Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala
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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". (1)
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 fully characterised".
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:
1. To 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)
DNA "polymerase III" (DNA polymerase g) is present in all cells.
2. 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 line.
3. HUT-78 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.
1. Adi 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).
2. 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 pp.7415-7419, (1980).
3. F. Wong-Staal et al. A survey of human leukaemias for sequences of a human retrovirus. Nature, 302 , 626 (1983)
4. 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, 850-853.
5. 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.
6. 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.
7. 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 for Clarification
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 unanswered.
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 affect interpretation.
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 retrovirus).
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:
1. The human genome contains DNA sequences, sometimes hundreds to thousands, of many retroviral families including HIV sequences. 1,2,3
2. New retroviruses may arise by combination between retroviral DNAs, DNAs of defective retroviruses or even by the rearrangement of cellular DNA.4
3. Cells 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
4. Gallo 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 stimulated.
5. Monocytes 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
6. An 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
7. Mixed lymphocyte cultures (cocultures) lead to retroviral expression.9
8. Extracts, even from normal unstimulated cells, when added to the cultures may increase retroviral expression.10
9. All 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:
1. As 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 is unknown.11,12,13 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 agent.
2. Thanks 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:
1. The retrovirus isolated from H9 cultures/cocultures is HIV and not HTLV-1 or even a combination of the two?
2. The antibody tests based on antigens derived from H9 cultures represent reactions with HIV antigens and not antigens of HTLV-1?
3. The 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 two?
4. Could both:
(a) Gallo's failure to detect the HIV genome in the T cells of AIDS patients17,
(b) and 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 Perth Hospital
David A Causer Perth, Western Australia 6001
Tel: (08) 9224 2500
Valendar F Turner Department of Emergency Medicine
Royal Perth Hospital
Tel: (08) 9224 2664
John M Papadimitriou Department of Pathology
University of Western Australia
Stirling Highway, Crawley
Wester Australia 6009
Tel: (08) 9389 276
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2. C.L. Parravicini et al, AIDS, 2, 171 (1988)
3. M.S. Horwitz, M.T. Boyce-Jacino, A.J. Faras, J. Virol., 66, 2170 (1992)
4. H.M. Temin, Harvey Lect. 69, 173 (1974)
5. S.A. Aaronson, G.J. Todara, E.M. Scolnick, Science. 174, 157 (1971)
6. G.J. Todaro, R.E. Benveniste, C.J. Sherr, in Animal Virology, D. Baltimore, A.S. Huang, C.F. Fox, Eds (Academic Press, New York, 1976), p.369.
7. J.A. Mikovits, N. Lohrey, R. Schuloff, F. Ruscetti, VII International Conference on AIDS, Florence 16-21 June 1991. Abstracts Vol.I, p151.
8. R.B. Stricker et al, Nature 327, 710 (1987)
9. M.S. Hirsch et al, Proc. Acad. Sci. USA. 69, 1069 (1972)
10. K. Toyoshima, P.K. Vogt, Virology. 38, 414 (1969)
11. G. Lecatsas, M.B. Taylor, S.F. Lyons, B.D. Schoub, SAMJ, 69, 793 (1986)
12. D.J. Hockley, R.D. Wood, J.P. Jacobs, A.J. Garrett, J. Gen. VIROL. 69, 2455 (1988)
13. H.R. Gelderblom et al, Micron Microsc. 19, 41 (1988)
14. R.R. Dourmashkin, C.M. O'Toole, D. Bucher, J.S. Oxford, VII International Conference on AIDS, Florence 16-21 June 1991. Abstracts, Vol. 1, p.122
15. R.C. Gallo, Sci. Am. 255, 78 (1986)
16. F. Wong-Staal et al, Nature, 302 , 626 (1983)
17. J. Lauritson, New York Native, 13th June (1994)
18. S.K. Arya, R.C. Gallo, B.H. Hahn et al, Science 927-930 (1984)
Competing interests: None declared