Posted on May 10th, 2009 No comments
This is about something I wrote in 1964, which was recently reproduced and is now available on line.
It can be seen by clicking on this link:
Seeing this 45 year old document prompted me to write this post.
It is about interferon and has nothing to do with AIDS, at least not in any immediately obvious fashion.
It is an interesting story, about at least one of the ways in which science progresses. It is a story of how an apparently insignificant change in an experiment can sometimes lead to very significant advances. In this instance, about how cytokines exert their effects.
Cytokines are protein or peptide molecules released by cells which then attach to the surfaces of other cells. As a consequence, the behaviour of the cells to which they attach is altered. In this respect cytokines are similar to hormones.
Generally, each cytokine will only attach to a specific receptor on the surface of the cell.
When a cytokine attaches to its matching receptor, a cascade of events is set in motion resulting in the activation of specific sequences in nuclear DNA.
Messenger RNA molecules are then transcribed from specific DNA sequences and these direct the synthesis of specific proteins that ultimately are the molecules that cause the particular effects produced by the cytokine.
Therefore, as the picture below demonstrates, cytokines are not themselves the molecules that directly mediate the effects they cause. Through a complex series of signalling events in the cell, set in motion by the binding of the cytokine to its receptor, specific proteins are made by the cell. These proteins are the actual mediators of the cytokine’s effects. 
In the illustration, the right angled arrow in the nucleus represents the messenger RNAs which will direct the synthesis of these proteins.
HIV DNA is integrated into host DNA. Should certain cytokines, IL-6 or TNF alpha for example, attach to their receptors on the cell membrane, a series of events follow, ultimately resulting in sequences in nuclear DNA being activated which in turn causes HIV DNA to make RNA which directs the synthesis of HIV proteins and ultimately of new HIV particles.
Since many of those cytokines that can activate HIV in this way are produced during the course of many different infections, this then is but one of the several ways in which HIV replication can be enhanced by many different concurrent infections. TB and malaria are among them, as are the bacterial diarrheal infections associated with a lack of sanitation and clean water. Controlling these many HIV enhancing infections, is with the exception of TB, a neglected target in the fight against the epidemic.
Interestingly, discoveries about the ways in which cytokines exert their actions have largely been made since AIDS was first recognized in 1981.
Thus HIV research has progressed in tandem with research on molecular cell biology. There have been reciprocal benefits. HIV research has both contributed to our understanding of molecular cell biology, as well as itself being advanced by discoveries in this field.
Interferon was the very first cytokine to be discovered. It was discovered by Alick Isaacs and Jean Lindenmann . Actually it was not really discovered as a specific molecule; the term interferon was coined by Alick Isaacs in 1957, to describe an activity – an antiviral activity released by virus infected cells. It was perhaps a bit premature to assume that this activity resided in a single molecule. But that was what we all thought at the time; it was nonetheless a concept that facilitated research as probably did the coining of the word “interferon” to describe this antiviral substance.
We now know that there are many types of interferon, and we therefore should properly speak of the interferons. Also, as is the case with cytokines generally, the interferons have multiple effects, but the antiviral effect is how it was first recognized and also measured.
Alick Isaacs was my mentor in the laboratory study of viruses; I shared a lab with him and worked on the mechanism of the antiviral action of interferon.
In 1963, we had no idea about how interferon exerted its antiviral effect. We at least knew that it did not directly inactivate viruses. Molecular biology – at least as far as eukaryotic cells were concerned, had hardly developed.
The 1964 article that can be seen by following the link at the beginning of this post resulted from the work of Joyce Taylor.1964 interferon article.
Joyce Taylor is a biochemist. She also worked in Alick’s lab in 1963. It was rather unusual, in those days for a biochemist to be working in a lab concerned with animal viruses. Animal virology was just beginning to employ biochemical methods.
Joyce was attempting to show that interferon blocked the synthesis of viral RNA. This of course required the use of biochemical techniques to identify and measure viral RNA.
She was able to demonstrate that viral RNA was not made in cells treated with interferon. This was accomplished by using a compound that blocked DNA directed cell RNA synthesis, actinomycin D. It was necessary to use actinimycin D because there is so much background cellular DNA directed RNA synthesis that unless this can be stopped it would be impossible to observer viral RNA synthesis. She used an RNA virus (SFV) that was unaffected by actinomycin D.
Joyce very clearly showed that the synthesis of SFV RNA was blocked in cells treated with interferon. as with the availability of actinomycin D, she was able to detect and measure viral RNA.
We are now coming to the happy, but at the time seemingly insignificant change in the sequence of steps in an experiment, that had such far reaching consequences.
This is how Joyce did her experiments. Cells were exposed to interferon for some hours, and then the SFV virus added with actinomycin D, to allow the measurement of viral RNA synthesis by removing the background of cellular RNA synthesis. As mentioned, in this way, Joyce was able to show very clearly that pre-treatment of the cells with interferon blocked the synthesis of SFV RNA.
One day, because Joyce had to leave early and she did not want her technician to handle actinomycin D, she added this drug with the interferon, at the beginning of the period of interferon treatment . Nobody at that time would have thought that this would make the slightest difference. It is this change in the time when the actinomycin was added that was critical, but it was not at all expected to have any effect.
But it did have an extraordinary effect. When cells were treated with interferon in the presence of actinomycin D it had no antiviral effect. At first it was thought that an inactive preparation of interferon was used, but the same result was obtained when the experiment was repeated.
The significance of the change in the order in which actinomycin was added was that now, while the cells were exposed to interferon, DNA directed RNA synthesis was also blocked.
The implications of this were quite extraordinary. At that time, 1963 and 1964, the foundations of our understanding of basic molecular cell biology were being worked out mostly in bacterial systems. The structure of DNA had been worked out, messenger RNA discovered (although there is some dispute as to who discovered it) and there was some understanding of derepression – that is the ability of certain molecules to cause the synthesis of specific proteins by bacteria.
The result of the changed order of Joyce’s experiment suggested that something similar might be happening in animal cells- that interferon was inducing the synthesis of a specific messenger RNA which in turn directed the synthesis of a protein responsible for its antiviral effect. This is what prompted me to write the short article that can be seen by clicking the link at the beginning of this post.
What was described in 1964 was in fact the first demonstration that cytokines exert their effect by attaching to a receptor on the cell surface, and as a result of this, specific regions on cellular DNA are activated,and RNA synthesized. Work showing that this RNA is responsible for the synthesis of proteins followed immediately.
Robert Friedman, was visiting the laboratory from the National Cancer Institute, and we worked together to show that not only RNA synthesis, but also protein synthesis was required for interferon action – and as was to be found, for the action of all cytokines.
Joyce Taylor remembers this story somewhat differently, but I trust that my version is correct. I have repeated it so frequently since the events in question, as an illustration of how science sometimes progresses.
Joyce changed the order of adding reagents. As a result we knew that interferon action needed cell the participation of cell DNA and the synthesis of RNA. Bob Friedman and I then showed that interferon action also required cell protein synthesis. Ian Kerr who was also in the lab around that time, and others then showed a part of what changes interferon induced in cells.
Interferon was the tool by which a signalling pathway was demonstrated that could account for the effects exerted on nuclear DNA by a molecule interacting with its receptor at the cell surface. Ian Kerr was a key contributor to this work.
This post was not directly connected with HIV/AIDS. But cytokines are most certainly connected with HIV/AIDS. This will be the subject of future posts.
 The genetic code is defined by the sequence of the four bases that make up genomic DNA. A particular sequence of three nucleotides can be regarded as a code component which ultimately defines a particular amino acid; amino acids are the building blocks of proteins. The DNA code is conveyed from the nucleus to the protein synthesizing apparatus in the cell cytoplasm in the form of messenger RNA. This RNA molecule is made from a DNA template and exactly reflects the nucleotide sequence of the section of DNA from which it is transcribed.
Posted on April 13th, 2009 2 comments
“Starting HIV Therapy Earlier Saves Lives”
“Study: Treatment for HIV Should Start Earlier”
“Starting Therapy Earlier Found to Improve Survival”
“Earlier HIV Treatment Boosts Survival”
With headlines like these you would think that there is a clear answer to the question of when is it best for HIV infected people to start antiretroviral treatment. There can be no doubt at all that starting antiviral therapy early – in this case at a CD4 count above 500 improves survival. These headlines, addressed to HIV infected individuals their physicians and the public are a unanimous response to a study that just appeared in the New England Journal of medicine (NEJM). http://content.nejm.org/cgi/content/full/NEJMoa0807252
But is this confidence justified?
Unfortunately, despite these headlines, the study which occasioned them was absolutely unable to justify the conclusion ; we still do not know when it’s best to start treatment.
The study examined data that had been previously collected. It was a retrospective observational study with all the problems inherent in such studies. These have been outlined in a previous post.
About a week after this study appeared in the NEJM, another large retrospective observational study was published in the Lancet (April 9th 2009
While both studies support the desirability of not delaying a start to antiviral therapy to a CD4 count below 350, they do differ with respect to the reported benefits of starting above that number. The Lancet study, whose lead author is Jonathan Sterne, finds a decreasing benefit at start times increasing above a CD4 count of 350, with nothing at starting around 400.
The authors of both reports agree that prospective randomized studies are the best way to approach a resolution of the “when to start” question – a question that might have already received a reliable general answer had we begun these studies in 1997, as some of us suggested we do at that time.
Obviously we cannot just wait for the results of randomized prospective studies. We do need guidelines now, but any recommendation based on available information must be regarded as provisional, until the results of prospective randomized studies are in. It is important that this be clearly stated. If we are ever going to be able to enrol a prospective randomized study then we cannot afford to delude ourselves that the answer to the when to start question is already known.
While the lead author of the New England Journal of Medicine did pay homage to prospective randomized trials – and a kind of ritualized homage is exactly what it sounded like, this gesture most certainly did not inhibit her from unreservedly recommending an earlier start to treatment, a start even at a CD4 count above 500, without conducting such a prospective study. Her conclusion:
“The early initiation of antiretroviral therapy before the CD4+ count fell below two
prespecified thresholds significantly improved survival, as compared with deferred
One of these prespecified thresholds was a count 500 CD4 lymphocytes.
This categorical statement, arrived at by the kind of study that cannot possibly justify such confidence, will have a negative effect on enrolment in proposed randomized trials, which are in fact the kind of study that can provide conclusions in which we can have justified confidence.
This study may well be the last coffin nail in any hopes there may have been for the completion of prospective randomized trials designed to address the “when to start” issue. It may now be impossible to enrol, and will never get off the ground. This difficulty is made so much worse by the kind of uncritical headlines shown above
I wonder how the commentators who rushed so uncritically to announce Dr Kitahata’s conclusion on the benefits of starting treatment at CD4 counts even greater than 500 will respond to the Lancet report, which did not find a benefit with starting at such high CD4 numbers? I hope I’m wrong in suspecting that this study will be largely ignored; the headlines trumpeting the survival benefit of starting treatment early – even above a CD4 count of 500 will not be marred by any doubt introduced by the study reported in the Lancet.
Among the problems with the New England Journal of Medicine study is that a significant number of people were left out of the analysis, because their HIV disease failed to cooperate with preconceived notions about the course of this disease.
This is a significant criticism and I will try to explain why. The study examined two groups of people, one with over 500 CD4 lymphocytes, and one with CD4 counts between 351 and 500.
Let’s just take the 351 to 500 group. Here, deaths in those starting at counts between 351 and 500 were compared with deaths in those starting below 350. Sounds reasonable? Maybe, until we learn that significant numbers of people with 351 – 500 CD4 cells who did not start treatment also did not progress to below 350 CD4 cells. So the authors just left these people out of their calculations. They in effect did not exist for the investigators.
The recommendations the authors make are meant for all people, including those who did not progress and were left out of the analysis. These people are also going to be treated with drugs they don’t need, as they cannot be identified.
I suppose this will do wonders for drug sales, but there will be individuals taking drugs for no reason and some may only suffer their ill effects as well as cost while deriving no benefit.
Here is another serious problem with this study.
Among those people with CD4 counts between 351 and 500, it is important to know just how long treatment was delayed in those who waited until their counts fell below 350. This information was provided; the median count at the time of starting treatment among all who waited was 286. But what was the CD4 count at starting treatment among those in this group who died?
This information was not given – at least I was unable to find it.
Could there have been those starting treatment with counts below 100, below 50 – maybe even below 20. In an extreme example, if a person waited to start treatment to a point close to death, there would not be much surprise that delaying treatment initiation is associated with a worse outcome.
Many physicians are proud that the field has abandoned uncritical authority as a guide to practice and has now embraced evidence based medicine. David Sackett, one of its originators, has stated that one pillar of evidence based medicine is the use of the best external evidence in making clinical decisions.
All too frequently physicians, while priding themselves on practising evidence based medicine, somehow are still able to make decisions based solely on their unproven beliefs, as if they have a private source to the truth, some special access to an oracle. I have heard one physician state that anyone with a viral load should be treated, another saying essentially the same thing in stating that he would treat every HIV infected patient no matter what the CD4 count. How on earth have they arrived at these conclusions? Patients might just as well seek advice from a palm reader.
As always you can’t beat the truth. No matter what the private sources of information to which some physicians and patients apparently have access, the truth remains that apart from people with under 200 CD4 cells the best time to initiate antiviral therapy is unknown.
I have once before faced this kind of opposition to conducting a randomized prospective study to address the question of when is it best to start treatment. In the early 1990s I participated in an effort to conduct a trial of early versus deferred treatment with AZT. A pilot study was initiated, and I participated with some statisticians in describing the study to numbers of physicians in New York City, with the hope of encouraging them to enrol patients. Despite expressions of enthusiasm, the response was so dismal that the trial could never take place. However there was one physician – just a single physician in San Jose who was able to recruit many more patients than all the others combined. He was so successful that we asked him to come to New York City to explain how he was able to enrol so many patients. His answer was simple. He told patients the truth. He did not know when it was best to start treatment, so he and his patients let the toss of a coin determine this, as a means of finding out what was best by participating in a study.
This means that the other doctors were unable to say they did not know. Maybe, as is the case today some actually felt that they did know, as they had complete faith in their intuition, or perhaps had some private access to the truth. For these physicians the practice of medicine is more akin to a faith based activity. Maybe other physicians did not know when it was best to start treatment, but might have felt unable to admit this; maybe some patients felt they knew and physicians acceded to their wishes.
The rational response to uncertainty – having first overcome the hurdle of being able to admit that there is uncertainty – is to try to resolve this by the best means available.
I fear we are not even close to recognizing that there is uncertainty about when to start treatment in people with over 200 CD4 cells. The NEJM article exacerbates the problem with its assumption of certainty, an assumption very sadly shared by some health care providers, some journalists and community commentators to whom HIV infected people turn to for advice.
In conclusion I cannot lose an opportunity to yet again bring attention to the need to individualize therapy. The rate of HIV disease progression is so widely variable that there are limitations in setting a fixed CD4 count as a guide to start therapy. A prospective appropriately designed trial can tell us if on average it is better to start above rather than below a certain CD4 count, or on average it is better to start treatment immediately or to defer it.
It is the “on average” limitation that needs fine tuning for each individual patient.
Not only will the rate of disease progression vary widely between patients, but there are other individual considerations that impact the decision to start treatment. For example, adequate housing, mental health issues, co morbidities and many other factors need to be considered.
These two aspects, the general and the particular, fit so very neatly into David Sackett’s description of evidence based medicine that I will quote a passage:
“The practice of evidence based medicine means integrating individual clinical expertise with the best available external clinical evidence from systematic research. By individual clinical expertise we mean the proficiency and judgment that individual clinicians acquire through clinical experience and clinical practice“.
BMJ 1996;312:71-72 (13 January) : Evidence based medicine: what it is and what it isn’t. David L Sackett, William M C Rosenberg, J A Muir Gray, R Brian Haynes, W Scott Richardson
The best available external evidence will be the results of a prospective randomized trial; these will provide general guidance. Individual clinical expertise will apply this to particular patients, taking into account many factors, not least of which is the patient’s rate of disease progression.
A previous post discusses the issue of individualization of treatment.
If we took individualization of treatment seriously, we could in fact come some way to identifying rapid and slow/non progressors. See previous post on individualization of treatment.
Posted on March 8th, 2009 1 comment
Why treatment of HIV infection must be individualized.
HIV disease is usually a progressive disease. That is, it has a starting point; the time of infection. The disease then progresses, and without treatment will generally end fatally. There are some very fortunate HIV infected individuals who are able to control viral replication and remain disease free. But for most, HIV disease does progress. But, for each individual, the rate at which it progresses varies widely. Disease progression is reflected in the fall in the numbers of CD4 lymphocytes.
So any single CD4 count measurement is really a point on a descending curve, one that does not necessarily proceed in a straight line, and falls at widely differing rates in different individuals.
Recommendations for the treatment of HIV infected individuals are issued periodically by DHHS and bodies such as the International AIDS Society. These recommendations, particularly those concerning when to start antiviral treatment, have always included a particular CD4 count as a signal to start or to consider starting antiviral treatment.
All individuals with a CD4 count of less than 200 should be on therapy. They are in great danger of acquiring a possibly fatal opportunistic infection and evidence derived from clinical studies makes it absolutely clear that antiretroviral treatment is life saving.
But what about people with higher CD4 counts? Here there is uncertainty about when in the course of HIV infection it is best to start treatment. Of course, if the drugs were completely harmless (including cost) it might be less important to have an answer to this question. However the drugs can have significant adverse effects, some of which only become evident after years of use. For people with fewer than 200 CD4 lymphocytes, the benefit of antiviral treatment overwhelmingly outweighs the risks.
For others, a very mixed group, with CD4 cells anywhere from 200 to over 1000, and each with a different rate of disease progression, we cannot, with any security, make a “one size fits all” recommendation as to when it is best to start treatment.
The best way to resolve clinical uncertainty remains randomized prospective clinical trials. By now we might already have obtained reliable evidence as to whether, on average, it is best for infected individuals with more than 200 CD4 lymphocytes, and who have no symptoms, to start antiviral treatment immediately, or to defer it. (A suggestion made in 1997 when the first guidelines were issued: http://aidsperspective.net/articles/guidelines1.pdf )
The current recommendations, regarding people with greater than 200 CD4 lymphocytes, and who are without symptoms, propose a CD4 count of 350 as a point to start treatment ( many believe this number should be 500). This recommendation is made for all individuals – it is a one size fits all approach. This kind of approach is appropriate for some aspects of treatment; for others it is very wrong.
Perhaps the most important example of a recommendation, where its application across the board is problematic, is that which deals with the time when antiretroviral treatment should be started in individuals with greater than 200 CD4 lymphocytes. This recommendation specifies a specific CD4 count at which to start. As noted, for individuals with a CD4 count below 200, there is no doubt that they will benefit from therapy. For others who have no symptoms, specifying a CD4 count for all is mistaken. It is here that individualization is necessary.
The reason is that no two HIV infected people are the same with respect to the rate of disease progression. During the early years of the epidemic, before antiretroviral treatment was introduced, we soon noted that the CD4 count declined at different rates in different people, and not necessarily in a straight line. As noted, at one extreme, there were the few fortunate individuals in whom there seemed to be no disease progression, at the other there were the few people whose CD4 cells fell very rapidly after infection, and who did not survive for more than 2-3 years, but most fitted somewhere between these extremes .
To illustrate this I have considered four possible situations. This is a picture of the possible rates of CD4 decline in four different individuals. . It is true that these pictures are constructs, but they do accurately reflect the observed variability in disease progression; real examples showing this variability would be easily found in my medical records, and of course in those of other physicians during the period between 1981 and about 1993.
The dip in CD4 cells following infection is usually seen when there is an opportunity to observe this. CD4 cells then rebound to a level called the set point, which will be different in relation to the pre infection level in different people. From then on it declines, but at a very variable rate, and can remain steady for varying periods before declining, again at varying rates.
Look at where three of them (A ,B and C) reach a count of 450 CD4 lymphocytes; A (an unusual rapid progressor) gets there in about one year, B in about 3 years, C in 7 years, and D, who is a fortunate non progressor is nowhere close after 18 years.
The arguments for starting early are not only to forestall reaching the dangerous level of 200 CD4 lymphocytes. The continuous deterioration of the immune system and diminished chances of recovery at lower counts are also arguments for an earlier start. There is also the possibility that there is a greater incidence of cancer, – other than lymphoma and Kaposi’s sarcoma, at higher CD4 counts in HIV infected people. If this is so then it remains to be shown how frequently these events occur and whether antiviral therapy can avert them.
Treatment itself, particularly if extended over many years, is not without risks, some of which cannot even be completely known yet, particularly with the newer antiviral agents. We have to do the best we can in making a risk benefit assessment. In order to do this we should attempt to obtain information on the rate of disease progress in any one individual. This may not be entirely possible, as the rate of disease progression in any one individual may not be steady; it may accelerate or slow down. But it is possible to obtain a good, if not perfect, picture of the course of HIV disease in any one person.
How might we obtain some information about a given individual’s rate of disease progression? Apart from obvious exceptions, and in people below 200 CD4 cells, there are no emergencies in HIV medicine. For each person we generally will have time to observe the CD4 count and viral load over a period of 6 to 12 months and obtain some idea of the rate of progress. A rapid fall in CD4 count might result in a decision to start in less than six months of observation. Or a consistent fall in CD4 count might lead to a decision to start treatment at CD4 numbers higher than even 500. This is far from perfect, as changes in CD4 cell numbers do not necessarily follow a straight line. But it is far better than basing a decision on a snapshot – which is what the experts are telling us to do.
Individualization involves more than considering the rate of disease progression. There are other factors, such as associated diseases, domestic and social circumstances such as a lack of housing, as well as mental health issues, and many other considerations that are involved in individualization. Observing people also provides the time to establish a doctor patient relationship and for the physician to become familiar with the patients particular circumstances.
The natural history of untreated HIV disease is relevant to the “when to start treatment” issue and will be the topic of the next post.
 Evidence supporting the recommendation is derived in part from retrospective observations. The reasons why these are unreliable guides are outlined in the previous post. It is critical to as far as possible, eliminate bias in study designs because this increases the probability that a particular outcome can be interpreted as indeed resulting from a particular intervention. In this case it would be that improved survival is due to an earlier start of antiviral therapy and that the medications mediate the effect – and not for example, from simply being under the supervision of a physician. Retrospective observations, that is, looking back at information already gathered cannot be free of confounding factors as described in the previous post. In a prospective study people would be randomly assigned to receive immediate treatment or to defer it. This will give us the most reliable answer to the question of which approach is better on average.
Examples of measures that should be taken in the treatment of every HIV infected person, irrespective of the rate of disease progression are the types of tests that are performed on the initial assessment of an infected person. For example, the initial assessment of an HIV infected person should always include not only CD4 counts and HIV viral load measurements, but also tests for hepatitis, toxoplasmosis, and many other investigations. Another example of an intervention that is appropriate for categories of infected people is treatment to prevent Pneumocystis pneumonia in people with less than 200 CD4 cells. And of course, people in this category must always be offered antiretroviral therapy.