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  • HIV disease and Positive feedback: An additional comment.

    Posted on August 31st, 2010 admin No comments

    HIV Disease and Positive Feedback.  An additional comment.

    A previous post focussed on the positive feedback interaction between HIV replication and immune activation.   HIV replication and immune activation reciprocally enhance each other.

    While HIV infection is an essential cause of the immune activation that’s characteristic of HIV disease, there are other factors that also contribute to it.   In that post as well as in the blog I write on the POZ magazine website, I described some of these additional factors that can add to immune activation.   As noted, viruses of the herpesvirus family, cytomegalovirus (CMV) in particular are the most important of these worldwide, while in parts of Africa certain endemic infections may be of great significance in contributing to immune activation.

    Since sustained immune activation, involving both innate and adaptive immunity is at the heart of the pathogenesis of HIV disease an understanding of how it is perpetuated is critical.

    Evidence for activation of innate immunity was noted in 1981, the year that AIDS was first reported, in the detection of large amounts of alpha interferon in the circulation of patients.  We even knew then that interferon alpha and gamma could  induce an enzyme, indole 2,3-dioxygenase  (IDO),  (IDO was known to be responsible for the inhibition of toxoplasma gondii by depletion of  tryptophan  in cells treated with gamma interferon) but we did not know then that this enzyme could contribute to the loss of T lymphocytes.   Another observation of historical interest is that even before AIDS was first reported in 1981, interferon was known to preferentially inhibit CD4 lymphocyte proliferation in mixed lymphocyte culture.

    Since immune activation and its effects, including  inflammation, are harmful if sustained,   there are mechanisms that can  dampen it.

    But in HIV disease, immune activation persists with continued deleterious consequences.

    The reason I’m revisiting this now is that there is a question that continues to be bothersome.

    HIV disease is not the only infection associated with long standing immune activation.

    Several endemic infections in Africa are also associated with sustained immune activation, certainly not all – some even have a dampening effect on immune responses. TB is another example of an infection associated with chronic immune activation.   In none of these conditions is there such a profound loss of CD4 lymphocytes as in HIV disease.  While individuals with active pulmonary  TB have been reported to have lower CD4 counts than healthy individuals, the numbers were well above 500.

    Is the difference between sustained immune activation associated with HIV and that associated with other chronic infections in HIV negative individuals a matter of degree – is it a quantitative difference?

    Could the  mechanisms that dampen and check  immune activation be impaired in HIV disease?   These mechanisms include the secretion of cytokines that have anti-inflammatory properties, such as IL-10, IL-13, and  TGF-beta, among others.  Specialized immune system cells can also dampen immune activation.  Tregs, a subset of T lymphocytes, have such a dampening effect.   Although there are conflicting reports on the relationship of Tregs to HIV disease, it is known that HIV targets some of  these particular T lymphocytes.

    This graphic comes from a previous post.

    In the diagram,  disease progression is represented by a circular  clockwise movement propelled by a positive feedback interaction between HIV replication and immune activation.   It can be accelerated by infections that contribute to immune activation, CMV in particular, but probably also  some endemic infections in parts of Africa.   CMV probably also has a positive feedback association with HIV in that it is more likely to be driven out of latency in the setting of HIV infection, and active CMV infections can enhance HIV replication by several mechanisms including their contribution to immune activation.   Some endemic infections probably also have analogous reciprocal interactions with HIV.    The influences that can slow the cycle are those mechanisms that dampen immune activation.   They include the effects of  Tregs, a subset of T cells with regulatory functions that  dampen immune responses, and the effects of cytokines with anti-inflammatory properties.

    In graphic terms, the speed of the clockwise circular movement will be the balance of forces that speed it up and those that slow it down.

    HIV disease progression is represented as moving clockwise in a circle, reinforced by sources of immune activation other than HIV and retarded by Tregs and other mechanisms that dampen immune responses.  Tregs  act as brakes, but HIV can directly make the brakes less effective.

    Could critical differences between HIV disease and other infectious causes of long standing immune activation where CD4 numbers are relatively preserved, be  the preferential targeting of Tregs by HIV and a different pattern of cytokine secretion?

    I wonder if this revised representation of HIV disease lends itself to a more formal modelling process.

    In this particular model a disease process is represented by a circular motion in a clockwise direction, with forces that both propel and retard it.  Some predictions can be made.

    The degree of immune activation at the time of HIV seroconversion would favour more rapid HIV disease progression.  The set point – the level from which CD4 lymphocytes decline following an acute HIV infection, would be lower, and the subsequent  rate of CD4 decline higher when HIV infection occurs in a person where there already is a higher degree of immune activation, compared to an individual where this is not the case.  There already is  some evidence in support of this possibility.

    It’s well established that HIV disease progresses more rapidly with increasing age.  Could an explanation for this be that immune activation increases with age – indeed, it’s been suggested that immune activation  contributes to the aging process.

    HIV disease progresses more rapidly in individuals with active TB.  CMV viremia was noted to carry an adverse prognostic significance in HIV disease very early in the epidemic.  There are but two  examples, but there are many more of  of a more rapid course of HIV disease in the setting of other  infections caused by bacteria, protozoa, viruses and helminthes.  Some are referred to in a previous post.

    Are Treg numbers at seroconversion and for a period immediately afterwards  related to subsequent disease progression?

    Could treatment with anti CMV agents during acute HIV infection retard subsequent disease progression?

    There already  is some evidence that treatment of HIV during acute infection might slow the subsequent course of HIV disease.

    The utility of any model of a disease process lies in its ability to provide a common explanation for disparate observations as well as to make predictions that can be tested by an analysis of available data or by experimentation.

    Viewing HIV disease as a process with a positive feedback interaction between HIV replication and immune activation with forces that both enhance and retard this interconnection,  provides a useful descriptive framework as well as testable predictions.

  • Interferon: Another Historical Digression

    Posted on May 10th, 2009 admin 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:

    1964 interferon article.

    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. [1]

    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.

    [1]  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.