Ending Plague

by Francis W. Ruscetti

  However, if you were a woman, you had to declare your doctoral thesis in advance and you COULD NOT work on those projects during your expected eight hours at the lab. You had to find additional time during the day to work on your own research, which probably meant an additional five to six hours of work, meaning you might only get about two or three hours of sleep a night.

  In 1987, the data were that only one in ten thousand CD4 T-cells were infected by HIV and yet all these cells were dying. The medical community called this “bystander effects,” a term used when the science does not know what is happening, like non-infectious inflammation that was called “sterile inflammation” until it was found to be caused by dysregulation of the microbiome. We had been working with Candace Pert and Mike Ruff on Peptide T, and I knew from their work on AIDS dementia, and that of Howard Gendelman, who would be on my thesis committee, that brain macrophages are involved in the disease. We hoped that blocking the interaction of the monocyte and T-cells could prevent the development of AIDS. I declared my doctoral thesis would be concerning HIV infection of monocyte/macrophages. When doing my technician work in Frank’s lab, I couldn’t be working on anything to do with that, but instead focused on research pertaining to TGF-beta, stem cells, and other assignments.

  During graduate school, this was my typical day: I’d arrive at Frank’s lab at around four or five in the morning, put in a few hours of work, and then drive an hour away to George Washington University for my classes. Then I’d return to Frank’s lab, put in several more hours on non-doctoral research, and then start my own experiments, often working until midnight or one in the morning. To put it bluntly, the men had no life, but at least they got eight hours of sleep a night.

  My molecular biology professor at George Washington, Dr. Ajit Kumar, a sweet, sweet man, literally cleared a bench for me in his lab so I could perform some of my lab work at the university. I was exhausted from literally working twenty-two hours a day.

  My doctoral thesis defense was November 14, 1991. My thesis was on HIV latency in monocytes and how the early administration of antiretroviral therapy could silence expression of the virus and those infected could be expected to live a normal life span if they followed this protocol. In fact, the well-known basketball player, Magic Johnson, had recently tested sero-positive for HIV, meaning he had an antibody to the virus. This was front page news a week earlier. The main question I was asked during my thesis defense was, based on my research, would Magic Johnson die of AIDS?

  I showed my data that demonstrated if he started the antiretroviral therapy early, maybe Peptide T or interferon alpha, and kept the inflammatory cytokines suppressed, keeping the virus latent in the monocyte macrophage, and thus prevent death of the CD4 T-cells, Magic would never develop AIDS, much less die of it. Nearly thirty years later, Magic Johnson is still with us, something that was unimaginable during the darkest days of the HIV/AIDS epidemic, when so many of these beautiful men were dying slow, horrible deaths.

  In 1992, Frank received the Distinguished Service Award from the NIH. It was a well-earned accomplishment and gave both of us the feeling that good science, as well as those who actually did the work, would eventually be acknowledged.

  My degree was awarded on February 17, 1992. After my successful thesis defense, Frank and I agreed it was best for me to do my post-doctoral work in the lab of Dave Derse. Derse was an accomplished molecular virologist working in the Laboratory of Genomic Diversity headed by Dr. Steve O’Brien, in a program in which they studied viral diversity in animals, with a specialty in big cats.

  Since all my graduate training was in cellular biology, I wanted to develop molecular virology skills. My main project at Derse’s lab was to create the first infectious molecular clone of HTLV-1. You can study a virus by producing great quantities of it, such as we did in fermentation chemistry, purifying HTLV-1 from human T-cells grown with IL-2. But that was a very labor-intensive process. Producing a molecular clone made such research a great deal easier. The problem with HTLV-1 is that it kept “popping out of the vector.” I was working on that project with a researcher from Bulgaria, a brilliant woman who spoke her mind, Dr. Maria Polianova. She always made me and Frank laugh because she knew little English when she came to the Derse lab in 1992 and, with her Eastern European accent, she always said she was working as a “post-dog.”

  That was a pretty accurate description of how I felt at the time.

  In addition to my “post-dog” work in Derse’s lab, I also had to continue my work in Frank’s lab. That meant my workload had shrunk from twenty-two hours a day to a scientific slacker pace of just sixteen hours. At least I was getting a good night’s sleep.

  A project which had grown out of my PhD thesis research was understanding DNA methylation, a main mechanism of silencing the expression of retroviruses. This work started as a collaboration with Dr. Stephen B. Baylin, a pioneer in the field of Epigenetics from Johns Hopkins University, only an hour drive from Fort Detrick. Although I was working long hours, I was basically commuting between Building 560 where the Biosafety Level 3 lab was located, and Frank’s lab located in Building 567. My PhD thesis, looking at the latency of viruses, suggested retroviruses could alter the DNA methylation machinery in order to persist. How the immune system might silence the expression of viruses had generated several intriguing avenues of research, but my personal favorite was DNA methylation. That’s mainly because I’m an identical twin. So, any differences between my sister and I are not due to genetics, but to epigenetics. Stephen Baylin reminded me of Frank, a brilliant thinker who could see patterns in the data and synthesize them into a new and revolutionary understanding.

  At George Washington University, I did all my work using blood from HIV-infected men who had not yet developed AIDS. I isolated the various blood cell subsets and the signaling molecules to distinguish the differences in the molecules and pathways between those who had not developed AIDS and those who had developed the disease.

  In addition to DNA methylation mechanisms of silencing the virus, my work showed inflammatory cytokines in patients with AIDS, but not in those HIV-infected men who had not developed the disease.

  What were the key off and on signals?

  I purified the nuclei of peripheral blood mononuclear cells from some HIV-infected men and showed that nuclear factor kappa B (NF-kB) was prominent in the nuclei of HIV-infected men who had developed AIDS. Nuclear factor kappa B (NF-kB or NF-Kappa-B) turned on the various proinflammatory cytokines, IL-6, IL-1, and TNF-Alpha, driving the development of AIDS. It wasn’t the infection. It was, in part, NF-Kappa-B, which was causing the cytokine storm that was leading to the deadly ravages of AIDS.

  We submitted that paper to a prestigious journal in 1992, and it was rejected. One reviewer wrote that everybody knows NF-Kappa-B is a cytoplasmic protein, so it was obvious my nuclei were contaminated with cytoplasmic protein, even though there was no evidence of such contamination. I had been down that contamination road before, with the earlier publication of HIV infection of monocytes/macrophages. In that paper, the reviewer rejected it, suggesting my monocyte cultures were contaminated with T-cells because everybody knew the HIV receptor is CD4 and monocytes don’t express CD4. Of course, later CCR5 was shown to be a coreceptor and people who had a thirty-two base pair deletion in CCR5 would not develop AIDS no matter how much HIV was in their blood.

  Frank taught me a valuable lesson as we filed the manuscript away, but not the knowledge of the data. Frank instructed me to ignore the arrogance of the gatekeepers of the scientific literature, and always show all the data, as the best papers should raise more questions than they answer. Other investigators may see clues to mysteries in our data from experiments they’ve done, perhaps using technology or methods unavailable to us at the time and solve the mystery.

  As government researchers, we didn’t have to “publish or perish” like many academic researchers at universities. However, to get a PhD at George Washington University, one needed two peer-revie
wed publications. Fortunately, those two were infection of a monocyte cell line and proof of primary human monocyte infection, published a year before my thesis defense.

  In the case of the DNA methylation and NF-Kappa-B papers, they took much longer to publish.

  About six years later a researcher at Harvard published in Cell, the cloning of I-Kappa-B, an inhibitor of NF-Kappa-B. I-Kappa-B held NF-Kappa-B in the cytoplasm until phosphorylation changed the shape of the inhibitor, releasing NF-Kappa-B into the nucleus of the cell and turning on the cytokine storm. I went back into the filing cabinet, pulled out our manuscript, attached a copy of the Harvard paper, and sent it back to the same journal, along with their rejection. I wrote nothing else. They published the article a few months later, exactly as it had been written years earlier.

  With the DNA methylation project, it was a different story.

  Baylin did not like to submit research which had any unanswered questions, and as a result, often held back any anomalous findings. If the author could not answer all the questions to the satisfaction of the journal, the journal would often reject the paper, and not allow it to be resubmitted at a different time.

  But Frank stood firm and said we had to submit all the data, even the parts we did not fully understand.

  That paper took several years of additional work to get published. We performed many experiments, using different techniques and technologies, and the results all supported our original findings. I thought the additional experiments were a tremendous waste of time.

  In 1998, the paper was finally published in an excellent journal, Molecular and Cellular Biology. That only happened because of Frank’s bulldog tenacity and brute force, and Steve Baylin’s patience and creative writing. That original paper on infection of a monocyte cell line raised far more questions than it answered and led to ground-breaking discoveries in four different fields of science.

  By comparison, it was simple and fast to publish my research creating the first infectious molecular clone of HTLV-1. My post-doctoral training in molecular virology was completed in less than two years, but the questions raised by my PhD thesis work took another several years to publish as we awaited, or created, the technological advances necessary to support the biological observations.

  ***

  As a staff scientist back in Frank’s lab in the mid-1990s, my work on inflammatory cytokines and monocytes was key to understanding an emerging, highly dangerous viral infection, Ebola.

  One project I was asked to collaborate on was to determine the difference between two strains of Ebola, the “Zaire” strain and the newly categorized “Reston” strain. These studies were being conducted in the Biosafety Level 4 laboratory at the US Army Medical Research Institute of Infectious Diseases (USAMRIID), just across the baseball field from our labs at the NCI.

  I conducted the studies in monocytes as a logical starting point based on my PhD thesis research. I reported the results in an abstract showing the differences in pathogenicity (disease causing) between the Zaire and Reston strains of the virus.

  To put it plainly, the Zaire strain was extremely dangerous, killing by creating an inflammatory cytokine storm in primary monocytes. But the Reston strain created no cytokine storm in primary monocytes and was thus of little danger to the public.

  However, the corrupt, old boys’ club of science, Robert Gallo, Tony Fauci, John Coffin, and Harold Varmus, to name a few, didn’t like the story emerging from our data. We were challenging the narrative, just as we’d done when we said that HIV infection didn’t have to cause AIDS. Simply stated, there could be HIV infection with no AIDS, and a diagnosis of AIDS with no HIV infection. It was all about the response of the immune system.

  Similarly, the Reston strain of Ebola was benign.

  ***

  Frank’s frankness got him into trouble with his superiors in 1998, although in typical government fashion, it wasn’t a direct assault. Instead, it was a sneak up from behind move to stab you in the back, and hope you bled to death.

  By this time, Frank was a lab chief and had several principal investigators working under him.

  They initiated an investigation of Frank’s lab, and although they found nothing wrong, they offered all of the principal investigators the option to leave and start their own independent laboratories. You see how sinister the power structure can be.

  No, we didn’t find anything wrong, they’d say to his investigators. But, if any of you want to leave, we’ll help you get started in your own lab, and we’ll take resources, money, and equipment from Frank’s lab to help you get started.

  Part of their campaign was starting a rumor that I was having an affair with Frank.

  Which was humorous because, despite the fact that I had boyfriends, I was also highly competitive, both in science and in our intramural softball games, which led to another rumor that I was a lesbian. When I complained about this attempt to marginalize my accomplishments by saying I was having an affair with my boss, some of my female colleagues replied with the deadpan response, “Well, at least you’re not gay.”

  And I hope this doesn’t sound self-serving, but I enjoyed decades-long relationships with a few absolutely gorgeous, principled, and intelligent men. We simply were never in a time and space to marry. Our goals diverged but our love remains strong to this day. It’s been my experience that strong men love strong women. It’s the sniveling cowards, the men without a spine, who are the most misogynistic. I believe the vast majority of men do not have trouble with strong personalities, be they male or female.

  This government proposal had the possibility of stripping Frank of all of his principal investigators, essentially decimating his laboratory. I’m sorry to say that all the male principal investigators took the opportunity to start their own lab.

  But two of the three female principal investigators said, “No, I’m staying with Frank.” Was that because he treated them with integrity and respect, and they knew they were unlikely to find that anywhere else? Frank was not simply an administrative lab chief. He was a mentor that a woman could trust.

  All of this came to a head after a softball championship after which I’d probably had a few too many beers. The powers that be were also telling the principal investigators who chose to leave that they could take our laboratory equipment. There was this one technician talking about what he might take from the lab, and I drew a line in the sand. I said, “Over my dead body will one piece of equipment leave our lab.” I freely confess I was emotional, and I may have also been crying over the injustice of it.

  This technician, who also happened to be in a wheelchair, took this as a physical threat. It was not. The other person who overheard me, a female technician named Cary, whose husband was on the team, backed me up.

  I was called into the office of Peter Fischinger, who was head of the contract lab. Frank told me once he’d told Fischinger something about Gallo that he’d told no one else. Within an hour, Gallo called Frank with the exact story. I knew not to tell Fischinger anything that could be used against me. Although the claim I’d threatened physical harm was dismissed, I was required to take a “sensitivity training” course and go see a psychologist a few times to deal with my “anger management” issues.

  I’m not sure if the sessions helped me, because even today, if you try to steal my stuff, I’m still going to defend my territory with every ounce of strength I have. It’s in my blood, and it’s how I was raised. I knew about the “Trail of Tears,” forced upon the Cherokee Nation long before they ever mentioned it in school. Take the guns away from the men, and then force the people on a long march where up to fifteen thousand are estimated to have died. No one would ever deceive me and steal my work to perpetuate fraud on the innocents, as had been done in previous centuries. Not on my watch. I plead guilty to having said more than once, “Over my dead body.” I meant it then, and I mean it now.

  Yeah, I know, it’s a character flaw.

  But I don’t ever remember claiming I was perfect.


  ***

  The attempt to undermine Frank’s lab also came at a time of great instability at the NCI.

  While Nixon had set up the structure under which a few powerful scientists could control the research community and demoted a bunch of government researchers to contractors for hire, Reagan put the system on steroids. Under his “Reduction in Force,” or RIF plan, government scientists often found themselves working in the same office, in the same lab, but with less money, no benefits, and no employment rights. This is what the government does when they want to hide a bunch of money so it can go to the higher-ups, while at the same time creating an entire group of second-class citizen scientists.

  The previous head of the Lab of Anti-Viral Drug Mechanisms, along with everyone else in the world-renowned contract lab, had finally had enough of this nonsense and realized they could make much more money in private industry. Most of the Lab of Anti-Viral Drug Mechanisms went to work for Celgene in sunny San Diego, California.

  You really couldn’t blame them.

  But it left the Lab of Anti-Viral Drug Mechanisms such a ghost town that they actually closed the lab while they figured out what to do. Peter Fischinger had a big problem with everybody leaving arguably the most successful lab under his direction.

  I was in Peter’s office being reprimanded for supposedly threatening that technician when I piped up and said, “I can run that lab! I’ve worked with them for many years.”

  That’s right. One minute I was in his office for allegedly threatening to beat up a guy in a wheelchair and the next I’m volunteering to rebuild a lab decimated by Reagan’s short-sighted policies.

  I got the job.

  For the first few weeks I called myself the eighty-thousand-dollar washerwoman because my primary job was to clean out the lab which had been left a ghost town. Then I had the privilege of hiring the entire team of researchers. Later, many commented that our lab’s personnel looked like the United Nations. I had researchers from China, Mongolia, and a Black woman from British Trinidad, and they were just great. I developed the study design to pursue mechanisms and treatments for AIDS-associated malignancies, starting with the thesis that aberrant expression of retroviruses was central to the immune system dysfunction and we needed to understand how to effectively intervene.