RNA in the Blood – A New Marker for Chronic Disease?
Chronic Illness Research Foundation — 05/01/1999
Table of Contents
Defining the Insult
Mutation or Adaptation? Why Chromosomes Shuffle Their DNA
A Novel Mechanism Underlying Chronic Disease?
How can you test for RNA in the Blood?
Glossary of Terms
The treatment of chronic illness is one of the greatest challenges facing modern medicine. In this century, we have witnessed great successes in the treatment of acute illnesses. Antibiotics produce amazingly swift recoveries from formerly fatal infections. Chronic diseases, however, progress more slowly and insidiously, and our successes with them have been far less dramatic. For most of the chronic illnesses, physicians offer only management rather than curative therapies. While we can find a specific cause for most acute illnesses, when we study chronic illnesses, we find ourselves faced with an interacting complex of genetic and environmental factors that start an individual along the lengthy and progressive course of a chronic disease. Cancer, chronic fatigue syndrome, Gulf War Syndrome, AIDS, neuropsychiatric disorders, multiple sclerosis, pemphigus vulgaris, Lou Gehrig’s disease, lupus and other autoimmune conditions, and multiple chemical sensitivities are conditions characterized by the interplay of a large assortment of different factors. Some of these factors work together to begin the development of chronic disease; others, again working in concert, cause disease progression.
Epidemiologists attempt to sort out disease causality and patterns and, occasionally, they find a common thread running between seemingly unrelated diseases. For example, an unusual and aggressive form of lymphoma in African children was described by Burkitt. Simultaneously, in another part of the world, a cancer that started in the nasal passages and throat was discovered to be common in Chinese men, but only rarely seen outside of Asia. It wasn’t until a member of the herpes virus family — the Epstein-Barr virus — was discovered that an association between activity of this virus and the development of these two malignancies was noted. The finding was all the more remarkable because almost all adults display evidence of Epstein-Barr virus infection, i.e. they have detectable antibodies to the virus.
In studying these two greatly different diseases, scientists were forced to examine many different factors to understand how an ubiquitous and normally innocuous viral infection could lead to a chronic malignancy, such as nasopharyngeal cancer, and a fatal one, like Burkitt’s lymphoma. Even more interesting to these scientists was the puzzle of how two greatly different diseases — one seen in adult men in China and the other among children in Africa — were related. We know that Burkitt’s lymphoma is associated with the profound immune dysfunction which can occur in conjunction with chronic malaria. We know less about the specific causes of the nasopharyngeal cancer found in Chinese men. We do know that Epstein-Barr virus infection alone cannot cause either condition, because most everyone is exposed, usually early in life, to the Epstein-Barr virus.
Equally confusing is the situation in which a disease is due to a virus, yet the disease can occur without the virus being present. Take, for example, the association of human immunodeficiency virus type 1 (HIV-1) and AIDS. The popular wisdom claims that HIV-1 is the sole cause of AIDS. This concept has been challenged by the growing literature describing “mysterious AIDS cases,” i.e. AIDS without any evidence of HIV-1 infection.
The fact that many chronic illnesses have such similar early symptoms has led some to postulate that their development depends on common factors, and further that a combination of inheritance, prior disease experiences, and life experience (i.e., cultural and environmental factors) determine an individual’s ultimate response to an initial challenge. An individual from a family with diabetes may never develop clinical evidence of the disease; on the other hand, if exposed to specific stressors, the same individual might develop full-blown symptoms. To date, no theory has satisfactorily explained either the potential variability of outcome in individuals or the observed commonalities among chronic diseases.
We propose here a common mechanism that is involved in the progression of many chronic illnesses. While chronic illnesses differ from acute illnesses by the many factors involved in their initial appearance, we believe there may be common events important in the progression of some of them. What do we know about the development of chronic illness?
1. Among susceptible people, an asymptomatic individual may begin to show symptoms of a chronic illness when sufficient aggravating factors are present. The development of many cancers, as well as numerous other chronic illnesses, has been associated with exposure to hazardous agents — not just to chemicals or radiation, but also to infectious agents.
2. Most chronic illnesses require decades to develop before the earliest symptoms appear. The pre-diabetic individual may lose a large fraction of the insulin-producing cells in the pancreas over a span of many years before any symptoms related to insulin deficiency are noted. Since the specific, essential insult may have occurred many years before symptoms are observed, it may be difficult to relate that insult to the ultimate development of symptoms.
3. The naturally-occurring immune system mechanisms that protect the body against hazardous challenges are encoded in genetic material in a number of very specific sites on the chromosomes. While each of these sites functions somewhat differently, they all share a common feature that immunologists believe to be unique to them: They are capable of rearranging or “shuffling” the genetic material within the site, generating new combinations of DNA which act like new genes. These “new genes” permit the body to produce diverse, novel proteins not previously coded for on the chromosomes. The areas that shuffle the genetic material, which have been called “hot-spots,” become active when challenged by toxic exposures (including infectious agents).
4. We know that these chromosome sites contain repeated regions of DNA that are quite similar to each other in structure. We have learned that the structure of these DNA regions allow them to become active in response to signals received by the cell’s nucleus (where the chromosomes reside). Immune challenges are thought to be the main cause for activation of these DNA regions. However, the diversity of challenges which may activate these areas may be broader than immune challenges as we generally understand them. Challenges such as “irritation” by hazardous exposures (chemicals and/or radiation are among these) are suspected as activators of the DNA “hot-spots.” It is well known that immune-related challenges can activate them. In response to this activation, new proteins, such as antibodies (which are tailored to the challenging substance), are produced.
5. How many of these chromosome hot-spots exist is not known. Over the years, numerous investigators have identified some of those involved in producing antibodies. Our findings suggest that other hot-spots exist. We do not know how they function, or if they even act protectively.
6. Data we have collected now suggest that there may be other such hot-spots, and that these appear to be active in individuals with certain chronic diseases.
7. While genetic reshuffling in these hot-spots serves an important function in producing specific responses to specific insults, chronic activation of these hot-spots may be deleterious. We do not know what damage can occur with prolonged genetic activity. We suspect, however, that prolonged activity in these hot-spots may lead to genetic damage.
8. Cells damaged by such prolonged genetic activity may affect other cells in unpredictable ways, ultimately having impact on the entire body.
Defining the Insult
We are still learning about ways in which chromosomes can be damaged, and the results produced by different types of genetic damage. For example, chromosomes can sustain direct damage, such as when exposed to radiation, which interacts with the chemical bonds of DNA, causing fragmentation and mutation. Many chemicals are also capable of direct interaction with DNA and produce similar effects (i.e., mutation). However, much of the damage DNA sustains is indirect, and some of it occurs naturally (for instance, aging is believed by some investigators to be the effect of a natural breakdown of our DNA). We have evolved enzyme systems that are devoted to maintaining and repairing these low levels of damage that occur naturally. When chemical or other insults affect the efficiency of our enzymatic maintenance processes — one type of indirect injury that DNA can sustain — the damaged DNA then goes unrepaired. When such indirect damage is coupled with direct effects, the total burden of damage is significantly increased.
Some substances that cause direct damage when present at high levels can also cause indirect damage with low-level exposure. In fact, such low-level exposures are thought to contribute to already-existing DNA damage in an indirect, cumulative fashion. While it was previously thought that chronic, low-level exposures cause damage to the enzymatic maintenance machinery, it is now suspected the damage they inflict involves a different mechanism. We believe that mechanism to be prolonged activity of a DNA remodeling machinery which, in normal states, is used to cope with acute challenges. When this machinery is chronically activated, however, it may result in the accumulation of genetic alterations that have deleterious effects on the chromosome.
Mutation or Adaptation? Why Chromosomes Shuffle Their DNA
A paradigm long held in biology maintains that the DNA contained in the cell’s chromosomes is never altered or changed except through damage that results in mutations. Within this paradigm, the DNA, under normal conditions, is immutable and acts as the blueprint for making all our proteins. To make proteins, DNA copies a message into a similar molecule known as RNA. The RNA is transported out of the nucleus, and the information contained in its sequence is translated into a specific protein. (It’s not entirely clear where the translation occurs, inside or outside the nucleus, but recent data suggest the translation from RNA to protein takes place outside of the nucleus.)
Recent studies of immune cells have shown us that this paradigm is incomplete. Immune cells make proteins which are tailored to bind toxic agents, including infectious ones, in a very specific manner. Because the potential diversity of these toxic agents is so enormous, the immune system is unable to store the information to code for every one of the proteins that interacts with every possible agent.
Fortunately, the immune system has developed a remarkable and surprising mechanism to solve this problem. Immune cells possess machinery that reshuffles the DNA components coding for proteins and, in the process, generates codes for an enormous array of new proteins. Additionally, the immune system possesses machinery that functions on an inter-cell level (i.e., between cells). This machinery selects the cells that produce the most successful DNA rearrangements. The selected cells are permitted to proliferate, becoming factories for their specific proteins (or antibodies). In addition to the factory cells, a small number of the selected cells are preserved within the immune system as a library or archive of that particular DNA rearrangement. This machinery works very well. Its constituent parts are finely tuned to shuffle highly evolved regions of DNA in a very precise manner.
We know that only small portions of the DNA in the chromosomes code for proteins (i.e. antibodies). What does the rest of the DNA do, and why does the cell maintain so much seemingly useless DNA? We’ve learned that chromosomal DNA comprises a far more complex structure than previously imagined. The stretches of seemingly-purposeless DNA have been called “junk DNA.” We now appreciate, however, that such DNA is very important to the proper functioning of the small amounts of coding DNA. Further, we are beginning to recognize regions within these long stretches of non-coding DNA that possess structures suggesting they are able to rearrange or reshuffle themselves.
We are uncertain how active or important these sites of rearrangement are in normal cells. Certainly their activity varies broadly. If these sites are active in the germ cells, which make up sperm and ova, the rearrangements will become part of the genetic inheritance of the newly-created individual. It seems obvious that rearrangement activity in germ cells needs to occur at a slow rate to enhance the chances of having healthy offspring.
Like the rearrangements that occur in germ cells, those that occur in other (non-germ) cells of the body are passed on to the descendants of the cell that underwent the transformation. In some cases, rearrangements may be so extensive that cells become non-functional. Severely compromised cells undergo a process known as “programmed cell death”; that is, they commit suicide. Another name used for this process is “apoptosis.” Apoptosis is a natural and healthy function. Cells which are very dysfunctional and of little utility to the body could utilize precious resources and prevent new cells from taking their place, were they not eliminated by apoptosis. There is also the possibility that these dysfunctional cells might not have lost their ability to reproduce themselves, and so their rearranged, damaged DNA would be passed on to their offspring. Further rearrangements and changes, including those leading to malignancy, can take place in these stressed cells unless they are eliminated by apoptosis.
When the immune system works properly, however, challenges from toxic agents, particularly infectious agents, drive the immune system to select those cells that have rearranged their DNA to produce specific antibodies. We suspect that toxic challenges also provoke changes in those areas of the DNA possessing the ability to reshuffle themselves (as described above). Such rearrangement of DNA fragments, we are beginning to understand, is a much broader and more pervasive activity than we previously appreciated. We’ve also made a finding that may be of even greater significance: DNA rearrangements that occur in areas outside of the antibody-producing regions may also be involved in protective responses (although it’s likely that these responses are more primitive and less developed).
Just as the rearranged DNA acts as a template for the production of proteins through the intermediary of RNA, we know that at least some of the rearranging DNA possesses RNA intermediaries as well. That is, areas of DNA rearrange themselves, code the rearranged area in RNA, and then translate the RNA back into DNA. This part of the story has been well documented. However, a new chapter is now emerging. Some of the RNA involved in this process has been found outside of cells. RNA has also been found circulating in non-cellular portion of the blood (the plasma). Biologists have spent little time or effort looking for RNA in the plasma because it has always been thought that unprotected RNA is unable to survive there, due to the presence of a variety of extremely efficient enzymes that destroy unprotected RNA. There are good reasons for rapidly disposing of RNA in the plasma. RNA is a rich source of potentially damaging information. The genetic core of a virus might be part of that information, for instance. The enzymes that destroy unprotected RNA in the blood plasma serve as a non-specific defense against viral infection. How, then, can RNA survive in the blood plasma?
It appears that our own RNA can survive in the blood plasma using the same mechanism that viruses have evolved to transport their genetic information from one cell to another. Viruses have evolved a variety of complex strategies to package their genetic information in a protective envelope that permits it to survive in hostile environments before entering a new host cell. The discovery of a protected form of endogenous RNA — that is, RNA not arising from an infectious agent — within the blood plasma strongly suggests that this RNA is similarly protected. The strong parallel between viral RNAs and the endogenous RNA now being found suggests that there may be a close relationship between the two, and that the RNA leaving a cell is purposefully protected for its ultimate use within another cell.
A Novel Mechanism Underlying Chronic Disease?
Have we identified a novel mechanism for transmitting important information from cell to cell, whereby a usefully rearranged bit of DNA is transmitted through the RNA intermediate to other cells of the body? It is too early to answer this very important question. However, we have observed the presence of detectable levels of RNA in the blood of individuals who have active, chronic forms of disease. The persistence of toxic stimuli may be associated with chronic rearranging activity in the DNA of these individuals’ chromosomes. We suspect that the persistence of this activity might lead to cellular dysfunction and thus to chronic illness.
The recent discovery of a protected form of RNA in the blood plasma suggests the existence of a shuttle mechanism, as has been described here, to transport genetic information in the form of RNA. Whether the mechanism is ultimately found to have a useful function, we are concerned that its chronic activation may have adverse effects. If this mechanism works as we have postulated during acute stress, it’s possible to also envision a system in which small, adaptive genetic changes are shared between cells. The small amounts of genetic material transported by this mechanism may be accommodated without causing any cellular dysfunction. However, the chronic activation of this mechanism may overload the adaptive capacity of the system.
We have also learned that the long, redundant areas of DNA in the chromosomes that do not code for proteins but which are able to rearrange themselves, thought to be “junk DNA,” possess similarities to certain viruses. These viruses, known as “retroviruses,” include such well known family members as HIV. The common feature of these RNA viruses — indeed, why they are called “retroviruses” — is that they copy themselves back into DNA, then inserting themselves into the chromosomes of a newly infected cell. There, integrated into the genetic material of the cell, they become a permanent part of the cell’s genetic legacy.
Retroviruses carry information within their genes that directs the construction of a protective envelope that permits their export into the hostile environment outside the cell and even outside of the host. This protective envelope provides the means by which the retrovirus infects a new cell or a new individual, beginning the cycle once again.
The retrovirus-like repetitive regions in our chromosomes’ DNA are far less complex than fully developed retroviruses. However, they are able to direct that their information be copied into RNA and subsequently back into DNA, which is then reinserted into a new chromosome area. Most of these “infections,” to borrow a term, occur within the cell from which the RNA was copied (rather than in a new cell, as is the case with retroviruses). The result of the process is that genetic material is permitted to jump from one region to another, all within the same cell. The progeny cells carry the newly copied information and will, in turn, pass the newly arranged genetic material to their progeny.
How can you test for RNA in the Blood?
Immunodiagnostic Laboratory (IDL), San Leandro, California, is making available a “Research Use Only” (RUO) plasma RNA test. RUO tests are used by clinical laboratories to provide important research use tests for health-care providers. One limitation is that these tests must be considered as having no benefit to the patient, since they have not undergone the true rigors of the scientific process. No diagnosis can be made from these tests. They are only to be used by health-care providers attempting to understand clinical presentation of mysterious ailments, but have no treatment implications. In addition, these tests are non-reimbursable and therefore their cost must be paid by the individual (in other words, insurance companies will not reimburse patients for the cost of this test). The laboratory has specimen kits available for individuals who are interested in determining if detectable levels of RNA are present in their blood. The kits contain all of the materials needed for collection and transport of the blood specimen back to the laboratory.
Chronic illnesses have been on the rise since the early 1950s. The mechanism of chronic disease development outlined here makes it perfectly clear why that is so: In America, people have been exposed to an extraordinary amount of toxic compounds in last 40-50 years. In 1945, the annual production of chemicals in the United States was eight million tons. By 1985, it had risen to 110 million tons — 950 pounds of chemicals for every US citizen. In addition to chemicals, we’re now being exposed to unprecedented numbers of foreign pathogens for various reasons, including the ease of international travel. On top of the exposures that are occurring as a result of societal change, the medical literature has documented that approximately 100 million Americans were injected with more than two dozen monkey viruses that contaminated the early polio vaccines.
The mechanism for the development of chronic diseases outlined here is supported by a significant amount of medical literature. In time, new research efforts should provide both hope and help to individuals who suffer from chronic illnesses. Identifying a disease-specific biological marker is a first step toward discovering and implementing effective treatments, and we believe that the identification of RNA in the plasma/serum of people with chronic diseases constitutes one of these first steps.