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Past Issues
Volume 16, number 5
May 2002
Illuminations
How do we know what we know and why does it matter?
In Vitro Veritas?
A look inside the Immunologist's toolkit
Remembering Fred
We do not approve. We are not resigned.
Interpretations
What you see is what you get
Looking Where the Light Is
By Bob Huff
There is an old science joke about a drunk who is searching for
his car keys under a streetlight. A cop happens along, and after
appraising the situation, asks the drunk where he had last seen
the keys. The drunk points toward a dark alley.
"Then why aren't you looking over there?" asks the exasperated
cop. The drunk looks up and replies, "Because the light is better
over here."
We know what we do about AIDS from two basic kinds of experience.
Soon after the syndrome was first reported, clinical observation
quickly revealed that people with the disease tended to waste away
or were stricken with any of a number of unusual infections. Most
eventually died. At the same time research scientists started looking
for abnormalities in blood and tissue samples from AIDS patients,
searching for a cause. But the kinds of tests available at that
time were limited and the conclusions researchers could draw from
their laboratory experiments were general and few. They were simply
looking where they could shine a light. After twenty years, new
assays and experiments, producing high quality streams of data,
continue to illuminate the underlying causes of this disease. Nevertheless,
the workings of immunity are so complex and so difficult to observe
in living persons that darkness still shrouds many crucial aspects
of what goes wrong when HIV interacts with the human immune system.
History Lesson
From the dim beginnings of the AIDS crisis, researchers tried to
find a laboratory assay that would predict what was in store for
an individual at risk for acquiring the immune deficiency. In particular,
clinical researchers needed some kind of surrogate for disease progression
to spare patients having to get sick or die before determining if
an experimental treatment was efficacious. Surrogate markers are
stand-ins for other kinds of data that are either inconvenient,
take too long, or are otherwise impractical to collect. Ideally,
a surrogate marker should be minimally invasive — such as a
blood test — and be cheap, quick and simple to perform. It
should provide an unambiguous numerical read-out to indicate with
a high degree of certainty, exactly how sick someone is, then tell
whether they are getting better or worse. An ideal marker should
warn if symptoms are about to occur and report promptly if a course
of treatment is going to be successful or not. Finally, this fantastic
marker should give the same reliable results for everyone, no matter
what stage of disease or genetic makeup they have.
An ideal surrogate marker still does not exist for HIV/AIDS. In
fact, after two decades of looking, only two assays, CD4+ T-lymphocyte
count and HIV RNA viral load, have been widely adopted as imperfect
surrogates for monitoring and predicting the course of disease in
people with HIV. These markers have been fairly well correlated
with the natural history of HIV infection and progression to AIDS,
but each has limitations. For example, routine viral load testing
does a good job of reporting what is happening to virus levels in
the blood, but not in lymphoid tissue where HIV interacts most significantly
with the immune system. And it's our ability to assess and analyze
the effects of HIV on immunity that is most critically in need of
improvement. We still lack reliable assays that can report on immune
reconstitution under the effects of HIV treatment or that can tell
when the body is capable of controlling HIV with its own immune
resources.
Since the first recognized AIDS cases involved very sick people,
early clinical reports detailed mostly non-specific markers of infection
and impaired immunity such as skin antigen recall tests, erythrocyte
sedimentation rates, or increased white blood cell counts. A few
curious researchers working at the frontiers of intercellular signaling
reported elevated levels of interferon and tumor necrosis factor
in people with AIDS. Others, specializing in virology and antigen
recognition, reported increased levels of cytomegalovirus and herpes
virus particles in their patients' blood. These observations were
made using the best tools available at the time and the data obtained
trickled into the sketchy literature that was slowly accumulating
about the mysterious syndrome.
When first described in 1981, it was recognized that the opportunistic
diseases attacking a growing number of young, homosexual men, were
also those that afflicted people with genetic or chemotherapy-induced
deficits in a wing of the immune system called cellular immunity.
This branch of immunity depends on T-cells — white blood cells
that earned their name because they were observed to develop in
the thymus — and is responsible for eliminating diseased cells
that have been invaded by outside pathogens.
In the seventies, someone noticed that T-cells could be identified
and separated from other white blood cells by mixing them with red
blood cells (erythrocytes) from sheep. The sheep erythrocytes would
then cluster around the T-cells and form rosettes (E-rosettes) that
could be viewed and counted under a microscope. An assumption eventually
emerged that the sheep cells attached themselves to certain cell-surface
proteins that were characteristic of a subtype of T-cells called
the T-helper cell. The various receptors and coreceptors we now
know as CD4, CD8, etc., were then simply thought of as cell surface
antigens — as late as the mid eighties some papers still referred
to the newly identified CD4 receptor as the E-rosette receptor.
But even this rudimentary technique revealed that, in AIDS, T-helper
cells were disappearing.
The need for a quicker and more reliable measurement of disease
progression was urgent. Fatefully, AIDS started to appear just as
a new wave of technology for marking and automatically counting
immune cells was becoming more available. CD4 cell staining with
fluorescent antibodies and flow cytometry machines that could rapidly
and accurately count the tagged cells soon led to the establishment
of the CD4 cell count as a standard marker for the severity of HIV
disease. The CD4 count eventually proved to be useful for predicting
disease progression, warning of risk for opportunistic infections
(OI), serving as a diagnostic milestone for the onset of AIDS, and,
after an initial debate, as a surrogate for demonstrating that an
experimental antiretroviral therapy was likely to have clinical
benefit. Today, most guidelines for making decisions about starting
and stopping treatment and initiating OI prophylaxis continue to
rely heavily on CD4 counts.
However, absolute CD4 counts (along with the ratio of the number
of CD4 cells to CD8 cells, another important immune system metric,
still preferred by some clinicians) are at best somewhat crude measurements
of the effects of HIV on an individual's immune system. Within the
broad category of CD4 cells, there are many subtypes, each with
particular characteristics and roles to play in the orchestra of
immunity. And while simple CD4 counts are invaluable for making
treatment decisions, they cannot describe which subsets of cells
are affected by HIV infection or how well they are functioning.
Despite the ability of HAART to raise CD4 counts for many people,
concerns about drug toxicities, resistance and the difficulties
of sustaining adherence to HAART have brought new urgency to improving
the ways we have of measuring and understanding the complexities
of the immune system and for finding new ways of treating it.
Immune Therapies
Although an enormous amount of attention has been paid to antiviral
drugs — well deserved given the success they've brought —
most people who think about the future of HIV realize that finding
some way of enabling the body's own resources to fight, disable
or ignore the harmful effects of HIV would be a step forward as
great as that achieved by HAART. Usually, this hoped-for breakthrough
is imagined to be a vaccine. Theoretically, a vaccine would work
by priming the immune system to react in multiple ways against multiple
physical features of HIV, thereby overwhelming an infection before
it could run out of control. A dream vaccine would prevent everyone
who took it from ever becoming infected. But as the difficulties
of HIV vaccine development have become painfully clear, many researchers
are now aiming for a vaccine that would simply help keep an established
infection under control by enhancing the body's HIV-specific immune
capacity. For now, the outlook for vaccines remains gloomy.
Who's Story Is It?
Theories about the mysteries of nature are essentially stories
that take the observations, experimental evidence, and conventional
wisdom of the day and stitch them together into a coherent narrative.
In biology, perhaps faster than in other fields, these explanations
are revised as new evidence is gathered. But the force of an appealing
pathogenesis narrative can shape research agendas (as well as political
agendas) for years. The danger is that valuable time and resources
may be squandered pursuing compelling but misguided strategies built
on insufficient data.
For example, observations about the density of E-rosettes fueled
some of the first speculation about the possibility of immune-based
treatments for AIDS. A 1982 paper reporting on in vitro findings
announced, "Drugs which are able to modulate T-cell functions, such
as thymosin, transfer factor, isoprinosine É, also increase the
percentage of active T-rosettes." (Wybran J) These agents and other
purported immune modulators were experimentally used and periodically
tested throughout the rest of the decade, although they failed to
show any consistent clinical value. Interest in IL-2, another immune
regulatory messenger recognized during the first years of AIDS,
has never gone out of favor and large trials continue to this day.
Nevertheless, some have argued that the early, vigorous, focus on
antiviral therapy for HIV may have slowed work on immune-based studies.
In the early eighties the basic narrative of immunity went something
like this: During a cellular immune response, T-helper cells sound
the alarm for antigen-specific immune cells to start multiplying.
When the attack has gone far enough, other T-cells, called T-suppressor
cells, sound the retreat and start eliminating the now superfluous
antigen-specific attackers. Although some early researchers hypothesized
that HIV-mediated autoimmune processes were responsible for the
resulting immune dysregulation, by mid-decade, conventional wisdom
held that AIDS was directly caused by the virus, which, in the words
of one famous researcher, "kills T-cells like a Mack truck."
In recent years, we've learned that most depleted T-cells are never
actually infected with HIV, and a more sophisticated view of pathogenesis
is emerging. There is growing understanding that immunity is a system
of give and take, which, when regulated, works very well. We know
that over five to ten years or more, T-cell counts decline at a
slow but steady pace. Yet the picture of what happens to the turnover
of the body's immune cell inventory is still developing. Although
it's obvious that something is seriously wrong with the system of
immune regulation in AIDS, surprisingly little is known about what
actually causes T-cell stores to dwindle. One theory says that T-cells
are destroyed at a consistently high rate but are also replaced
at a similar rate, thus maintaining a rough balance. Over time,
the theory goes, the body's capacity to replace T-cells starts to
wear down, leading to their eventual depletion. But which part of
the T-cell replacement mechanism is wearing out and what is responsible
for wiping out the T-cells that disappear? New research tools are
slowly getting at these questions.
In this issue, Daniel Raymond catalogs some of the latest assays
that immune system scientists are using to pick apart the mysteries
of T-cell depletion. He also describes what their experiments are
telling us about how HIV might be doing its damage. Although these
studies are still in fairly early stages, it's possible to imagine
that, one day, a new approach to treating HIV could be developed,
perhaps using a drug that interrupts a crucial, very specific step
in HIV-mediated immune dysregulation.
Yet despite accelerating progress, new assays are not born and
accepted overnight. It may take years to validate initial observations
in competing labs. Then standards must be established before test
results can be used diagnostically or correlated with other assays.
So, although new assays may be casting new light onto the workings
of T-cell creation, destruction and replenishment, many of the explanations
constructed from this emerging evidence remain tentative and sometimes
contradictory. In 2002 we continue to look where the light is best
and make up stories for what we find that fit with what we already
know. Hopefully this dawning generation of new immune assays will
help us locate the sorely needed key to immune control of HIV.
Stalking HIV Immune Dysregulation
By Daniel Raymond
Immune assays are laboratory tests that attempt to measure various
aspects of the immune system such as function, quantity and stage
of maturation. Researchers are seeking correlations between these
markers and health in a desire to test and describe the potential
benefits of immune-based HIV therapies such as IL-2 and other immune
regulators, as well as vaccines that might have therapeutic potential.
Newer assays are also starting to shed light on the mechanisms behind
the immune depletion that occurs during HIV progression and those
involved with immune reconstitution after therapy. While these new
generation assays are still used exclusively in research settings,
the findings that they've made possible have already influenced
clinical practice by justifying experimental treatment strategies
such as Structured Treatment Interruptions (STIs) and antiretroviral
(ARV) therapy during acute HIV infection. Several of the assays
expanding our knowledge of HIV pathogenesis and HIV-specific immunity
are also contributing to important research on the role of immune
function in other diseases such as cancer. Because of these broader
implications, it is important to understand what these assays measure
and what they can tell us — as well as their limitations. While
immune assays have provided a wealth of data, the interpretations
of these data often remain contested.
Types of Immune Assays
The emergence of AIDS in the early 1980s created a number of challenges
for researchers and doctors. The first of these was to identify
the cause of AIDS, which eventually led to the isolation of HIV.
Subsequently a screening method to detect HIV infection was developed,
and the HIV antibody test entered into widespread use. But long
before there was any agreement that a virus was responsible for
the new syndrome, doctors had recognized that patients were losing
immunity to common pathogens because one of the crucial components
of a healthy immune systems was being selectively depleted: the
T-helper cell, now known as the CD4 cell.
One of the first and still most important tools for studying cells
was the fluorescence activated cell counter or sorter (FACS). These
devices let scientists count and separate populations of immune
cells by tagging them with special antibodies. These machines are
now in routine use in diagnostic labs around the world producing
thousands of CD4 counts for doctors and patients daily. But the
elements of FACS technology remain at the forefront of nearly every
immunologist's research toolkit.
Newer immune assays — some of which are not new at all, but
are only recently coming into use for HIV research — are allowing
scientists to probe deeper into the number and function of T-cells
and ask increasingly incisive questions. For example:
What kind of T-cell is it? Is it a naive or a memory cell? Can
the T-cell recognize and respond to HIV? How many of each of these
kinds of cells is present at any particular time?
What does the cell do? Is it in a resting or activated state? Is
it proliferating or dying? Is it functional — can it respond
to pathogen? Or is it a useless imposter masquerading as a viable
T-cell?
These newer immune assays are built on the key technologies that
made FACS technology possible in the late 1970s: monoclonal antibodies
and flow cytometry. Monoclonal antibodies were first developed in
1975 by fusing an antibody-producing B-cell with a long-lived cancerous
cell called a myeloma. The fusion created a hybridoma, an immortal
cell that could continually reproduce a single antibody specific
for a particular antigen. These antibodies could then be used to
detect the presence of a target antigen through various methods,
including the enzyme-linked immunosorbent assay, or ELISA test.
The standard HIV test uses ELISA to detect HIV antibodies in blood
or saliva. A tiny plastic well is coated with bits of antigen —
fragments of common HIV proteins that HIV antibodies will stick
to. Then the sample (e.g., blood) being studied is added; if HIV
antibodies are present in the sample, they will bind to the antigen.
Next, a monoclonal antibody, custom made to bind to HIV antibodies
and linked to a special enzyme, is added. Then the well is washed
out leaving only the enzyme-linked antibodies that have attached
to the patient's HIV antibodies. Finally, the enzyme's substrate
is added to the mix and wherever the enzyme remains, a color change
occurs, thereby revealing the presence of HIV antibodies. This is
a positive result. If the sample does not contain HIV antibodies,
the secondary antibodies (the monoclonal antibodies) will have nothing
to bind to, and the color won't change.
Monoclonal antibodies have a myriad of diagnostic and therapeutic
applications, including the ability to characterize cell populations
by sticking to cell surface proteins that are unique to certain
classes of cells. For example, some monoclonal antibodies can tag
CD4 proteins while others identify CD8 cell surface markers. CD
stands for cluster of differentiation, in that the CD4 molecule
differentiates T-helper cells from cytotoxic (cell-killing) T-cells,
marked with CD8. An array of multiple types of antibodies used in
combination can further identify a cell as having naive or memory
markers, resting or activated markers and so on.
Both the absolute and relative frequency of different cell types
in a blood sample can be measured by various elaborations of the
basic flow cytometry techniques developed in the 1970s. Antibodies
labeled with a fluorescent dye are mixed with a blood sample containing
all kinds of cells. But only the kind of cell that is being counted
is tagged with the fluorescent antibody. The fluid of the sample
is passed down a very narrow channel that is only wide enough for
one cell to pass through at a time. A laser beam briefly illuminates
each cell in the column. When the laser strikes a fluorescing antibody
stuck to a cell, light will be emitted at a signature wavelength
that can be recorded by a detector and counted as one cell. More
advanced versions of this technology actually allow the counted
cells to be shunted into a separate channel for collection. This
is called cell sorting and is an extremely powerful function of
FACS technology. In HIV research, FACS analysis can be used to isolate
particular subsets of cells for further analysis and manipulation.
The newest generation of cell counters and sorters use more than
one colored laser and can detect several subtypes of cells all in
one pass by using multiple kinds of antibodies and special fluorescent
dyes. Monoclonal antibodies and flow cytometry have revolutionized
molecular biology and over the years increasingly detailed portraits
of cell subsets in HIV infection have been produced using variations
on these methods.
The next revolution, now underway, will come from the development
of extensive matrices of tiny DNA detectors called microarrays that
can reveal which genes are active in cells and when they become
activated. (See Microarrays
on the Horizon) This promises to bring previously unimagined
resolution to what actually happens inside cells as they mature
and adopt new functions. While the full potential of this technology
is years away, in the meantime, researchers have a variety of methods
at their disposal to measure the dynamics of immunity.
The ELISA Test
Enzyme Linked Immunosorbant Assay
Characterizing T-cell Populations
As we've seen, T-cells can be separated and counted based on cell
surface markers. Besides binding to the manmade antibodies in the
assay systems, these molecules on the surface of T-cells have functions
in the normal life of the cell. Cell surface proteins may bind to
messenger proteins produced by other cells, which in turn sends
a series of signals that may activate a gene in the cell's nucleus.
Take the CD4 and CD8 cell surface markers. As a normal part of immune
function, both of these CDs work as coreceptors helping the main
T-cell receptor to recognize and respond to foreign antigens that
ought to be eliminated. In HIV infection, the typical ratio of CD4
to CD8 cells in a healthy person (about 2:1) becomes inverted as
CD4 cells are depleted. And as CD4 cells dwindle, the risk of falling
victim to one of the opportunistic infections of AIDS is increased.
While CD4 and CD8 are two of the best-known cell surface markers,
currently, over 200 different CDs have been described — with
certainly more to come. The job of figuring out what all of these
proteins do will take a great deal of additional research with new
techniques and assays.
Lymphocyte Traffic
Naive, Effector, and Memory Cells
T-cells, like all blood cells, start life in the bone marrow as
progenitor stem cells. The T-cell line spins off and migrates to
a specialized collection of cells called the thymus, a kind of finishing
school for immune cells. In the thymus, each individual T-cell develops
a receptor with a highly specific affinity for the size and shape
of a particular protein fragment. The look and feel of this fragment
is called its epitope. After leaving the thymus, before the cell
has encountered its antigenic mate, the T-cell remains naive.
When a naive T-cell encounters an epitope that matches its receptor,
a process of activation and proliferation takes place. The cells
produced by proliferation are called effector cells. CD4 effector
cells are cells that help guide the immune response; CD8 effector
cells are called cytotoxic T-lymphocytes (CTLs) and are responsible
for killing infected cells. After an infection has been controlled,
most effector cells die, but a small number of memory T-cells are
left behind. The memory cells now have hair triggers for when they
next encounter their antigen and are able to react with a stronger
and more rapid response than their naive predecessors. naive, effector
and memory T-cells are distinguished in the laboratory by the particular
subtype (called isoform) of CD45 cell marker on its surface. Cells
with the CD45RA isoform are naive; effector and memory cells express
the CD45RO isoform. Immunologists use a kind of shorthand to refer
to the two types of cells. naive cells are described as CD45RA+
(+ as in positive), or alternatively as CD45RO-, or lacking the
RO isoform. This ability to distinguish between naive and effector
or memory cells has opened the door to observations that HIV infection
causes a significant depletion of the pool of naive CD4 cells, while
at the same time, most of the HIV-infected but surviving cells are
memory cells. This surprising finding has raised an intriguing question:
If HIV is not present in substantial numbers of naive cells, then
how and why is the naive pool being depleted? The classic assumption
that T-cells die as a direct result of HIV infection — with
depletion occurring as either HIV or the immune system kill off
the infected cells — can't explain the deficit in naive cells.
This observation has opened up much research and speculation about
the role of immune activation and thymic output
But the picture becomes even more complicated as some features
of some memory cells confound this interpretation. Under certain
conditions, memory cells seem to revert to the naive type (CD45RA+),
though there is other evidence this reversion may be illusory. Furthermore,
a different cell surface protein called CD27, a costimulatory molecule,
has been proposed as a possible means of differentiating between
naive, effector, and memory cells. At this point we need a map:
naive: CD45RA+ CD27+
Effector: CD45RA+ CD27-
Memory: CD45RO+ CD27+
The CD45RO+ CD27- cells are terminally differentiated and unable
to proliferate; increased numbers of these dead-end cells are associated
with certain immune disorders as well as with aging. Another complication
is the discovery of two types of memory cells — central memory
cells, which can travel through lymphoid tissue and are marked by
the chemokine receptor CCR7, and effector memory cells, which do
not enter the lymph nodes and are CCR7-. However, it remains unclear
whether these represent two distinct subsets of memory cells or
two different phases, since memory cells may acquire and shed cell
surface markers as needed.
Activation Markers
Activated T-cells express various markers, which play different
roles in the activation process; for instance, some markers provide
a costimulatory signal when the T-cell receptor binds to antigen,
while others help the cell to bind more strongly to another cell,
as in the case of CTLs binding to a cell targeted for destruction.
T-cell activation can be studied by looking for activation markers,
though again there is not always a direct correlation between activation
and the presence of a marker. Some markers are expressed during
different stages of activation, so that an assay measuring a marker
expressed late in activation may underestimate the amount of activation.
Other markers are also expressed in low levels on resting cells;
during activation their expression increases and they appear on
the cell surface in higher concentrations
Given the variety of activation markers, some studies use more
than one marker to get a fuller picture of activation rates. A growing
body of research is demonstrating that in HIV infection, overall
activation levels are higher than those seen in HIV- controls. This
has led to the hypothesis that activation-induced cell death rather
than direct cell killing of HIV-infected cells drives T-cell depletion.
Activation-induced cell death (AICD) occurs following rapid proliferation;
most of the effectors cells are short-lived, surviving only for
the time it takes to perform their role in bringing an infection
under control. In turn, the AICD hypothesis has prompted speculation
that drugs that suppress immune activation, such as cyclosporin
A, may paradoxically slow T-cell depletion. Studies are underway
to explore this possible therapeutic strategy.
Other common activation markers:
CD25 (high levels): the alpha chain of the IL-2 receptor,
which stimulates proliferation
CD38 (high): regulates activation and proliferation
CD69: early activation marker
CD70: binds to CD27; may have costimulatory role
CD95: also known as Fas; mediates cell death signals
CTLA-4: Cytotoxic T-lymphocyte-associated protein 4, also
classified as CD152; downregulates activation and proliferation;
competes with CD28, a costimulatory molecule — both bind
to B7 (CD80, CD86)
HLA-DR: Major histocompatibility complex molecule; used in
antigen presentation |
Recent Thymic Emigrants
Another open question is whether T-cell depletion is solely due
to death of T-cells, or might also reflect a decrease in the production
of new T-cells. Recall that T-cells are produced in bone marrow
and mature in the thymus. All T-cell progenitors, or thymocytes,
start with the same set of genes for the T-cell receptor. These
genes contain all of the possible varieties of T-cell receptors
that the individual can produce; the potential tremendous variety
means that a huge number of antigens can be recognized and dealt
with. While in the thymus, T-cell progenitors undergo a random shuffling
of genes to select a specific T-cell receptor for each cell, a process
that results in an enormous diversity of T-cell receptors. As a
byproduct of this shuffling, the unused T-cell receptor genes assemble
into loops of waste DNA called TRECs, or T-cell receptor rearrangement
excision circles. TRECs remain inside the naive cells after they
leave the thymus and begin circulating through the blood and lymphatic
tissue. Since TRECs are not reproduced during cell division, their
presence can help distinguish between new cells straight from the
thymus and cells subsequently reproduced through proliferation.
Because the size of the thymus was observed to shrink with age,
it had long been believed the thymus no longer actively produced
new T-cells after adolescence. But tracking TRECs has helped show
that the thymus can continue to produce new cells throughout adulthood,
though output slowly decreases with age. Still, analysis of TRECs
in HIV infection has yielded conflicting results, with some studies
finding a decline in thymic production over the course of HIV infection
that is reversed with HAART, and others observing no change other
than the normal slowdown that comes with age. Some researchers have
raised the possibility that the thymus compensates for T-cell loss
by increasing its production of replacement cells, although this
theory remains controversial.
Part of the difficulty lays in interpreting TREC levels. Since
TRECs are measured as the proportion of T-cells containing the excision
circles, their ratio relative to the overall number of cells becomes
diluted as existing cells proliferate, thereby reducing the relative
number of TRECs found. If cells are proliferating rapidly, then
declining TREC levels would not necessarily reflect a decrease in
thymic output. This means TREC results have to be analyzed in the
context of immune activation. TREC levels may also be affected by
cell death; in untreated HIV infection T-cell half-lives are shorter,
and TREC levels in HIV-positive populations may underestimate thymic
output. TREC data, suggestive as it is, has not yet provided conclusive
evidence for an impairment of thymic production in HIV infection.
Many questions concerning the role of thymic output in HIV disease
remain unresolved despite evidence for direct HIV infection of and
replication in the thymus. Enlarged thymic tissue has been observed
in some (but not all) people with HIV — although very often
those who experience stronger CD4 rebounds following HAART. This
supports the idea that, in at least some cases, there may be a mechanism
that allows the thymus to increase production in response to T-cell
depletion. The potential benefits of treatments proposed to increase
thymic production, such as IL-7 and recombinant human growth hormone
may be evaluable by determining whether these treatments increase
TREC levels.
TREC Analysis
TREC dilution shows clonal expansion
Examining T-cell Dynamics
When T-cells encounter antigen, they become activated and begin
proliferating. Ultimately, most of the newly produced cells will
be removed by programmed cell death, or apoptosis. Assays that measure
proliferation and apoptosis are also yielding new insights into
pathogenesis.
Proliferation
When a cell divides it begins a cycle with several distinct phases
as the cell's DNA is unwound, copied, separated into two nuclei
and ultimately into two new cells. These stages are referred to
as G1, S (the DNA synthesis phase), G2, and M (the mitosis, or cell
division, phase). Ki-67 is a nuclear protein expressed during late
G1, S, G2, and M phases of the cell cycle; following proliferation,
the cell ultimately returns to a resting, or G0, state, where Ki-67
is not detectable. Detection of the Ki-67 antigen by monoclonal
antibodies has therefore been used as a generalized marker of proliferation.
One limitation of this marker is that cells arrested during the
cell cycle will also contain Ki-67 even though they are not actively
proliferating. HIV has been observed to cause cell cycle arrest
in infected cells; its effect on the validity of Ki-67 as a marker
for proliferation is unknown.
Ki-67 staining has demonstrated that proliferation rates increase
during HIV infection, and decrease upon treatment with HAART. These
studies shed light on an important question in HIV pathogenesis:
Is increased proliferation an attempt to compensate for cell death
and maintain a homeostatic balance of cells? Or does proliferation
result from increased activation, sending cells into the cell cycle?
Homeostasis describes the normal dynamics of T-cells: cell death
and the creation of new cells (by the thymus or through proliferation)
balance each other out, so that the overall number of T-cells remains
stable. Even in HIV infection, the immune system remains in a relative
state of homeostasis and T-cell decline is generally slow —
it takes, on average, 10 years following infection to completely
lose the balance between influx and destruction and progress to
AIDS. Therefore, at any given time, losses due to cell death are
roughly offset by gains in new cells.
Ki-67 levels increase as CD4 counts decrease, indicating that as
CD4 cells are removed, proliferation speeds up. While this could
suggest that cells are proliferating as a result of depletion (arguing
for a homeostatic response), when people with low CD4 counts begin
therapy with HAART, the Ki-67 signal, and therefore proliferation,
immediately begins to decrease, even before substantial gains in
CD4 counts are observed. This would argue for HIV-induced activation
as the cause of increased proliferation.
Improved techniques have been developed to directly label cells
produced during proliferation. Bromodeoxyuridine (BrdU) and deuterated
glucose assays were developed as alternatives to the classic [3H]thymidine
test, a radioactive assay long considered the "gold standard" for
measuring proliferation. The newer assays can be used with flow
cytometry techniques that allow for sorting and quantifying the
proliferating cell types. BrdU and deuterated glucose are typically
administered for a set period of time, during which all new cells
undergoing synthesis incorporate the labeled molecules into their
DNA. Thus labeled, proliferation can be quantified (using monoclonal
antibodies or mass spectrometry) and the longevity of marked cells
can be measured by sampling over time. BrdU labeling correlates
well, though not perfectly, with Ki-67 levels. Recent studies have
demonstrated that proliferation rates are elevated in people with
HIV, but begin to normalize upon treatment with HAART. Decay rates
(the disappearance of labeled cells, implying cell death) are also
increased for CD4 cells associated with HIV infection, suggesting
rapid turnover, at least among a subset of proliferating cells.
The extent of proliferation can also be measured indirectly by
examining telomere lengths to gauge the replicative history of cells.
Telomeres are DNA sequences that cap the ends of chromosomes to
preserve their physical integrity during cell division. Telomere
lengths tend to shorten after each cycle of DNA synthesis. This
can ultimately lead to replicative senescence — when the telomeres
become too short to guarantee that the cell can successfully divide.
Once again, this picture is complicated by the ability of an enzyme
called telomerase to extend telomere length during DNA synthesis
and offset the losses during replication.
Telomeres can be measured to determine an average length, thus
providing an estimate of how many replication cycles a cell has
undergone — the shorter the average telomere length, the more
replication it has seen. Telomerase activity can also be measured
in cells, to determine whether there is compensation for telomere
shortening. Assays have compared telomere lengths in people with
and without HIV, finding that while telomeres are shortened in CD8
cells during HIV infection, no significant shortening occurs in
CD4 cells, with other assays ruling out increased, compensatory
telomerase activity as an explanation. This contradicts one proposed
model explaining CD4 cell depletion as a consequence of exhaustion
due to rapid turnover in HIV infection; if that were the case, telomere
lengths should be significantly shortened. Another factor that would
influence average telomere length is thymic production of naive
cells. Since naive cells haven't undergone proliferation, they will
have the longest telomere lengths. Therefore, a change in thymic
output could affect average telomere length — decreased output
would lead to a shorter average length, while increased output would
extend the average length. This suggests the importance of using
multiple assays — i.e., combining telomere assays with TRECs
— in order to guide the interpretation of results.
The Cell Cycle: Four Phases of Cellular Division
| Overview
of Immune Assays |
|
| T-cell Subsets
Assay |
What it measures |
Technical notes |
|
|
|
Naive
Effector
Memory |
CD45RA+CD27+
CD45RA+CD27-
CD45RO+CD27+ |
Uses monoclonal antibodies and flow cytometry;
measures absolute number but not function |
| Activation Markers |
CD25 (high levels); CD38 (high); CD96; CD70;
CD 95; CTLA-4; HLA-DR |
Uses monoclonal antibodies and flow cytometry;
tagging multiple markers increases sensitivity |
| TRECs |
Recent thymic emigrants |
Uses PCR; numbers may be diluted against a background
of increased cell proliferation |
| T-cell Dynamics
Assay |
What it measures |
Technical notes |
| Ki-67 |
Proliferating cells |
Uses monoclonal antibodies; provides a snapshot
of levels of proliferation; also stains non-proliferating cells
arrested in cell cycle |
| BrdU; deuterated glucose, deuterated water |
Cell proliferation |
Directly measures cells produced through proliferation;
requires multiple visits and blood draws |
| Telomere length |
Replicative history |
Indirect measure of the extent of prior cell
proliferation; interpretation complicated by potential telomerase
activity, or changes in thymic output |
| TUNEL; Annexin V |
Apoptosis |
Can be used with flow cytometry to characterize
apoptotic cells |
| T-cell Function
Assay |
What it measures |
Technical notes |
| Delayed-type hypersensitivity (DTH) |
T-cell memory response to recall antigen |
In vivo assay; requires two visits 2 – 3
days apart; measurement somewhat subjective, and therefore hard
to standardize; does not necessarily predict effectiveness of
immune response |
| Lymphoproliferative response (LPR) |
Proliferative capacity of antigen-specific T-cells |
Storage and treatment of samples can negatively
affect results, so samples should ideally be analyzed at an
on-site lab; requires a control to determine non-antigen-specific
proliferation (background proliferation); time-consuming |
| Tetramers |
Antigen-specific CD8 T-cells |
Highly sensitive; measures both functioning and
non-functioning cells; requires knowledge of HLA alleles and
matching epitopes; may not pick up responses restricted through
other alleles |
| ELISpot; Intracellular Cytokine (ICC) staining |
Antigen-specific T-cell response |
Measures only functioning antigen-specific CD4
and CD8 cells; does not detect anergic cells; may detect partially
functional cells; ELISpot may be more sensitive, though ICC
staining can be used with flow cytometry. |
Apoptosis
Apoptosis (programmed cell death) can be triggered in multiple
ways but follows a tightly scripted series of events once induced.
These events include chromatin condensation, changes in membrane
permeability, release of cytochrome-c from the mitochondria, caspase
activation, and DNA degradation. All of these can be measured with
varying degrees of sensitivity and specificity. Again, using two
or more assays can increase the reliability of results.
TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)
measures fragmented nuclear DNA, which is a late-stage event in
apoptosis following cell surface and nuclear morphological changes
— meaning that this assay may not detect cells in early stages
of apoptosis. However, it is considered very sensitive. DNA fragmentation
is not unique to apoptosis; it also occurs in necrotic cells (necrosis
is "accidental" cell death resulting from injury), so TUNEL assays
used alone may not be highly specific; specificity is increased
when used in combination with another apoptosis-specific assay,
such as immunohistochemical labeling of active caspase-3 (Cytosolic
Aspartate-Specific Protease 3) — an enzyme involved in the
disassembly of apoptotic cells. One review of various apoptosis
assays found that TUNEL was more reliable based on specificity and
its correlation with microscopic observation of cells. Another common
assay uses Annexin V, a protein that binds with high affinity to
phosphatidyl serine, a molecule that moves from the inner cell membrane
to the outer membrane during cell death. Both sensitive and specific,
Annexin V can detect cells in early stages of apoptosis.
Researchers generally agree that the majority of apoptosis in HIV
infection happens in uninfected cells, with a variety of potential
mechanisms proposed. Viral proteins vpr, tat and nef have been implicated
(though vpr may also inhibit apoptosis), as has the binding of gp120
to the CD4 receptors of uninfected cells. HIV protease may play
a role in apoptosis of infected cells, perhaps by increasing degradation
of Bcl-2 (a key apoptosis inhibitor). HIV infection can also increase
Fas expression, Fas susceptibility, and Fas ligand expression (Fas,
or CD95, is a major pathway inducing apoptosis). HAART reduces apoptosis
levels, with some question as to whether protease inhibitors in
particular play a direct role (perhaps by inhibiting caspases).
Despite the increasing body of knowledge about the role of apoptosis
in HIV infection, many questions remain.
Measuring Immune Response
Many of the assays discussed so far can distinguish between different
kinds of T-cells, but they don't measure T-cell function —
whether the cell can mount an effective response to antigen. When
T-cells respond to antigen, they proliferate and produce various
chemical messengers called cytokines and chemokines, several of
which can be measured under laboratory conditions. In HIV research,
these lab assays are of particular interest for evaluating the activity
of immune cells that would normally be expected to attack and eliminate
infected cells. Defective HIV-specific immunity — the flawed
ability of T-cells to react to HIV — may be the main reason
that humans ultimately fail to control HIV infection. Assays of
HIV-specific immunity are also expected to become useful for the
evaluation of anti-HIV vaccines and other immune-based therapies.
Nonetheless, these tests can only provide an indirect picture of
immune function, and conclusions about their meaning must be approached
with caution.
Delayed-Type Hypersensitivity
Delayed-type hypersensitivity, or DTH, was one of the first tests
developed to assess immunological memory. The DTH response was discovered
over 100 years ago in an attempt to develop a tuberculosis vaccine.
Robert Koch observed that about two days after injecting a killed
tuberculin preparation under the skin, an inflammation in the form
of a small swelling, or hard bump called an induration appeared
in people who had previously been exposed to the tuberculin bacillus.
Later it was determined that indurations were caused by T-cells
and other white blood cells rushing to the injection site to attack
the invader. The DTH test in current use measures the diameter of
the induration to gauge the presence and extent of immune memory.
The absence of an immune response, indicated by little or no induration,
is referred to as anergy (a state of immune system nonresponsiveness).
DTH tests can measure general immune responsiveness as well as
responses to particular antigens. General immune responsiveness
evaluates the ability in vivo to mount a reaction to several common
antigens that virtually everyone has been exposed to sometime in
their past. Theoretically, anyone with a healthy set of memory T-cells
will trigger a recall response. Tetanus toxoid, candida albicans,
and mumps are commonly used as recall antigens in a general test;
one version, the Multitest CMI, probes for responses to seven different
antigens. The progression of HIV disease can sometimes be predicted
by DTH response, with diminished responses, or anergy, signaling
an increased risk for experiencing an opportunistic infection. By
the same token, the reappearance of DTH response has also been used
to demonstrate immune reconstitution following antiretroviral therapy.
In theory, DTH tests using MAC and CMV antigens could help determine
when it is safe to discontinue OI prophylaxis following a HAART
associated rise in T-cells, as long as a recall response indicated
that the immune system has regained the ability to keep these infections
under control. This approach remains hypothetical, however; DTH
tests for these pathogens are not routinely available, and no potential
for guiding clinical practice has been established.
DTH tests may also prove useful for examining responses to HIV
vaccine candidates. If a vaccine has properly primed an individual's
memory T-cells with its antigens, injecting a protein fragment under
the skin similar to that expressed in the vaccine should elicit
an immune response; the appearance of an induration at the injection
site would demonstrate that the vaccine has had some effect. However,
while DTH testing might be useful in eliminating vaccines that fail
to generate immune responses, the simple presence of a DTH reaction
can not demonstrate that a vaccine will be protective — just
because a few T-cells respond to one isolated protein fragment doesn't
mean that the entire system can successfully fight off an HIV infection.
Lymphoproliferative Response
Lymphoproliferative response (LPR) is another way of measuring
the ability of CD4 cells to respond to antigen. In this assay, an
individual's T-cells are cultured in the lab then mixed with an
antigen plus a radioactively labeled building block of DNA called
3H-thymidine. If the antigen stimulates the cells to proliferate,
the new cells will incorporate the radioactive thymidine into their
chromosomes. The amount of labeled thymidine detected in the cells
gives a quick indication of the power of the antigen to stimulate
a proliferative response. This measurement is reported as a stimulation
index. LPR can assess the general function of one's immune response
by culturing cells with multiple antigens. A decrease in LPR generally
correlates with HIV disease progression while immune reconstitution
following HAART is associated with an increase in LPR. Improved
LPR after immune reconstitution supports the view that CD4 cell
recovery represents both a quantitative and qualitative restoration
of immune function; in other words, not only are more T-cells available
than before treatment, but they also do a better job of responding
to antigen. This observation underlies the rationale for discontinuing
prophylaxis for OIs such as PCP, MAC, and CMV following sustained
improvement of CD4 counts.
LPR assays can also be used to assess HIV-specific immune responses.
To do this, CD4 cells are cultured with one or more viral proteins
and the ability of the cells to respond and proliferate is measured.
It is generally thought that HIV-specific immune response is lost
during early infection, perhaps due to HIV preferentially infecting
and killing HIV-specific CD4 cells. The rationale for initiating
ARV treatment during acute HIV infection gained support after research
showed that early treatment might help preserve a strong HIV-specific
CD4 response. It is hoped that if this response can be preserved,
then progression to disease may be delayed. Several studies have
measured whether HIV-specific immune responses can be influenced
by certain treatment strategies, including periodic treatment interruption.
Although some research has indicated increased HIV-specific LPR
in people with chronic HIV infection after undergoing structured
treatment interruptions (STIs), these responses tended to quickly
disappear, apparently providing only limited control of viral replication.
Treatment with HAART during chronic infection also fails to restore
HIV-specific immunity as measured by LPR; in fact, LP responses
to HIV antigens tend to decrease to the point of disappearance as
long as HAART mediated viral suppression is sustained. This finding
suggests that the survival of HIV-specific memory cells requires
at least some antigenic exposure to HIV and that during effective
treatment, there may not be enough circulating HIV to keep the HIV-specific
cells in play. Research currently underway will explore whether
therapeutic vaccines or repeated cycles of treatment interruption
that allow periodic, controlled viral rebound might possibly stimulate
more durable HIV-specific CD4 cell responses.
Tetramers
Tetramer staining assays are producing a revolution in our understanding
of cellular immune responses. This technique allows antigen-specific
CD8 cells to be directly detected and quantified. Introduced in
1996, the tetramers assay used a new method to bind antigen to CD8
T-cell receptors. Major Histocompatibility Complex (MHC) is a cell
surface molecule that displays antigen and is required for binding
with T-cell receptors in vivo. Under laboratory conditions CD8 cells
bind only weakly to antigen complexed with MHC. However, a specially
constructed four-part complex of the antigen with MHC — the
tetramer — allowed the antigen complexes to bind avidly with
CD8 cell receptors, which can then be tagged and counted by flow
cytometry. The technique is now widely used to identify and count
HIV-specific cytotoxic T-lymphocytes (CTLs), especially in studies
of HIV progression. Although tetramers able to bind to CD4 cells
have been synthesized, their ability to effectively measure T-helper
response has not yet been established.
Tetramer assays were crucial in establishing the role that HIV-specific
CTLs play in the body's immune control of HIV. Studies in HIV-infected
long-term nonprogressors and in monkeys suggest correlations between
greater numbers of HIV-specific CTLs, slower disease progression,
and lower viral loads. Over the course of infection, CTL numbers
tend to decline as viral load rises. According to one study, although
CTLs specific for HIV and Epstein-Barr virus persisted during chronic
infection and could be detected by tetramer staining, the lymphocytes
lacked the ability to proliferate and kill infected cells, possibly
due to a lack of sufficient numbers of functional CD4 cells to trigger
them. Yet, as we've seen, HIV-specific CTLs virtually disappear
during successful HAART treatment, suggesting that HIV-specific
CD8 populations are not maintained in the absence of circulating
virus. Interrupting HAART has been shown to increase the number
of HIV-specific CTLs in circulation, which some have hoped can bolster
at least partial immune control of the virus.
ELISPOT and Intracellular Cytokine Staining
ELISpots tests are derived from ELISA techniques and are used in
HIV research to quantify antigen-specific CD4 and CD8 responses
by measuring the amounts of various cytokines produced. When a T-cell
is stimulated after encountering its antigen, it begins to produce
several messenger proteins. In an ELISPOT test, a plastic well is
coated with antibodies to the cytokine of interest (for instance,
interferon-gamma, or IFN-g, which is secreted by CD4 and CD8 cells).
A layer of cells that have been exposed to the antigen in question
are placed in the wells and incubated. If any IFN-g is produced
in response to the antigen, it will bind to the antibodies attached
to the plastic well. After washing the well to remove excess cells,
a chemical is added that binds to the IFN-g forming colored spots
that highlight the areas in which cytokines have been secreted.
The number of spots counted gives an indication of the number of
antigen-specific T-cells found in the original sample.
Intracellular Cytokine (ICC) staining goes one step further by
measuring cytokine production on the inside of CD4 and CD8 cells.
Cells are chemically frozen and then treated with a detergent that
makes their membranes permeable to fluorescent monoclonal antibodies.
Once inside the cell, the antibodies tag the cytokines the researcher
is looking for. Cells are exposed to antigen then processed to determine
which have produced cytokines in response. As an additional step,
the internally tagged cells can be sorted and counted by flow cytometry.
This allows responding cells to be characterized in multiple, highly
specific ways.
Unlike tetramer assays that can distinguish between CD8 cells that
may or may not be able to kill their targets, the ELISpots and ICC
staining assays only identify functional cells. For this reason,
ELISpots and ICC assays are used in conjunction with tetramers to
develop a more complete picture of HIV-specific CTLs. Tetramers
identify a cell's specific antigenic match and the ELISpot and ICC
tests report on the strength of the cell's response to that antigen.
Although ELISpot and ICC assays are most commonly performed on blood
samples, a recent study found that the majority of HIV-specific
CTLs reside in the lymph nodes, and that the number of those cells
declined less than those in the blood during treatment with HAART.
ELISpot and ICC staining are revealing much about the role of cell-mediated
immunity in HIV infection. According to one study that measured
IFN-g production in response to p55 antigen (gag), on average, about
0.12% of circulating memory CD4 cells from HIV infected individuals
were HIV-specific. This proportion was about one-tenth the frequency
of CMV-specific CD4 cells, implying a significant deficit in the
HIV-specific CD4 subset. While nonprogressing individuals tended
to have a somewhat higher average proportion of HIV-specific CD4
cells (0.40%) than did progressors, the correlation was neither
absolute nor exclusive. This suggests that cell counts alone cannot
fully account for individual differences in resistance to disease
progression or immune control of the virus. Another study measuring
IFN-g production found that a population of HIV-specific CD4 cells
persisted during chronic infection but that they were unable to
proliferate in an LPR assay. This inability to proliferate was correlated
with disease progression. In contrast, HIV-specific CD4 cells from
nonprogressors retained the ability to proliferate in response to
antigen.
Further evidence of functionally impaired HIV-specific CD4s came
from a study that examined production of IFN-g (through ELISPOT)
and IL-10 (through ICC staining) in response to gag antigen. IL-10
production was higher in people with progressive HIV disease compared
to non-progressors and seronegatives, and high levels of IL-10 production
were associated with little or no production of IFN-g in response
to gag. IL-10 has been shown to induce T-cell anergy, downregulating
IFN-g production and the proliferation of activated cells. In this
study, the IL-10 producing cells were not necessarily HIV-specific,
which leaves open the possibility that the gag antigen itself may
induce IL-10 production. Treatment with HAART reversed this effect,
restoring IFN-g production and decreasing IL-10 levels.
ICC staining in combination with tetramer analysis has also identified
functional defects in HIV-specific CTLs. When CTLs are activated
by antigen, they secrete IFN-g, tumor necrosis factor a (TNF-a),
macrophage inflammatory protein b (MIP-b), and perforin, a protein
that punches holes in the membranes of infected cells targeted for
CTL-mediated killing. One study compared expression of these substances
in CMV-specific and HIV-specific CD8 cells, finding that while cytokine
secretions were similar, HIV-specific CTLs expressed much lower
levels of perforin. A cell-killing assay was then used to measure
the cytotoxic function of the CTLs. In this lab assay, target cells
are labeled with 51Chromium (51Cr) then mixed with antigen (HIV
or CMV) to infect the target cells. CD8 cells are then added and
their ability to kill the infected cells is assessed. As target
cells are killed, they release 51Cr, which can be measured to determine
how many infected cells were killed by CTLs. In this study, HIV-specific
CTLs were less effective at killing infected cells than the CMV-specific
cells were, a finding consistent with reduced perforin expression.
These HIV-specific cells also carried high levels of a cell surface
protein called CD27, a marker indicating that these CD8 cells had
not become fully mature effector cells capable of killing their
target cells.
Limitations of Functional Assays
Assays such as tetramers, ELISpots, and ICC staining have been
widely used in research aimed at strengthening or restoring HIV-specific
immune function, particularly in STI and vaccine studies. However,
these assays do not measure immune response in the body, and there
is significant controversy about how relevant these in vitro models
are to the dynamics of immune function in vivo. For instance, in
light of the possible defect in perforin expression, studies measuring
IFN-g production by CD8 cells in response to stimulation with HIV
antigen may overestimate their functionality. Similarly, tetramer
analysis requires identification of well-characterized human leukocyte
antigen (HLA) types that will produce MHC molecules capable of presenting
specific HIV antigen fragments. This means that the value of tetramer
assays is currently limited to studying populations of individuals
with HLA types that are clearly identified and relatively homogenous.
This also requires a clear understanding of which MHC molecules
are able to present specific antigenic fragments. To complicate
matters, the ability of HIV to develop mutations able to escape
CTL response has been observed. Finally, all of these assays use
laboratory standard, wild-type viral antigens, which may not be
representative of the viral strains existing in study patients.
This too could possibly result in an overestimation of functional
immune response.
Dr. Allan Landay of Rush Medical College, chair of the Federal
AIDS Clinical Trials Group's (ACTG) immunology committee, has suggested
that an ideal measure of HIV-specific immunity might be an integrated
assay of immune function that examines all of the cellular players
— antigen-presenting cells, CD4 cells, and CD8 cells —
simultaneously. Dr. Landay also emphasizes the desirability of culturing
the viral populations actually present in a patient's body to determine
whether individuals can mount an effective immune response to their
own HIV infections. While work toward this ideal is underway, such
a comprehensive assay does not currently exist and may not appear
in clinical use for many years.
Microarrays on the horizon
The sequencing of the human genome has opened up vast new
areas of scientific inquiry. Microarray assays are one of
the pioneering methods for using genomics data for new research
opportunities. When a gene is expressed, cellular machinery
transcribes messenger RNA (mRNA) from the DNA sequence encoding
the gene. This mRNA is then translated into a specific protein
or enzyme, according to the function of the gene. All cellular
functions begin with DNA transcription. By looking at which
mRNAs are present in a cell, you can determine what the cell
is doing, assuming that you know the function of the gene
encoded. This is the concept behind microarrays, sometimes
referred to as gene chips or DNA chips by analogy to the fabrication
process for computer chips. There are different methods for
constructing a microarray and different techniques for applying
genetic information to the chip, or surface, but they all
follow similar principles. In one version, small samples of
DNA representing the coding regions of various genes in question
are bound to a glass microscope slide to create a microarray.
Thousands of genes can be represented on a single slide in
this manner. Then, mRNA is extracted from the cells being
studied and reverse-transcribed into complementary DNA (cDNA),
which is labeled with a fluorescent dye. The cDNA preparation
is hybridized with the slide, with each cDNA binding to its
respective gene and forming fluorescent spots. Gene expression
is then measured with a scanning laser microscope and analyzed
by computers; the intensity of fluorescence indicates the
level of gene expression.
HIV research is only beginning to incorporate microarray
assays with early work focusing on the role HIV plays in regulating
gene expression. One study found that in infected cells, HIV
progressively takes over the transcription machinery, leading
to increased production of HIV mRNA while host-cell mRNA production
declines. Simultaneously, genes that promote apoptosis and
activate cell death associated-proteins called caspases are
also increased. Another study found that the viral protein
Nef could induce virtually the same transcriptional processes
as occur when a T-cell is activated (at least artificially,
or in vitro, by anti-CD3 antibodies), with differences only
in factors regulating HIV transcription (Nef upregulates transcription,
while anti-CD3 antibody activation favored factors downregulating
HIV transcription). These results should be interpreted with
caution, given that they may not fully reflect in vivo processes;
for instance, apoptosis may be more easily triggered under
in vitro conditions. But these findings do lend support to
other research suggesting a critical role for Nef, and for
studying its value as a potential target for drug and vaccine
development.
Other studies underway will help elaborate both the effects
of HIV infection on cells as well as on more general immune
processes. Microarrays can clarify differential gene expression
by comparing two conditions or states of cells, such as treated
vs. untreated, resting vs. activated, naive vs. memory, or
cells at different points in time during proliferation. An
obvious limitation is the need to understand as fully as possible
the role and function of the different genes being observed.
Despite the successful sequencing of the human genome, many
questions persist about the functions of the many proteins
encoded. Microarray assays also produce a huge volume of data,
demanding an increased role for the techniques of bioinformatics
to manage the flood of information produced by these studies.
While other techniques for measuring differential gene expression
exist, in some cases predating microarrays, these new assays
will allow for rapid, simple, simultaneous comparison of an
enormous number of genes. The trick will lay in figuring how
to best use these tests — and what to do with the results. |
| Fred
Gormley 1951 – 2002
Early on the beautiful spring morning of May 19, a few hours
before 42,000 people gathered in Central Park for New York's
annual AIDS Walk, Fred Gormley, a longtime friend of GMHC's
Treatment Issues, passed away.
Fred died from a cascade of complications beginning with
weight loss and persistent thrush which led to ports for amphotericin
infusions and parenteral feeding, culminating in a staph infection,
hospitalization, and kidney failure. Fred died from AIDS.
Fred was responsible for managing TI's mailing list
and monthly production, but better known for his darkly humorous
articles about one gay New Yorker's odd life with AIDS. He
also took delight in crafting a sassy table of contents and
in finding piquant pull quotes for each issue. Our readers
will miss him.
In Central Park, Tyne Daly quoted Edna St. Vincent Millay:
"Quietly they go, the intelligent, the witty, the brave."
Fred was an admirer of Ms. Daly and all things theatrical;
he would have enjoyed pronouncing the poet's name. We will
miss him. |
The Perils of Interpretation
By Daniel Raymond
Immune assays have generated several key findings about HIV pathogenesis:
The selective depletion of naive CD4 T-cells is a hallmark of progressive
HIV disease. Chronic immune activation results in increased proliferation
and apoptosis rates, which may play a major role, perhaps overshadowing
the contribution of the direct cytopathic effects of HIV and/or
cell-mediated destruction of HIV-infected cells. The possibility
of impaired thymic production, and the prospects for stimulating
the thymus to compensate for CD4 loss, represent important but contested
areas of research. HIV-specific CD4 and CD8 T-cell responses, which
are significantly impaired in untreated progressive HIV disease,
continue to decline during the course of antiretroviral therapy.
Studies of interrupted suppressive therapy suggest that periodic
antigenic stimulation may temporarily awaken these responses.
Research indicates an association between HIV infection and multiple
forms of immune dysfunction, processes that may operate independently
or synergistically. Still other research has focused on the innate
immune response and a perceived shift in the course of HIV infection
from a predominantly Th1-type response (where effector CD4 cells
prime cell-mediated immunity, including CTLs) to a Th2-type response
(in which CD4 cells direct their support to humoral immunity, stimulating
B-cell and antibody production). As of yet, however, there is no
"smoking gun" revealing the precise mechanism(s) of HIV pathogenesis.
Pools of consensus ebb and flow around various hypotheses, and while
research has led to a range of proposals for therapeutic strategies,
no new agents have been approved to treat the immune system rather
than the virus.
Immunity research has had its most direct impact on clinical practice
by justifying and guiding the treatment of acute HIV infection with
a combination of HAART and treatment interruption. This approach
has yielded promising data, which in turn has influenced thinking
on vaccine development, but a demonstration of long-term clinical
benefit is years away. Moreover, such early-stage treatment could
only benefit the relative few who are diagnosed very soon after
infection, and are willing and able to begin treatment immediately.
Ultimately, the applicability of in vitro assays to in vivo immune
system dynamics and virus-host cell interactions must be questioned.
For instance, the extensive research into the mechanisms of HIV-mediated
apoptosis has generally been conducted in vitro, yet there is ample
evidence that in vitro cell cultures can have different requirements
for activation, signal transduction, and transcription.
Furthermore, it may be questioned if demonstrating that HIV affects
the induction of apoptosis would necessarily mean that this mechanism
plays a role in disease progression. Would anti-apoptotic agents
in development to treat other diseases have any benefit in HIV treatment?
Perhaps questions about how and why cells are dying in HIV disease
are less important than looking at which cells are dying —
too many of the useful cells and not enough of the infected or functionally
impaired cells. Or perhaps lowering activation rates or increasing
thymic output would be more effective in countering T-cell depletion.
Immune assays and the researchers who use them have become increasingly
sophisticated at answering certain questions about the immune system
and T-cell dynamics: What kind of cells? How many? What are they
doing? Are they functional? However, these assays are only valuable
for the kinds of questions they know how to answer; the whys and
hows of pathogenesis and immune reconstitution can only be addressed
through the vicissitudes of interpretation and speculation. The
models or assumptions already in place at any given time tend to
guide the research, and preconceived assumptions have certainly
been overturned more than once in the history of HIV research. Yet
the gradual accumulation of data inevitably shapes the scientific
consensus on what we can say we know about HIV; certain theories
are judged more or less consistent with the evidence, as other hypotheses
and lines of inquiry gain support or are ruled out. Finally, as
our investigational tools continue to evolve, perhaps the advent
of research informed by genomics will afford us the luxury of worrying
that our ability to ask questions will increase faster than our
capacity to make sense of the answers.
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