To Resign over an Editorial Decision You Disagree With

What would you do if you were serving on the editorial board of a scientific journal which had just published a manuscript that you knew was seriously flawed. Moreover, you knew that publication of the manuscript might seriously undermine global public health? That was the circumstance of cell biologist Klaudia Brix, Professor of Cell Biology, Jacobs University Bremen, Germany, when, in 2011, the Italian Journal of Anatomy and Embryology (IJAE)—the official publication of the Italian Society of Anatomy and Histology—published a paper by infamous AIDS denialist, Peter Duesberg, which reiterated his already discredited argument that HIV (the human immunodeficiency virus) does not cause AIDS (1). Brix resigned in protest from the IJAE editorial board. But why is that noteworthy? Remarkably, she was, for a time, the only member of the journal’s 13-person editorial board to do so, despite other members having similar misgivings over the decision to publish the paper. Afterwards, Heather Young, an anatomy and neuroscience researcher at the University of Melbourne, likewise resigned from the IJAE editorial board. Here is the background to this state of affairs.

Peter Duesberg is not the only AIDS denialist. However, he has been the most infamous of the AIDS denialists. HIV is a retrovirus, and Duesberg is the only AIDS denialist who also happens to be an expert retrovirologist. In fact, Duesburg was at one time a highly esteemed retrovirologist. In 1985 he was elected to the U.S. National Academy of Sciences; mainly for his 1970 discovery, with Peter Vogt, of the first known retroviral oncogene—the Rous sarcoma virus v-src.

AIDS denialist, Peter Duesberg

Duesberg first put forward his denialist view in a 1987 paper in Cancer Research (2), which asserted that AIDS results from drug abuse, parasitic infections, malnutrition, and antiretroviral drugs. In Duesberg’s assessment, HIV is just another opportunistic infection. He has maintained that view since then, despite overwhelming evidence to the contrary. Consequently, he is looked upon as a pariah by the scientific community.

Even though Duesberg’s denialist views have been rejected by AIDS experts, Duesberg’s standing as a retrovirologist enabled him to yet influence some public health officials. In 2000, Duesberg was serving on a panel advising Thabo Mbeki (President of South Africa after Nelson Mandela) on how to manage the South African AIDS outbreak. Although Mbeki was an able and intelligent leader, he accepted Duesberg’s denialist view that HIV was not the cause of the South African AIDS epidemic. Thus, Mbeki allowed the South African outbreak to get completely out of control (3). Two independent studies later concluded that over 300,000 South African AIDS deaths would not have occurred if the Mbeki government’s public health policy had not followed the denialist view. Many thousands of South African AIDS victims, including infants, would have been spared infection if the government had publicized that AIDS is an infectious disease, and if it had made antiretroviral drugs available, particularly to pregnant women (1).  See Asides 1 and 2.

[Aside 1: The reasons why Mbeki assented to Duesberg’s denialist view are not clear. One possibility is that Mbeki held strong anti-colonialist and anti-West sentiments—born of having come of age during South Africa’s apartheid era—which led him to see his country’s AIDS crisis as a means by which the West sought to exploit his nation. To that point, he may have doubted the efficacy of expensive antiretroviral drugs, which were available only from large Western pharmaceutical companies. Moreover, the cost of treating the 5 million or more HIV-infected South Africans with those drugs would have exceeded the annual health department budget of his poverty-stricken nation by a factor of ten. Mbeki did accept that AIDS is the consequence of a breakdown of the immune system. But he was inclined to believe (or at least claimed) that poverty, bad nourishment, and ill health, rather than a virus, led that breakdown; a stance that enabled him to justify treating poverty in general, rather than AIDS in particular. Duesberg defended Mbeki in his publications, denying that hundreds of thousands of lives were lost in South Africa because of the unavailability of anti-retroviral drugs. But in 2002, after Mbeki suffered political fallout from the consequences of having acceded to Duesberg’s views, he tried to distance himself from the AIDS denialists, and asked that they stop associating his name with theirs.]

[Aside 2: The 2000 International AIDS Conference was taking place in Durban (a city in the South African province of KwaZulu-Natal) at the same time that Mbeki’s AIDS panel was convening in Johannesburg. Consequently, the denialist views expressed by Mbeki’s panel were also being heard in Durban. This prompted the so-called “Durban Declaration,” signed by over 5,000 scientists and physicians, and published in Nature, which proclaimed that the evidence that HIV causes AIDS is “clear-cut, exhaustive and unambiguous”.]

Well before Duesberg submitted his paper to IJAE, the arguments put forward in the paper had already been appraised and rebuffed by the scientific community. Indeed, the paper had previously been rejected by several other journals. The first submission was to the Journal of Acquired Immune Deficiency Syndromes (JAIDS), a peer-reviewed medical journal covering all aspects of HIV/AIDS. The JAIDS editors found that Duesberg’s contentions in the paper were based on a selective reading of the scientific literature, in which he dismissed all the vast evidence that HIV is the etiologic agent of AIDS. Not surprisingly, JAIDS rejected the paper, with one peer reviewer even warning that Duesberg and co-authors could face criminal charges if the paper were published.

After JAIDS rejected the paper, Duesberg  submitted a revised version to Medical Hypotheses (4). Like the original paper sent to JAIDS (as well as the version accepted by IJAE), the paper submitted to Medical Hypotheses contained data cherry-picked to cast doubt on HIV as the cause of AIDS. Nonetheless, Medical Hypotheses accepted the paper. However, the paper never went to press. But first, what was the explanation for the seemingly bizarre decision to accept the paper?

The answer laid in the fact that Medical Hypotheses was the only journal of its parent publisher, Elsevier, that did not use peer review; instead relying on its editorial board to select papers for publication. In any case, before the accepted paper went to press, prominent AIDS researchers, including Nobel laureate Francoise Barre-Sinoussi (co-discoverer that HIV is the cause of AIDS, 5), complained to Elsevier that the paper would have a negative impact on global healthcare, and requested that the paper be withdrawn.

Elsevier responded to these protests by asking the editors of another of its journals, The Lancet, to oversee a peer review of the paper. The Lancet editor sent the paper to five external reviewers, each of whom found that it contained numerous errors and misinterpretations, and that it could threaten global public health if it were published. Elsevier then permanently withdrew the paper.  Elsevier also instituted a peer-review policy at Medical Hypotheses (and fired the journal’s editor, who resisted the change).

The Medical Hypotheses incident resulted in more notoriety for Duesberg when the University of California, Berkley, where Duesberg is still a professor of molecular and cell biology, bought charges of misconduct against him for making false scientific claims in the paper, and for a conflict-of-interest issue. Apropos the latter, Duesberg did not reveal that co-author David Rasnick had earlier worked for Matthias Rath, a German doctor and vitamin entrepreneur, who sold vitamin pills as a therapy for AIDS. Duesberg was later cleared of both charges. But the next iteration of paper, to IJAE, did not respond to these allegations.

Duesberg regarded Elsevier’s actions as another example of “censorship” imposed by the “AIDS establishment.” Undeterred however, he submitted a revision of the paper to IJAE, which that journal then accepted, prompting Klaudia Brix and Heather Young to resign from that journal’s editorial board. The IJAE paper contained the same cherry-picked data and discredited assertions that were rejected earlier by JAIDS and Elsevier.  Moreover, publication of the paper still posed a threat to global public health. What then was behind the IJAE decision to publish?

Here is what happened. The paper was “peer-reviewed” by IJAE, but by only two reviewers; one of whom was Paolo Romagnoli, the IAJE editor-in-chief, who is neither a virologist or an epidemiologist but, instead, a cell anatomist. Consequently, the paper underwent only one external review, and there is no information regarding whether the lone external reviewer was an AIDS expert. One board member (who did not resign) later commented: “Only one [external] reviewer in my mind is not enough for manuscripts of a sensitive nature… (6)” [But this comment too is a bit troubling. Bearing in mind that the paper contained numerous errors and misinterpretations, would those have been okay if the paper were not of a “sensitive nature”?]

One also might ask why a journal that specialized in anatomy and embryology would consider a paper about the cause of AIDS. To that point; Klaudia Beix gave, as a reason for her resignation from the IJAE board, her belief that a journal should function within its scientific “scope” (6). So how did Romagnoli, the IJAE editor-in-chief, justify his decision to consider the paper?  He did so by asserting that it dealt with “issues related to the biology of pregnancy and prenatal development and with the tissues of the immune system (6).” But despite Romagnoli’s contention, the only mention of embryology in the paper was a short comment in the abstract: “We like to draw the attention of scientists, who work in basic and clinical medical fields, including embryologists, to the need of rethinking the risk-and-benefit balance of antiretroviral drugs for pregnant women, and newborn babies (1).”

As for Romagnoli’s reliance on only two reviewers, he justified that stance on the fact that the reviewers had concurring opinions. Moreover, he claimed that his criteria for selecting reviewers—apparently irrespective of their expertise—was to choose individuals (himself included) who he believed would not reject a paper merely because it challenged prevailing opinion.

But is there any possibility that Duesberg might be right? The answer is virtually none whatsoever. An earlier post noted: “…the evidence that HIV causes AIDS is, without exaggeration, overwhelming. Consider just the following. Data from matched groups of homosexual men and hemophiliacs show that only those who are infected with HIV ever develop AIDS. Moreover, in every known instance where an AIDS patient was examined for HIV infection, there was evidence for the presence of the virus. These data have been available for years, and Duesberg should have been aware of them. What is more, there has been the enormous success of antiretroviral therapy in changing AIDS from a nearly invariably fatal disease, into a manageable one, for many HIV-infected individuals (3).”

Even so, Duesberg is not regarded as a pariah by AIDS experts merely because his views concerning the connection between HIV and AIDS challenge accepted wisdom.  Instead, as asserted by Harvard University AIDS epidemiologist, Max Essex, Duesberg has sustained a “dangerous track of distraction that has persuaded some people to avoid treatment or prevention of HIV infection (6)”.

A scientist mounting a long-time challenge to the “establishment,” and being ridiculed for his views, before eventually being vindicated, makes for a very good story. However, such instances are very rare. Exceptions include Howard Temin (7) who hypothesized reverse transcription, and Stanley Pruisner (8) who hypothesized prions—infectious agents that contain no nucleic acid genome. Both researchers had to endure widespread ridicule for several years. But, and importantly, irrefutable evidence eventually accumulated to support their hypotheses. And, finally, both were awarded Nobel Prizes. But Duesberg has not been vindicated and, almost certainly, he  never will be.

References:

1. Duesberg PH, et al., 2011. AIDS since 1984: no evidence for a new, viral epidemic – not even in Africa. Italian Journal of Anatatomy and Embryololgy 116:73–92. http://fupress.net/index.php/ijae/article/view/10336/9525

2. Duesberg P, 1987. Retroviruses as carcinogens and pathogens: expectations and reality. Cancer Res. 47:1199–220. PMID3028606.

3. Thabo Mbeki and the South African AIDS Epidemic, Posted on the blog July 3, 2014.

4. Duesberg PH, et al., 2009. WITHDRAWN: HIV-AIDS hypothesis out of touch with South African AIDS – A new perspective. Medical Hypotheses. doi:10.1016/j.mehy.2009.06.024. PMID19619953.

5. Who Discovered HIV, Posted on the blog, January 24, 2014.

6.  Corbyn Z. 2012. Paper denying HIV–AIDS link sparks resignation: Member of editorial board quits as editor defends publication. Nature doi:10.1038/nature.2012.9926.

7. Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.

8. Stanley Pruisner and the Discovery of Prions: Infectious Agents Comprised Entirely of Protein, Posted on the blog December 15, 2016.

 

Tony Hunter and the Serendipitous Discovery of the First Known Tyrosine Kinase: the Rous Sarcoma Virus Src Protein

In 1911 Peyton Rous, at the Rockefeller Institute, discovered the Rous sarcoma virus; the first virus known to cause solid tumors (1). Although Rous’ eponymous virus also would be known as the prototype retrovirus, his discovery generated only scant interest at the time, and would not be recognized by the Nobel Committed until 65 years later! [Nobel prizes are not awarded posthumously. Fortunately, Rous had longevity on his side. He died 4 years after receiving the prize, at age 87.]

In 1976 Harold Varmus and J. Michael Bishop, then at the University of California San Francisco, discovered that the Rous sarcoma virus oncogene, v-src, as well as the oncogenes of several other tumorgenic retroviruses, actually were derived from cellular genes that normally play an important role in controlling cell division and differentiation (2). Moreover, Varmus and Bishop showed that these cellular “proto-oncogenes” can be altered by mutation, to become “oncogenes” that contribute to cancer. [Varmus and Bishop received the 1989 Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.]

But what is the actual activity of the protein coded for by the normal cellular c-src, and by v-src as well? The story of that discovery is rather delightful and begins as follows.

In 1978, Raymond Erikson and coworker Marc Collette, then at the University of Colorado Medical Center, were the first researchers to isolate the Src protein. They accomplished this by first preparing lysates from avian and mammalian cells, which had been transformed in culture into tumor cells by Rous sarcoma virus. Next, they precipitated those lysates with antisera from rabbits that bore Rous sarcoma virus-induced tumors. The premise of their strategy was that antibodies from the tumor-bearing rabbits would recognize and precipitate proteins that were specific to cells transformed by the virus .

With the Src protein now in hand, Ericson and Collette next sought its function. They initially asked whether Src might have protein kinase activity (i.e., an activity that adds a phosphate group to a protein.). This was a reasonable possibility because protein phosphorylation was already known to play a role in regulating various cellular processes, including cell growth and differentiation.

Ericson and Collette tested their premise by incubating their Src immunoprecipitates with [γ-³²P] ATP (i.e. ³²P-labelled adenosine triphosphate). In agreement with their proposal, they found that the antibody molecules in the Src immunoprecipitates had been phosphorylated. [Note that Src’s protein kinase activity was simultaneously and independently discovered by Varmus and Bishop.]

Ericson and Collette also carried out control experiments that were particularly revealing. When the same rabbit antisera was used to immunoprecipitate extracts from normal cells, or extracts from cells infected with a transformation-defective mutant of Rous sarcoma virus, no signs of protein kinase activity were seen in those immunoprecipitates. What’s more, the protein kinase activity was found to be temperature sensitive in immunoprecipitates from cells infected with a mutant Rous sarcoma virus that was temperature-sensitive for transformation.

These control experiments confirmed that the protein kinase activity in the immunoprecipitates was coded for by the virus. What’s more, they confirmed that the kinase activity of the retroviral Src protein plays an essential role in transformation. Furthermore, when taken with the earlier findings of Varmus and Bishop, they implied that the kinase activity of the cellular Src protein plays a key role in the control of normal cell proliferation.

While Erickson and coworkers were carrying out the above experiments in Denver, Walter Eckhart and Tony Hunter, at the Salk Institute, were looking into the basis for the transforming activity of the mouse polyomavirus middle T (MT) protein. [Unlike Rous sarcoma virus, which is a retrovirus, the mouse polyomavirus is a member of the Polyomavirus family of small DNA tumor viruses. SV40 is the prototype Polyomavirus.]

Tony Hunter

Since Erickson’s group was finding that Src expresses protein kinase activity, Eckhart and Hunter asked whether the polyomavirus MT protein might likewise be a protein kinase. Thus, as Erickson and Collette had done in the case of Src, Eckhart and Hunter examined immunoprecipitates of MT to see if they too might express a protein kinase activity, and found that indeed they did.

Interestingly, it was not known at the time of these experiments that MT actually does not express any intrinsic enzymatic activity of its own. Instead, MT interacts with the cellular Src protein to activate its protein kinase activity. See Aside 1.

[Aside 1: For aficionados, MT is a membrane-associated protein that interacts with several cellular proteins. Importantly, the phosphorylation events carried out by MT-activated Src cause a variety of signal adaptor molecules [e.g., Shc, Grb2, and Sos] and other signal mediators [e.g., PI3K and PLCγ] to bind to the complex, thereby triggering a variety of mitogenic signaling pathways. These facts were not yet known when Eckhart and Hunter were doing their experiments.]

At the time of these experiments, serine and threonine were the only amino acids known to be phosphorylated by protein kinases. In fact, Erikson and Collette, as well as Varmus and Bishop, believed that threonine was the amino acid phosphorylated by the Src kinase (see below). Consequently, Hunter asked whether the polyomavirus MT protein likewise would phosphorylate threonine. [Recall that MT actually does not express any intrinsic enzymatic activity of its own.]

Hunter’s experimental procedure was relatively straightforward and reminiscent of Erikson’s and Collette’s. It involved incubating immunoprecipitates of MT with [γ-³²P]ATP, hydrolyzing the immunoglobulin, and then separating the amino acids in the hydrolysate by electrophoresis. But, to Hunter’s surprise, the position of the labeled amino acid in his electropherogram did not correspond to that of either threonine or serine.

Hunter was well aware that tyrosine is the only other amino acid with a free hydroxyl group that might be a target for the MT kinase activity. And, while there was no precedent for a tyrosine-specific protein kinase, Hunter proceeded to ask whether the polyomavirus MT protein indeed might phosphorylate tyrosine.

Hunter began by synthesizing a phosphotyrosine molecule that could be used as a standard marker against which to compare the labeled amino acid in a repeat of his earlier experiment. And, to his pleasure, Hunter found that the amino acid that was phosphorylated by the MT kinase activity ran precisely with the phosphotyrosine standard marker in his new electropherograms.

But why had other researchers not detected tyrosine phosphorylation earlier? It was partly because phosphotyrosine accounts for only about 0.03% of phosphorylated amino acids in normal cells. The remaining 99.97% are phosphoserine and phosphothreonine. But, again, that is not the entire explanation. The rest is truly precious.

In Hunter’s own words, he was “too lazy to make up fresh buffer” before doing his experiments. Had the buffer been fresh, its pH would have been the usual 1.9; a pH that, unbeknownst to all at the time, does not separate phosphotyrosine from phosphothreonine during the electrophoresis procedure. The pH of the old buffer that Hunter used in his experiment had inadvertently dropped to 1.7; a pH at which phosphotyrosine is resolved from phosphothreonine. That fact enabled Hunter to discriminate phosphotyrosine from phosphothreonine for the first time. Thus, Hunter attributes his hugely important discovery to his laziness.

The finding that tyrosine is the amino acid phosophorylated  by the polyomavirus MT protein kinase activity led Hunter and his Salk Institute-colleague Bart Sefton to ask whether Src too might phosphorylate tyrosine, rather than serine or threonine (4). Indeed, they found that the retroviral Src protein, as well the normal cellular Src protein, function as tyrosine-specific protein kinases. [Recall that it became clear only later that MT actually has no intrinsic enzyme activity of its own and that it acts through Src.] Moreover, the levels of phosphotyrosine were 10-fold higher in cells infected with wild-type Rous sarcoma virus than in control cells, consistent with the premise that Src’s protein tyrosine kinase activity accounts for the altered growth potential of those cells.

Subsequently, Stanley Cohen, at Vanderbilt University, discovered that the epidermal growth factor (EGF) receptor contains an intrinsic protein-tyrosine kinase activity, further underscoring the importance of protein-tyrosine kinases in the normal control of cell proliferation. [Cohen shared the 1986 Nobel Prize in Physiology or Medicine with Rita Levi-Montalcini for their discoveries of growth factors, including EGF.] Subsequent studies identified additional receptor protein-tyrosine kinases, such as the fetal growth factor (FGF) receptor, and non-receptor protein-tyrosine kinases, such as Abl, each of which activates a mitogenic intracellular signaling pathway.

Tony Hunter and coworkers went on to demonstrate that protein-tyrosine kinases play key roles in additional crucial cellular processes, including cellular adhesion, vesicle trafficking, cell communication, the control of gene expression, protein degradation, and immune responses. Moreover, discoveries regarding the role of protein-tyrosine kinases in cell transformation and cancer gave rise to a promising new rational approach to cancer therapy; i.e., the targeting of protein-tyrosine kinases. For example, the drug Gleevec, which inhibits activation of the Abl and platelet-derived growth factor (PDGF) tyrosine kinases, was approved by the U.S. Food and Drug Administration for the treatment of chronic myelogenous leukemia and several types of gastrointestinal tumors.

References:

1. Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.
2. Harold Varmus: From English Literature Major to Nobel Prize-Winning Cancer Researcher, Posted on the blog January 5, 2016.
3. Collett, M. S. and R. L. Erikson, 1978. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc. Natl. Acad. Sci. USA 75: 2021-2024.
4. Hunter, T., and B. M. Sefton. 1980. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77:1311–1315.