiently diluted
before testing (less than 1:20 for IFA and less than 1:80 for ELISA).
Other specific cross-reactivities with cellular antigens, such as those
of B cells, may also be responsible for the higher findings among blood
donors. However, both the present study and Lennette's find nearly all
classic or endemic KS patients without HIV were positive for HHV-8
antibodies. Lennette et al reported 100% of her classic cases
positive, and we have found only five of the 72 samples reported to us
as classic KS with no antibody. Their finding for KS/HIV seroprevalence
is higher (96%) than our finding of 92%; perhaps this difference is
due to their larger study population.
Twenty-six of our HIV-1/non-KS sera were HHV-8 antibody positive,
which is more than double the general population (11%), but lower than
the pre-KS patients (84%). The samples in this HIV-1/non-KS category
are sera from the late 1980s and early 1990s, with known histories,
including both homosexuals and intravenous drug abusers. Any patients
who were diagnosed later with KS were not included in this category,
but were included in the pre-KS group. If we did not have this
retrospective data on the sera, the percentage of positive
HIV-1/non-KS samples would have undoubtedly been higher. This is
demonstrated in the "HIV-1/non-KS homosexual" category where the
HHV-8 prevalence is 50% when the KS status was not known at the time
of testing. Subsequent follow-up found that four of the seven
homosexual patients who had antibody to HHV-8 were diagnosed with KS
within 5 years of the time the tested sera were drawn. If later serum
samples from these patients can be obtained, they will be assayed to
see if they show the same pattern of elevated antibody titers after the
development of KS as seen in Fig 3. The percentage seropositivity in
heterosexual and perhaps homosexual non-HIV-1/non-KS groups suggest
they would probably approach the same percentages as the blood donors
if larger numbers of samples were to be tested. The absence of any HHV-8 antibody in Hodgkin's patients suggests that HHV-8 plays no role
in this disease.
The peptides used for ELISA and blot techniques by other
investigators8,11-13 were carefully chosen so they did not
contain sequences that might cross-react with EBV. However, by using
discrete peptides, the assays may well miss antibodies to other
immunogenic viral proteins in the virus, especially because it is not
yet known which viral proteins are immunodominant. Despite the fact that HHV-8 shows some sequence homology with EBV,16 the
data presented here indicates that the antibodies to HHV-8 detected by
the ELISA to viral lysate are specific and do not cross-react with EBV
or other human herpesviruses. To assess the applicability of various
immunologic assays for HHV-8, a well-designed study is needed where the
same sera panel are tested by the various available assays.
The fact that classic KS patients have much higher average titers of
HHV-8 than do KS patients with HIV-1 would indicate that conditions
that allow KS to manifest itself are more likely to occur in
HIV-1-infected patients. Biggar17,18 has traced the explosive increase in the number of KS cases in acquired
immunodeficiency syndrome (AIDS) patients. Before 1980, the relative
risk of KS for all men was 0.29/100,000.17 In 1990, the
relative risk of developing KS for homosexual/bisexual men with AIDS
had risen to 106,000 times more than the non-AIDS population and for
nonhomosexual/bisexual men with AIDS, the relative risk was 13,000 above the non-AIDS population.18 Some of this increased
risk is certainly due to the impaired immune system of HIV patients and
increased exposure through risk behaviors; but a growing body of
evidence points to the increased expression of cytokines and growth
factors found in HIV-1 patients that contributes to initiation or
maintenance of KS lesions, as well as the presence of the HIV-1 Tat
protein.19-21 In general, it can be stated that those
patients coinfected with both HIV-1 and HHV-8 are at high risk for
developing KS.
The five classic KS patients and the five HIV-1/KS patients without
antibodies to HHV-8 hold open the possibility that some agent(s) other
than HHV-8 can also initiate the cascade of cytokines and growth
factors that leads to KS lesions. However, HHV-8 DNA is certainly found
by polymerase chain reaction (PCR) in nearly all classical KS
lesions,22 and perhaps these patients were not able to
mount an IgG antibody response to the virus. We have not yet addressed
the question of the presence of IgM antibodies to HHV-8, but it should
prove to be a very interesting study. It is also a possibility that
these samples were misidentified or misdiagnosed. Indeed, we received
one sample, not included in our data, that was identified as a classic
KS and had no HHV-8 antibody titer. A biopsy of this patient was
subsequently reexamined and was found to have been misinterpreted and
the diagnosis of KS was removed.
The limited preliminary data from the longitudinal studies seems to
show a pattern of HHV-8 IgG antibody titers that appears similar to
that of EBV-VCA where significantly elevated antibody titers are found
in Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC).
Elevated EBV-VCA titers have been attributed to the pathogenesis of BL
and NPC.23,24 Therefore, a significant increase in IgG
antibody to structural viral proteins suggests that HHV-8 plays a role
in the pathogenesis of KS. Consistent follow-up of HHV-8-infected
individuals for IgG antibody would add to the knowledge of its role in
HHV-8-associated malignancies.
Our HHV-8 whole virus ELISA showed elevated HHV-8 IgG antibody in the
sera of KS patients and is able to detect antibodies to HHV-8 before
patients develop KS symptoms. Because we used a whole virus lysate,
most if not all, of the latent and lytic proteins are represented, and
therefore the assay is more likely to detect most sera with antibodies
to HHV-8. The data we obtained from the HHV-8 IgG antibody whole virus
ELISA correlated very well with our IFA data. Our ELISA is specific,
reproducible, and ideal for large-scale seroepidemiologic studies, as
well as for longitudinal follow-up of KS patients and those at risk for
KS, such as HIV-1 positive homosexuals. The ease of running these assays allows not only a qualitative positive or negative reading, but
also the ability to easily titrate samples.
Based on the data presented here, we concluded that in comparison to
other alpha, beta, and gamma human herpesviruses (HSV-1, CMV, HHV-6,
HHV-7, and EBV), the prevalence of HHV-8 in the general population is
substantial (11%), but not as widespread as other human herpesviruses,
which have prevalency rates of over 80% in the general population.
Elevated IgG antibody to HHV-8 in KS patients adds to the growing body
of evidence that HHV-8 plays a role in the etiology and pathogenesis of
KS.
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FOOTNOTES |
Submitted October 30, 1997;
accepted February 12, 1998.
Address reprint requests to Louise G. Chatlynne, PhD, Advanced
Biotechnologies Inc, 9108 Guilford Rd, Columbia, MD 21046.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract