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Immune Enhancement and Health: An interview with Dr. Ronald Pero
Richard A. Passwater, Ph.D.
Our immune systems protect our health by recognizing and rejecting or destroying any foreign agent or mutated cell. I like to say in my basic lectures to lay people who are newcomers to the health food movement that the immune system is a security force that separates what is ďyouĒ from what is ďnot you,Ē then tries to destroy every thing that is ďnot you.Ē Anything invading the body such as bacteria or viruses Ė as well as mutated or cancer cells Ė are targets for the immune system. These things are not you and will do harm to you if left alone to multiply by themselves. The immune system is a complicated system having several components that we are beginning to understand more and more each year. I try to keep track of our new knowledge by yearly updating a large color-coded chart which details various immune system components, including eleven types of white blood cells and 53 of the cytokines that they produce. Cytokines are protein factors or messengers that regulate many cell functions of cell of the immune system. For our purposes here, all that needs to be said is that our immune system depends largely on the effectiveness of various white blood cells.
In this chat, we will discuss how a family of compounds called carboxyl alkyl esters (CAE) significantly improves our health and resistance to disease by enhancing the immune system. While CAE can be extracted from an herb, the action of CAE is far different than the action of the herb itself which is primarily due to a greater abundance of the indole alkaloid and triterpinoid compounds in the herb. The herb by itself has comparatively little immune-enhancing capability, but is important for its anti-inflammatory action.
Dr. Ronald W. Pero middle name is William, but it might as well be ďDNA repair.Ē Dr. Peroís peer-reviewed articles are pushing the 200 mark, with 113 listed in the Medline file. Of those, approximately 100 deal with DNA repair. Many of his publications deal with genotoxicity.
Dr. Pero received his Ph.D. degree in Biological Sciences from the University of Rhode Island in 1968 and did his Post-doctorate at North Carolina State University in 1969. After serving as Adjunct Professor at North Carolina State University and New York University, Dr. Pero became a Docent in Biochemistry at the University of Lund (Sweden) in 1979, and has been involved with several Swedish and Swedish-American projects. Dr. Pero became a Professor of Molecular Ecogenetics at the University of Lund in 1987, and now is the Head of that Department. He is also Professor of Experimental Therapeutics at the Boston University Medical Center
Passwater: Dr. Pero, why did you become interested in biochemistry?
Pero: When I was a child, I used to baby-sit my cousin. When she became three years old, she developed lymphoblastic leukemia and died within six months. This made a lasting impression on me, and I decided right then I wanted to do what I could to fight this disease. Because I didnít feel enough emotional dedication to deal with the day-to-day aspects of patient care, I decided to concentrate my efforts into mechanistic studies of cancer. Having this as a background, what better career than biochemistry?
Passwater: Why did you choose academia over industry?
Pero: While it is true that I have concentrated my career into academic science, this has not meant that I have ignored the industrial applications of science. Currently, I have founded and serve as chief scientific officer of Oxigene, a publicly traded company listed on the NASDAQ, as well as president of CampaMed, a private company dedicated to the scientific development of proprietary nutraceuticals. I believe the best way to develop nutraceuticals for the market place is to ground their research and development to well-established academic scientific principles that result in peer-reviewed approval
Passwater: What have been your main research interests?
Pero: My main research interests have evolved around the role of deoxyribonucleic acid (DNA) repair and DNA damage in regulating toxicological mechanisms important to normal and tumor cell survival.
Passwater: Since DNA is the physical carrier of our genetic information that governs the production of all proteins and cells in the body, DNA repair has long been an interest of mine in terms of slowing the aging process as well as preventing the mutations that lead to cancer. I often discuss ways of slowing DNA damage from free radicals and other reactive oxygen species in this forum, but rarely discuss DNA repair. Please review the importance of DNA repair for our readers.
Pero: Our genes are encoded in our DNA. Healthy DNA/genes allows for the proper expression of our genes which permits us to be healthy and live to our genetic potential. As our genes become damaged and oxidized, the expression of those damaged genes translates into a variety of degenerative diseases including immune dysfunction.
Our DNA is wound around proteins called histones. While DNA is in contact with the histones, it can become damaged, but not repaired. The body produces an enzyme called **** POLY Adenosine Diphosphate Ribosyl Polymerase (PARP) which loosens the DNA cords allowing the DNA to move away from the proteins. When this happens, enzymes are then able to remove the damaged portion of the gene which allows for the repair process to take place.
Passwater: What is the relationship between DNA damage and immune system health?
Pero: The connection between DNA damage and immunity is via the knowledge that immunity is dependent on antigenic stimulation of growth (proliferation). Cell proliferation cannot proceed in the presence of DNA lesions (damage) because the enzymes called polymerases responsible for incorporating the DNA precursors into DNA only recognize the building block nucleosides thymidine, deoxycytosine, deoxyadenosine and deoxyguanosine. It follows then if immune cells cannot grow, there will be immune suppression.
Passwater: So, since we canít decrease the time that DNA is in contact with the histones, we have to increase apoptosis to destroy the cell, otherwise mutated cells will survive and produce ďdaughterĒ mutated cells. Apoptosis forces immune system cells to either differentiate (mature to increase immune specialization) or die, instead of continuing to proliferate. Letís pick up on that later, but first, lets look at what you have elucidated about a phospholipid-like class of compounds called carboxyl alkyl esters (CAE). What is the generalized structure for this family of compounds?
Pero: CAE can be generalized as an ester of carboxylic acid having a long-chain fatty acid or alcohol, and a conjugated aromatic group. The structure can be written as R1-COO-R2, where R1 is a water-soluble condensed aromatic ring system with or without a carboxylic acid group and R2 is a long-chain fatty acid or alcohol. Thus, CAE have two major parts Ė a water-soluble part and a fat-soluble part, which enable CAE to behave as a biological soap. Soap works because it has an affinity for both fats (grease and oil) and water.
CAE are normally not classified as antioxidants, but they do have antioxidant properties. If the water-soluble portion is highly aromatic and hydroxylated, they can resemble bioflavonoids. Itís not important for most people, but for any readers that are chemists, the water-soluble portion contains an aromatic ring system, ranging from a substituted benzene ring to a conjugated five- or six -ring system. As you increase the aromatic side, the molecule would be expected to become more and more water-insoluble, but since these rings are highly hydroxylated, they remain water-soluble. The fat-soluble portion is a long-chain fatty acid or alcohol.
Passwater: You mentioned phospholipids. Let me remind our readers that phospholipids and membrane structure were recently discussed by Dr. Maret Traber and I in this column in November 1997. Basically, phospholipids are lipids (fats) that contain, in addition to fatty acids and an alcohol, a phosphoric acid group. Phospholipids are the main lipid constituents of cell membranes. Lecithin is an example of a phospholipid. It often is an ingredient in foods because it offers the property of linking both water and fat. Other examples of phospholipids are phosphatidylcholine and phosphatidylserine. Phospholipids have a "head" that has an affinity for water and two "tails" that have an affinity for fats.
Cell membranes have a lipid bi-layer consisting of phospholipids that are arranged so they have a polar head group that likes the water phase facing outwards towards the water-based cytosol or plasma, and tails that face into the membrane center. Then on the opposite side of the bi-layer, the tails are arranged in the same manner, only now they are going towards the tails from the other side of the bi-layer. Interspersed are other lipid molecules such as vitamin E and other fat-soluble nutrients and proteins that act as sensors or transporters. Cholesterol is also present to give some firmness or appropriate rigidity to the membrane. The exact degree of firmness/fluidity is critical to membrane and hence, cell, function.
You have done a lot of pioneering work with CAE and cell membrane fluidity. How does CAE affect membrane fluidity?
Pero: CAE are like molecular soaps that cleanse cell membranes by competing with phospholipids in the membrane and improve microviscosity and the cholesterol-to-phospholipid ratio within the cell membranes. (Rivnay, B.; Bergman, S.; Shinitzky, M. and Globerson, A. Correlations between membrane viscosity, serum cholesterol, lymphocyte activation and aging in man. Ageing and Development 12:119-126, 1980)
Passwater: What led you to look into the role of CAE in immune system health?
Pero: When I started my work on Catís Claw (Uncaria tomentosa or Una de Gato), I began with the enormous discrepancy existing in the literature between its historical medical use and the commercial preparations being offered to the public. The Indians native to the Amazon basin prepare water-soluble teas, whereas most commercial products are either encapsulated pulverized plant parts or ethanol extracts. In 1967, Dr. Klaus Keplinger from Austria discovered the presence of indole alkaloids in Catís Claw. (Keplinger, K., Cytostatic, contraceptive and antiinflammatory agents from Uncaria tomentosa. PCT int. appl. WO8201, 130, 1982 and US Patent 4,940,725) Since these data were disclosed, the effectiveness of Catís Claw products have been evaluated against this well-known class of biologic modifiers.
No efforts have been made to determine whether the indole alkaloids or triterpinoids can explain the medicinal properties of Catís Claw. Factors such as bioavailability of alkaloids in Catís Claw, or even what percentage of the biological effects generated from Catís Claw were due to alkaloids, have never been determined. When we looked into these questions surrounding Catís Claw products at CampaMed, we discovered that there was no relationship between alkaloid content and the immune stimulating properties of Catís Claw. Hence, we sought to identify another new class of immune-stimulating substances in our Catís Claw extract, which we call C-Med-100.
Passwater: So you decided to ignore the alkaloids, triterpenes, polyphenols and sterols, all of which have utility in their own right, and extract and concentrate the CAE found in Catís Claw, an herb which has historical usage for the treatment of several human disorders including cancer, inflammatory and infectious diseases. In doing this, do you believe you have produced a dietary supplement that brings about a greater immune enhancement and DNA repair than can be provided by any level of Catís Claw itself or other extracts of Catís Claw?
Pero: Definitely! Not to take anything away from the herb which has provided help for people over thousands of years, but the CAE extract offers health benefits not obtainable with Catís Claw itself. We have shown by comparative testing that this specific mix of molecules produces a degree of immune enhancement and DNA repair not achievable with the herb alone or with conventional extracts. The action of CAE is separate and distinct from indole alkaloids. The adverse effects and toxicity of Catís Claw and other extracts that occurs at the high dosage rate needed to obtain the required amount of CAE is a limiting factors in their use to obtain this level of immune enhancement. Even the hot teas made from Catís Claw prepared by the Campa Indians contain large molecular weight compounds that contribute to toxic side effects. These large molecular weight compounds are removed in the process that we use to produce C-Med-100.
Passwater: So the goal is to ďnourishĒ our cell membranes with CAE to improve the cholesterol-to-phospholipid ratio. Why is that important to our general health and our immune systemís health?
Pero: Cell membrane integrity is controlled by the ratio of cholesterol-to-phospholipids in the membrane. If the membrane is too fluid due to its high phospholipid content, then the membrane receptors that regulate nutrient and hormone transport and absorption are impaired. Nutrient uptake by cells is important to our overall health.
In addition, membrane integrity is critical to optimal immune cell responsiveness. The cholesterol-to-phospholipid ratio in cell membranes determine whether you have efficient transport of chemical messengers or signals into cells that govern growth, function (e.g., DNA repair and immune cell metabolism) and/or toxic responses of the exposed cells. When immune cell membranes are too stiff, cell-to-cell chemical messenger receptors in the membranes do not function properly and thus, the immune cell does not function efficiently. CAE strongly influences and regulates DNA repair and apoptosis. (Sheng, Y.; Pero, R. W.; Amiri, A. and Bryngelsson, C. Induction of proliferation in human tumor cells treated with extracts of Uncaria tomentosa. [Anticancer Research 18: 3363 - 3368 (1998)] As you mentioned earlier, apoptosis is the programmed destruction of damaged cells -- as opposed to cell lysis of necrosis -- before the damage is replicated as a mutated cell. This is critical in preventing Ė and in overcoming -- cancer. Our immune systems Ė if healthy Ė can destroy cancer cells.
Therefore, we have examined the C-Med-100 extract for the presence of CAE, and have found this class of compounds to account for most of its immune-enhancing properties. Phospholipids are only a type of CAE which occur in nature in many forms and molecular weights (sizes). Those present in C-Med-100 obviously can modulate immune cell functions.
Passwater: Would such action be considered nutritional or nutraceutical?
Pero: In my opinion, the extract should be considered a nutraceutical not a nutrient. What it does is enhance protective cellular metabolic processes and stimulate nutrient uptake. The extract itself, C-Med-100, is not known to be essential to nutrition that drives life processes but it can enhance some of them.
Passwater: Why do you call your CAE extract ďC-MED-100?"
Pero: The name ďC-Med-100" acknowledges the appropriate components of the extract to help differentiate it from other extracts of Catís Claw and from Catís Claw itself. The active ingredients of this water-soluble extract are virtually all CAE and the extract is free of indole alkaloids and other compounds that present toxicity problems at the dosages needed to fully activate the DNA repair and other immune-enhancing properties of CAE. ďCĒ stands for the Campa Indian Tribe located at the headwaters of the Amazon and who were extremely important in helping us define the historical medical uses of Catís Claw. ďMedĒ stands for the physiological properties of the product that are useful as a natural medicine in the treatment of human disease. ď100" stands for the 100% bioavailability of this nutraceutical preparation.
Passwater: I notice that your experience with the Campa Indian tribe goes back quite a while. You were the founder of Oxigene in 1994, but earlier, in 1986, you founded CampaMed. Therefore, I have the feeling that you have applied for patent and trademark protection.
Pero: Of course. C-Med-100 has been granted a trademark and we applied for novel composition and use patents in the U.S. in 1997 and internationally in 1998. The trademark was applied for in 1996.
Passwater: Aside from Catís Claw, what are other rich sources of CAE?
Pero: So far, we have looked into more than eight different plant species for which preparations are commercially available. All of them had significant amounts of CAE present, although they differed substantially in their chemical structure, bioavailability and quantity. However, none of them approached the quantitative level or 100% bioavailability of C-Med-100.
Passwater: Share with us some of the points along your learning curve while studying Catís claw to isolate the active components. Are there limitations with the use of Catís claw extracts or teas that preclude their use at the dosages needed for optimal immune health?
Pero: One point that may be of interest is that we have confirmed what has long been known about the higher molecular weight compounds. As long ago as 1967, Dr. Keplinger emphasized that it was important to remove tannins from Catís Claw preparations to minimize adverse effects such as palatability and up-set stomach. Our comparative testing has shown that our CAE extract is devoid of toxicity where other Catís Claw products have shown toxicity.
Passwater: What is the practical significance of the action of C-MED-100? Does it reduce the chances of cancer? Does it help the body fight cancer? Will it help protect against the common cold or flu?
Pero: The practical consequences of C-Med-100 that we have documented so far are three-fold:
1) The process of DNA repair can be enhanced, thus improving the chances of an individual to avoid DNA (genetic) damage, which in turn, is recognized as the most common origin of chronic disease and aging in man.
2) The immune cell function can also be enhanced by preventing DNA damage accumulation in immune competent cells, which in turn, can better proliferate in response to antigenic stimulation.
3) Because C-Med-100 contains CAE, membrane integrity can be fine-tuned so that more appropriate nutrient uptake and metabolic-directed signal transduction can occur, thus permitting a more healthy life as we age.
Passwater: Can C-MED-100 over stimulate the immune system as in autoimmune disease such as rheumatoid arthritis?
Pero: As I understand autoimmunity, at least some immune competent cells continue to receive growth and function signals, even though they should not. Because C-Med-100 should affect signal transduction pathways via membrane integrity, then in principle, C-Med-100 should be useful in relieving autoimmune disorders, not the opposite. However, we have no animal or human data indicating that C-Med-100 could be useful in patients with autoimmune disease. This is an aspect we intend to look into, but as yet have not initiated.
Passwater: Walk us through some of your research. After you elucidated the mechanism of action of C-MED-100, what studies have you done to verify that C-MED-100 does indeed boost the immune system
Pero: We have shown immune stimulation in vivo (studies in living systems) in rodents and humans in several different ways;
Rats supplemented from 4 - 6 weeks with C-Med-100 have (i) elevated w blood cell counts and (ii) increased growth response to mitogens such as
Rats treated with a chemotherapeutic agent, doxorubicin, have a 50% reduction in their white blood cell count and often are not able to recover to normal levels in 15 - 20 days. However, rats first treated with doxorubicin and then supplemented with C-Med-100 recover after 3 - 5 days of supplementation.
Humans supplemented with C-Med-100 also increase their white blood cell counts after four weeks of supplementation.
Passwater: What do your in vitro (studies done in chemical glassware rather than in living systems) cell line studies tell us?
Pero: Our in vitro studies establish one clear mode of action of C-Med-100 and that is induction of apoptosis. As we have discussed earlier, apoptosis is a naturally occurring programmed cell death, and it is one of the main ways in which normal cells can alter their growth into a differentiation process where functional cellular aspects are emphasized. Thus, compounds which can induce apoptosis in general have the possibility of increasing cell function or eventually leading cells to die for lack of functional definition. As a consequence of showing apoptosis induction by C-Med-100, we have determined its usefulness in killing tumor cells that lack differentiating capabilities and by stimulating immune cells that can accept differentiating signals.
Passwater: An infection can cause the body to produce more white blood cells to attack the cause of the infection. What is the significance of increasing the white blood cell counts in the absence of infection?
Pero: Amplifying white blood cell counts within the normal range of the individual distribution in the population has the advantage of giving you more white blood cells with which to fight diseases. Of course, increasing white blood cell counts without increasing white blood cell function would be a more modest positive advantage against fighting disease. However, our animal studies have also shown that the increase in white blood cell count is also paralleled by an increase in immune cell function.
Passwater: Have you done an in vivo study to look at DNA repair?
Pero: Yes, we have conducted several in vivo DNA repair studies which have demonstrated the effectiveness of C-Med-100 in enhancing DNA repair of radiation induced DNA damage.
Passwater: One of the techniques that you have used to measure immune enhancement involves measuring the uptake of thymidine. Thymidine is a nucleoside made up of the pyrimidine base thymine linked to deoxyribose. What is the significance of increasing thymidine incorporation into lymphocytes?
Pero: One of the primary ways to evaluate immune cell responsiveness is to estimate their growth response. When white blood cells called lymphocytes sense an appropriate antigenic stimulus, they must grow and divide in order to produce antibodies against the antigen which normally is an invading organism or an abnormal cell such as a cancer cell. This growth response can be estimated by measuring the incorporation or radiolabeled thymidine into DNA, which occurs during cell replication. Such studies have indeed verified the effect of C-Med-100.
Passwater: Chemotherapy uses chemicals that kill cells hopefully a greater percentage of cancer cells than healthy cells, but not always. Also, chemotherapy often suppresses the immune system and can be counterproductive when what the body really needs is to enhance immunity to selectively kill cancer cells. This is evident with leukopenia, the drug induced depletion of white blood cells. The immune suppression can be life-threatening. Have you looked into whether or not C-MEd-100 can reduce the leukopenia associated with chemotherapy?
Pero: We have studied whether C-Med-100 could reduce chemotherapy-induced leukopenia in the rat. Our data were quite clear. C-Med-100 enhanced considerably the ability of leukopenic rats to recover normal levels of white blood cells.
Passwater: How about human studies?
Pero: We have done one human study. The design was to baseline four individuals by measuring their white blood cell counts for four weeks, and then to supplement with C-Med-100 and again measure weekly their white blood cell levels. The data have shown that C-Med-100 supplementation significantly increased their white blood cell counts.
Passwater: What kind of statistical significance did you obtain? Were the average values separated sufficiently when considering their standard deviation to be meaningful?
Pero: With regard to all our studies that we have discussed, I have mentioned only data that were statistically significant. Naturally, this demands consideration of standard deviation among groups to be taken into consideration.
Passwater: Did all subjects show the same improvement or were some greatly affected, some not affected and others negatively affected?
Pero: There was individual variation in the human study which is to be expected. Three of four individuals showed increases in white blood cell counts whereas one individual remained unchanged. However, as a group, the white blood cell levels were significantly increased.
Passwater: If C-MEd-100 is so effective, is it safe? What toxicity studies have you done? How does C-MED-100 compare to other immune enhancing agents in terms of safety?
Pero: We have not compared the safety of C-Med-100 with other immune enhancing nutraceuticals. However, we have compared C-Med-100 with other Catís Claw products. We have seen no toxicity in rats at ten times the concentration of an effective dose that enhances DNA repair and immune cell response. However, other Catís Claw products were toxic at those levels. Moreover, the human C-Med-100 supplementation study was devoid of any side effects. Hence, we at CampaMed are quite convinced of the safety of C-Med-100, especially in comparison with other Catís Claw products where there is a surprising lack of knowledge available about toxicity.
Passwater: How long does it usually take (and what dosages are required) to optimize the immune enhancing effect of C-MED-100?
Pero: Our animal data support that four-to-six weeks of daily supplementation at the equivalent human dose of two 350 milligram tablets daily is optimal to demonstrate enhanced DNA repair and immune cell responsiveness. After this, our human maintenance study has shown efficacy is achieved with one 350 milligram tablet daily.
Passwater: There seems to be a host of products coming on the market that claim to boost immunity. There are Pycnogenol, vitamin E, beta glucans, ďpolysaccharide phytoproteins,Ē IP-6, immunoglobulin-containing milk, and various herbs, etc. How do we sort all of this out. Do we need them all? Are some more important? How does C-MED-100 fit into the total picture?
Pero: I think that this is a very important question. There are a lot of nutraceuticals in the market place that claim immune-enhancing properties. We have just begun to look at some of them in more detail. So far we have been able to show Pycnogenol to have immune-stimulating effects, but not through the same pathway.
One thing that concerns me is that people may erroneously believe that the immune enhancement from Catís Claw is due to its alkaloid content and think that because a Catís Claw product is high in alkaloids that it is a great immune enhancer. Catís Claw is a very good herb for several conditions, but it has nowhere near the immune enhancing and DNA repair power of C-Med-100. Or in the case of Pycnogenol where Dr. Ron Watson has demonstrated immune enhancement. If someone then puts some bioflavonoids in a formula it doesnít necessarily follow that the product will actually stimulate the immune system. Another area of caution is products that contain small amounts of compounds having immune enhancement properties when used in larger quantities and then claiming a synergistic action that makes the product effective. Where is the evidence that the product works? Instead of synergism, the product may not have enough of any compound to measurably improve any one component of the immune system, let alone produce synergism. Like the lady used to say in the hamburger commercial Ė whereís the beef? Since differences in preparation form (herb tea, solvent extract or dried herb powder), fraction of natural part (whole plant, root, bark, leaves) used and dosage are critical, whereís the evidence that the product works as claimed?
I believe that one thing the consumer should do is to start asking the companies supplying these products for scientific evidence that their product really does what they say. This means that providing evidence that an immune-stimulating agent is present in a product is not good enough. They must show that it is active in the preparation that they want to sell. The nutraceutical must actually work in the quantities provided according to label suggested dose.
This is what we have tried to do with C-Med-100. We feel we have provided evidence in animals and humans that what we intend to market in fact has immune stimulating properties. In addition, C-Med-100 can at the same time protect you against DNA (genetic) damage. No other nutraceutical products have made these claims nor is there any demonstration of this health benefit for any other nutraceutical. For these reasons, we believe C-Med-100 should be the measuring stick against all other nutraceuticals with similar claims. In such a way, the consumer and the health food industry could sort out in a scientific manner which products work for which indications involving immune stimulation.
Passwater: Where will your research take you next?
Pero: CampaMed intends to direct its short-term research goals on further defining a role for C-Med-100 in prevention through demonstrating health benefit from enhancing DNA repair and immune stimulation. We hope to accomplish this endpoint by utilizing case control studies in humans at risk to chronic diseases.
Passwater: Thank you for discussing your research with us.
© 1999 Whole Foods Magazine and Richard A. Passwater, Ph.D.
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