THE CELL LINES HUT102 AND HUT78
In 1980, describing the establishment of these two
cell lines Adi Gazdar et al wrote:
"Because T cells failed to replicate in unsupplemented growth
medium, one or more mitogens were added to replicate flasks. PBMCs from patient 1 with SS replicated
after the addition of Con-A (10 mg/ml), and lymph node cells
from patient 2 with MF proliferated in the presence of LyCMM (20%). Properties of the cultures, named HUT78 and
HUT102, respectively, are presented below.
While mitogen-induced proliferation was immediate, growth rates varied,
with periods of vigorous growth (tenfold increases in cell numbers/week)
alternating with periods of slow growth and cell death (crises). Cells surviving the second crisis (after
approximately 12 wk of culture) have proliferated vigorously and continuously
for more than 52 wk without requiring mitogen supplementation; however, there
was an absolute requirement for the mitogens during the first 10-12 wk".
In another paper published by Gazdar and his
associates including Gallo, in the same year one reads: "Cell line HUT102 was derived from the
patient’s lymph node biopsy; CTCL-3 was established a year later from his
peripheral blood cells. Both have been
No mention is made of the HUT78 cell line. Describing the isolation of HTLV from the
HUT102 line, they wrote: "From
passage 4 to passage 50, the production of virus particles required induction
with 5-iodo-2'-deoxyuridine (IdUrd).
However, constitutive production occurred by passage 56 and has
persisted up to and including the current passage 200…. Peak virus budding and
subsequent release into the extracellular fluid, as measured by electron
microscopy and DNA polymerase activity, occurred 24 hr and 72 hr, respectively,
after induction by IdUrd." (2)
In 1983 Gallo wrote: "Another cell line HUT78, of a mature T cell that express no
HTLV antigens or viral particles contained HTLV proviral sequences". (3)
In a paper published in 1986, Gallo and his
colleagues gave no data but stated:
"uninfected HUT-78 and JM cells do not express any RT activity in
the culture supernatant before or after stimulation with PHA." (4)
This claim is at odds with the following:
determine the RT activity in HUT-78 cells, Gallo used the template-primer
An.dT. As far back as 1974 Gallo
wrote: "In addition, Fridlender et
al discovered another DNA polymerase activity in HeLa cells and later in
PHA-stimulated human lymphocytes. They
named it R-DNA polymerase [DNA polymerase III] because under certain assay
conditions it best utilised polyadenylic acid [(A)n] as a template
in synthesising polydeoxythymidylate [(dT)n] a property it shared
with viral reverse transcriptase and with a reverse transcriptase in human
leukaemic cells." (5)
III" (DNA polymerase g) is present in all cells.
In 1973 cancer researchers, based mainly on the detection of
RT activity in leukaemic cells, concluded:
"Thus for leukaemia, breast cancer, and now for human lymphomas,
including Burkitt's disease, the evidence for involvement of an RNA tumour
virus in human neoplasia is becoming more compelling." (6)
HUT-78 is a leukaemic cell
was established from a patient with mature T4-cell leukaemia, a disease which
Gallo claims is caused by HTLV-1.
Since at present it is common knowledge that RT
activity is no more specific to retroviruses then the enzyme ATPase, which
before the 1970's was used to detect and quantify retroviruses, the reported
lack of RT activity in HUT-78 is difficult to understand.
In one of the four 1984 papers published in Science
where Gallo and his colleagues described the isolation of HIV, he wrote: "One neoplastic aneuploid T-cell line,
derived from an adult with lymphoid leukaemia, was found to be susceptible to
infection with the new cytopathic virus isolates….The cell line HT was tested
for HTLV before being infected in vitro and was negative by all criteria
including lack of proviral sequences.
Continuous production of HTLV-III was obtained after repeated exposure of
parental HT cells (3 x 106 cells pretreated with polybrene to
concentrated culture fluids harvested from short-term cultures of T cells
(grown with TCGF) obtained from patients with AIDS or pre-AIDS….The parental
T-cell population was extensively cloned in order to select the most permissive
clones that would preserve high rates of growth and virus production (for
example, see clones 4 and 9 in Table 1).
A total of 51 single-cell clones were obtained by both capillary and
limited dilution techniques using irradiated mononuclear cells from peripheral
blood of a healthy donor as a feeder". (7)
In other words the HT cell line from which
"HIV" was isolated, but not the parental cell line, was stimulated
with many factors including highly oxidised irradiated death cells. Furthermore, thanks to the Gallo probe we
know that the HT (H9) cell line is no other than HUT78.
The realisation that HT (H9) and HUT-78 are one and
the same cell line and its use in
"HIV" research creates several problems which we addressed in a
letter (see below) sent to Nature and Science.
Both journals rejected it.
F Gazdar, Desmond N Carney, Paul A Bunn, Edward K Russell, Elaine S Jaffe,
Geraldine P Schechter & John G Guccion.
Mitogen Requirements for the In Vitro Propagation of Cutaneous T-Cell
Lymphomas. Blood, Vol.55, No.3 (1980).
Bernard J Poiesz, Francis W Ruscetti, Adi F Gazdar, Paul A
Bunn, John D Minna & Robert C Gallo.
Detection and isolation of type C retrovirus particles from fresh and
cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA, Vol.77, No.12
F. Wong-Staal et al. A
survey of human leukaemias for sequences of a human retrovirus. Nature, 302 , 626 (1983)
Zagury, D., Bernard, J., Leonard, R. et al. (1986), Long term
cultures of HTLV-III infected cells: a model of cytopathology of T-cell
depletion in AIDS. Science, 231,
Brian H Lewis et al. (1974).
Human DNA Polymerase III (R-DNA Polymerase): Distinction from DNA
Polymerase I and Reverse Transcriptase.
Science, Vol 183, 867-869.
D Kufe et al. (1973).
Evidence for the involvement of RNA tumour viruses in human lymphomas
including Burkitt's disease. In :
Possible Episomes in Eukaryotes, Editor Luigi G Silvestri, North-Holland
Publishing Co., Amsterdam – London.
Popovic M, Sarngadharan MG, Read E, Gallo RC. (1984). Detection, Isolation and Continuous
Production of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and
Pre-AIDS. Science 224: 497-500.
Gallo's HIV Isolation : Unanswered Questions
and a Plea
The enquiries on some of Gallo's scientific contributions
(Science, July 15, 1994) have clarified the ethical issues regarding his
isolation of HIV, however some basic scientific questions still remain
For the isolation and continuous production of HIV,
Popovic, Gallo and their colleagues used a T-cell line, HT. By culturing this lymphoid cell line with
tissue derived from AIDS patients, they claimed to have isolated and continuously
produce HIV. This "parental"
HT cell line "was tested for HTLV before being infected in vitro and was
negative by all criteria including lack of proviral sequences". Irrespective of what the term
"HTLV" includes, "all criteria" cannot be more than reverse
transcriptase (RT), retroviral particles as seen with the electron microscope,
immunofluorescence reactions with patients' sera and proviral nucleic acid
detection by hybridisation.
While the HIT cultures/cocultures used to isolate HIV were
subjected to a number of stimulants including polybrene and radiation, they
were compared only to the "parental" HT cell line which was merely
"left alone" to serve as control.
Furthermore, unlike the "parental" HT culture, all the HT
cultures/cocultures, including the cultures infected with "pure" HIV,
contained, in addition to HIV, non-HIV material (including cellular and non
cellular products), which originated from the AIDS patients. These are all significant variables and
As far back as 1972, Gallo himself was the first to show
that RT which in sucrose density gradients bands at 1.16g/ml can be found in
"PHA stimulated (but not unstimulated) normal human blood
lymphocytes". Elsewhere he
states: "that human leukaemia
carried type C virus-related information.
An examination of blood samples from normal donors showed the absence of
any specific R-MuLV-related information although this clearly depended on the
metabolic state or the type of cells used as control since these sequences were
found in phytohemagglutinin-treated human blood lymphocytes". (MuLV = Murine leukaemic virus, a
In the proper evaluation of a scientific experiment,
appropriate controls are a basic requirement.
In the case of retrovirus detection, controls are an essential element
for the reasons listed below:
human genome contains DNA sequences, sometimes hundreds to thousands, of many
retroviral families including HIV sequences. 1,2,3
retroviruses may arise by combination between retroviral DNAs, DNAs of
defective retroviruses or even by the rearrangement of cellular DNA.4
in culture, sooner or later, spontaneously start to release retroviruses. The appearance can be accelerated and the
yield increased a million-fold by stimulating the cultures with a number of
agents, including those used in the HT AIDS cultures/cocultures.5 In fact, as far back as 1976, George Todaro,
a well known retrovirologist, and his colleagues at the laboratory of Viral
Carcinogenesis, National Cancer Institute, emphasised "that the failure to
isolate endogenous viruses from certain species may reflect the limitations of in
vitro cocultivation techniques".6
and Montagnier agree that no HIV (RT, retrovirus-like particles, antigen
antibody reaction, HIV genomic sequences) can be detected in T-cell
cultures/cocultures with tissue from AIDS patients, unless the cultures are
from HIV+ patients in which no HIV DNA can be detected, even by the
use of polymerase chain reaction, become positive for HIV RNA after
cocultivation with normal ConA activated T cells, leading the authors to
conclude that "HIV expression can be activated in monocytes which lack
detectable HIV DNA".7
antibody found in AIDS patient sera binds to an 18kD antigen in: HIV infected peripheral blood T cells and
HIV infected HUT78 cells and because of this, the antigen is considered to be
an HIV protein. However, PHA stimulated
normal, uninfected peripheral blood T cells and PHA stimulated non infected
HUT78 cells, but not unstimulated peripheral blood T cells and HUT78 also have
a protein of the same molecular weight which react with AIDS patient sera.8
lymphocyte cultures (cocultures) lead to retroviral expression.9
even from normal unstimulated cells, when added to the cultures may increase
the cultures/cocultures from tissues from AIDS patients contain non HIV
material including cellular derivatives.
Regarding the HT cell line and thus its clone (H9)
used by Gallo and his colleagues for the "isolation" and
"continuous" production of HIV, one must also keep in mind that:
far back as 1972, Gallo and Todara, presented evidence "that human
leukaemia cells contain reverse transcriptase with antigenic properties of the
polymerase of type C virus".
Before the AIDS era many researchers detected "retroviral"
particles in several malignant tissues.
HT is a leukaemic cell line.
Gallo, Popovic and their colleagues reported virus-like particles with
morphological characteristics which they ascribed to HIV in the HIV infected HT
cell line and no particles in the "parental" HT line. However, many authors have observed that the
HIV infected HT (H9) cell line contains, in addition to the particles with
morphological characteristics ascribed to HIV, several other viral-like
particles with morphological appearances unlike those of HIV and whose nature
Moreover, British and American researchers presented evidence that
non-HIV infected H9 (HT) cultures contain budding virus-like particles.14 These two omissions cast doubt on the
reliability and significance of Gallo's electron microscopy studies and his
conclusion that the detection of virus-like particles with the morphological
characteristics ascribed to HIV is proof of an externally derived infectious
to the "Gallo probe", it became known that the HT line is in fact
HUT78, a cell line established from a patient with mature T4-cell leukaemia, a
disease which Gallo claims is caused by HTLV-1.15 Although Gallo and Popovic claimed to have
tested the "parental" (HUT78) cell culture for HTLV before being
infected in vitro and it "was negative by all criteria including lack of
proviral sequences", one year earlier, in 1983, Gallo claimed to have
shown that HUT78 "contained HTLV proviral sequences".16
Gallo and his colleagues "isolated" HIV by
density gradient banding, a method which cannot distinguish between
retroviruses. Since the H9 (HUT78)
cultures contain a retrovirus, HTLV-1, even when not infected with HIV (that
is¸ cultured with HIV infected tissues from AIDS patients or contaminated with
LAV), how can one be confident that:
retrovirus isolated from H9 cultures/cocultures is HIV and not HTLV-1 or even a
combination of the two?
antibody tests based on antigens derived from H9 cultures represent reactions
with HIV antigens and not antigens of HTLV-1?
retroviral genetic sequences derived from the H9 cell line which are used to
synthesise both the probes and primers for the hybridisation/PCR studies are
indeed genomic sequences of HIV and not those of HTLV-1 or a combination of the
failure to detect the HIV genome in the T cells of AIDS patients17,
his claim that the nucleic acid sequences of "the genome of HTLV-III are
homologous to the structural genes (gag,
pol and env)" of HTLV-1.18
be explained by the
inadvertent use of the HTLV-1 genome (or a combination of the HIV and HTLV-1
genome), instead of HIV in his hybridisation studies?
The above questions must be answered not only because they
address basic scientific problems but, and most importantly, because they have
profound diagnostic and clinical implications.
Eleni Papadopulos Department
of Medical Physics
Bruce Hedland Thomas Royal
David A Causer Perth,
Western Australia 6001
(08) 9224 2500
Valendar F Turner Department
of Emergency Medicine
(08) 9224 2664
John M Papadimitriou Department
of Western Australia
(08) 9389 276
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Aaronson, G.J. Todara, E.M. Scolnick, Science. 174, 157 (1971)
Todaro, R.E. Benveniste, C.J. Sherr, in Animal Virology, D. Baltimore, A.S.
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Wong-Staal et al, Nature, 302 , 626 (1983)
Lauritson, New York Native, 13th June (1994)
Arya, R.C. Gallo, B.H. Hahn et al, Science 927-930 (1984)
Competing interests: 2.