Chronic Fatigue Syndrome

Symptoms of chronic fatigue syndrome (CFS), joint pain (arthritis), back pain, etc. are symptoms of extreme chronic inflammation. Recent studies of blood cells of CFS patients show patterns of gene expression consistent with systemic inflammation.

Chronic fatigue syndrome (CFS), Lyme disease, fibromyalgia, post traumatic shock, multiple chemical sensitivity (MCS), irritable bowel syndrome and many other syndromes share the same symptoms that are linked by inflammation. These symptoms may also blur into arthritis, back pain, heart disease and numerous psychological symptoms. Thus, depression is considered by some to be a symptom of inflammation. Many of the symptoms in this large group of related syndromes also respond to anti-inflammatory diets and treatments. The close association between inflammation and infection also leads to surprising responses to antibiotics. The complexity of symptoms, however, makes the labeling of the syndromes problematic. Some new technical approaches may clarify both the causes and status of these diseases.

Inflammation of tissues results from relaxation of walls of blood capillaries. This relaxation or dilation of capillaries results from the proteins and chemicals released from the cells near the capillaries. These adjacent cells have detected infecting bacteria (TLR), or responded to some type of injury or damage. The endothelial cells lining the capillaries also respond to the inflammatory signals by displaying proteins on their surfaces that snag immune cells from the blood. Thus, inflammation also results in colonization of the inflammation site with white blood cells (in contrast to the hemoglobin-containing red blood cells) that we see as pus.

The immune cells circulating in the blood and going in and out of tissues advertise the quality and quantity of inflammation by the pattern of proteins that they display on their surfaces and secrete. The presence of these inflammatory proteins in turn reflects the fact that those cells have expressed particular patterns of genes, i.e. each gene in the group was converted into messenger RNA (mRNA) that was in turn translated into protein. Gene expression can be measured either by the amount of each protein accumulated, or more conveniently by the amount of each mRNA present. The cells of each tissue are different because they express different genes and have a different mRNA population.

It is now possible to approximate the concentration and action of each cell type in a sample of blood. Viral infections produce different patterns than bacterial infections and the patterns differ at the various stages of an infection. These are observations using new technologies that are approaching a global expression fingerprint, but are still to some extent based on sorting the cells within a blood sample into groups based on their surface proteins and then analyzing the mRNAs of each group of cells.

A recent research article (below) announced the identification of unique patterns of chronic inflammatory gene expression in the blood cells of chronic fatique syndrome patients. These patterns permit both a peek into the immune processes behind CFS as well as providing an explanation of the current symptoms of the syndrome.

(Aspler AL, Bolshin C, Vernon SD, Broderick G. Evidence of Inflammatory Immune Signaling in Chronic Fatigue Syndrome: A Pilot Study of Gene Expression in Peripheral Blood. Behav Brain Funct. 2008 Sep 26;4(1):44. [Epub ahead of print] PMID: 18822143)

Vagus Nerve Controls Intestinal Inflammation

Macrophages release inflammatory signals (TNF, IL-1, IL-6, IL-18) that result in tissue inflammation. Nicotine is anti-inflammatory by acting on the acetylcholine receptors normally responsive to acetylcholine released by the vagus nerve. Acupuncture is anti-inflammatory by stimulating the vagus nerve-mediated effects on macrophages.

The relationship between the nervous and immune systems has been accepted as a reality, but has been elusive. Numerous examples in alternative medicine appear to show that a variety of treatments have immunological impacts, but explanations based on cellular biology have been slow to materialize. Here I will discuss some of the recent experiments that reveal obvious connections between nerves and macrophages that may explain in medical terms at least part of the efficacy of acupuncture.

Dilation of blood vessels that causes reddening, swelling and warmth of tissue inflammation results from changes at the cellular level. If the sentinel cells of a tissue, macrophages, are exposed to a bacterium, for example, receptors on the surface of the macrophages bind fragments of the bacterial cell wall, i.e. lipopolysaccharide (LPS) or endotoxin, and signal the expression of five dozen genes. Among these genes are inflammatory mediators, TNF, IL-1, IL-6, IL-18, that are released from the macrophages and trigger behavioral changes in the surrounding cells of the tissue, which are observed as inflammation.

Expression of the inflammatory genes is controlled by a master transcription factor, NFkB. Thus, LPS will signal a macrophage, NFkB is activated, inflammatory genes are expressed, mediators are secreted and tissue inflammation is observed. Dozens of different inputs determine if NFkB will be activated or quieted. Nicotine for example has been observed to block inflammation by LPS.

It has been shown that macrophages also have receptors for the neurotransmitter acetylcholine that is released by branches of the vagus nerve in the intestines. It has also been recently shown that excitation of the vagus nerve releases acetylcholine and blocks the response of intestinal macrophages to LPS. Thus, vagus stimulation is anti-inflammatory and blocks NFkB activation through a competing transcription factor, STAT3. Nicotine acts by binding to the acetylcholine receptors of the macrophages and is similarly anti-inflammatory.

Acupuncture appears to work by needle stimulation of the vagus nerve that sends signals to the brain. Returning nerve impulses via the vagus nerve subsequently release acetylcholine back into the surrounding tissue and block inflammation. In this context, acupuncture would be exploiting an existing inflammation dampening system, that would serve to localize spreading inflammatory signaling and emphasize the source of inflammation for action by the circulating elements of the immune system.

Obscene Science

Typical science sometimes reveals the stupidity of some business practices. This is displayed in stark reality in the case of how animals are manipulated to mimic human diseases. Common human cosmetic procedures are applied to mice to cause arthritis and high fructose corn syrup is fed to mice to give them diabetes.

It is now possible to visualize the inflammation in mouse joints with special dyes that fluoresce in the infrared, outside of the absorbance wavelength of hemoglobin, but how do you give mice arthritis? The answer is obscenely easy -- inject them with collagen.  Then you can watch as their joints become inflamed.

I previously performed lots of Google searches on different aspects of collagen metabolism, so I know that the first ten thousand hits on any search with collagen as a search term result in the how-to’s of collagen injections for the high estrogen lip look.

Let’s go one step further. From my work on developing special methods for highly selective antibody production, I know that mice and other animals produce the most heightened, extreme reactions to the smallest amount of antigen when injected in critical areas such as toe pads or .....lips.

Putting this all together, shouldn’t injecting collagen into lips also be a great way for producing arthritis? At one point I put the pieces together for myself and did a search of collagen injection + side effect + arthritis, and guess what? As you might expect, if you inject your lips with collagen, you can produce arthritis. From my work on inflammation, I would suggest that you could use lip injection as a way of measuring the level of chronic inflammation. Inject collagens in the lips and measure the length of time until joint pain starts -- the shorter the time, the higher the level of chronic inflammation.

People ask why I am afraid of high fructose corn syrup. Let’s turn to animal models of disease once again for another example of obscene science. This time we are trying to study type II diabetes. How can I get mice to exhibit diabetes, so that I can study treatments that block the development or reverse diabetes? The answer is just feed them HFCS and they develop diabetes. That doesn’t happen with sucrose. Clearly HFCS is a dangerous sweetener, if you are concerned with diabetes. It is interesting that mice can be protected against HFCS by prior treatment with omega-3 fatty acids. This indicates that type II diabetes development is an inflammatory process.

IBD treatment 2008

Inflammation of the bowel responds to the anti-inflammatory diet differently from other inflammatory diseases, such as arthritis or allergies. Special attention must be paid to absorption problems, e.g. B12, methionine, and intestine-specific interactions -- glucosamine and heparin.

There has been a flurry of reports in the last few months on new therapies for inflammatory bowel diseases (IBD). The emphasis has been on delivery directly to the bowel, as opposed to through the blood circulation and a return to earlier observations on the relationship between thrombosis and antiphospholipid disease and IBD. Both of these conditions bring up the use of therapeutic heparin. Clotting is usually associated with heparin, since controlling clotting is the major clinical use of heparin, but heparin is taken completely out of its natural context -- the gut.

Heparin is a fragment of heparan sulfate (HS), a long polysaccharide synthesized sugar by sugar on a protein. Thus all heparin starts as a heparan sulfate proteoglycan (HSPG) and all HSPGs pass through the secretory system of cells, but the proteins are embedded in the inside of the membrane of the secretory vesicles, so instead of being released into the extracellular environment, the HSPGs are displayed on the outside surface of the cells. The cell surface is dominated by a forest of HS chains. Most extracellular proteins, including the inflammatory interleukins, bind to the HS chains. Receptors that are needed to respond to the hormones bind in pairs to pairs of hormone proteins with the HS between them, like two pairs of buns on an HS hot dog. The HS is an integral part of the signaling systems and also many other complex processes such as clotting.

As HS passes through mast cells the HS is enzymatically cleaved into fragments of heparin. The heparin is negatively charged and the histamine and enzymes secreted by the mast cells are positively charged, so they neutralize each other. When a mast cell releases histamine in response to allergens binding to its surface, histamine and heparin are released. Heparin is produced for drug use from scrapings of hog and cattle intestinal mast cells. A major feature of IBD is enhanced activity of intestinal epithelial mast cells.

IBD is frequently associated with other inflammatory conditions, such as thrombosis, the tendency to produce clots, or antiphospholipid antibody production (APA). Both thrombosis and APA are treated with heparin and aspirin. Aspirin might appear obvious to inhibit COX-2, the enzyme that produces inflammatory prostaglandins from omega-6 fatty acids, unfortunately these same prostaglandins are needed for the integrity of the gut. That is why aspirin can cause bleeding of the gut. This is also why aspirin inhibits muscle growth following exercise, since muscle growth requires inflammatory prostaglandins. COX-2 inhibitors also block production of anti-inflammatory prostaglandins from omega-3 fatty acids (fish oil).

Heparin would be a natural treatment for IBD, but it is usually administered by injection or intramuscularly and has a systemwide impact on many signaling systems, including clotting. Since bleeding is already a symptom of IBD, keeping administered heparin out of the blood would be an advantage. The other alternative is oral administration. Bathing the stomach and intestines in heparin on the way to its application in the lower bowel is a problem, because HSPGs are rapidly cycled on the surface of cells and any heparin in contact with a cell is rapidly internalized.

The answer to IBD drug administration is to use a system that delivers the drug to the affected bowel. There are several systems recently reported that encapsulate and selectively release drugs. The drugs of choice seem to be heparin and Mesalazine. Another new approach is the use of glucosamine. Since glucosamine acts directly on the gut to produce systemic anti-inflammation, it is a natural choice. Acupuncture is also highly effective in some cases of IBD and it is particularly useful, because it also produces a very strong placebo effect to which IBD seems to be very sensitive. From my experience in other areas of pain relief and anti-inflammation connected with acupuncture, I would also expect castor oil packs to be very useful for IBD. If you have had experience with these anti-inflammatory approaches, I would enjoy your comments.

Open Up Your Brain

The brain is fed and oxygenated by capillaries that penetrate its grey matter, but the cells and proteins of the blood are kept separated from the brain by the cells and matrix of the vessel walls, the blood brain barrier (BBB). That barrier can be breached by inflammation that lowers the heparan sulfate composition and stretches the matrix.

The blood can be considered a large organ with liquid surrounding the cells of the tissue instead of extracellular matrix composed of proteins and polysaccharides. Organs are typically surrounded by a basement membrane made of laminin, entactin (nidogen), collagen and perlecan (a heparan sulfate proteoglycan). These components are secreted by the adjacent cells, and the proteins and polysaccharide (heparan sulfate) bind to each other in multiple ways and hold the tissue together. The cells lining blood vessels, endothelial cells, are connected to each other by tight junctions and the space between the cells is filled with the extracellular matrix of the endothelial cells, the equivalent of the basement membrane of the blood.

Inflammation leads to an alteration in the structure of blood vessels that produces the gross symptoms of inflammation: swelling, reddening, warmth and pain -- the blood vessels dilate or stretch. Persistent or chronic inflammation of vessels can lead to changes in the composition of basement membranes and one notable change is a decrease in the heparan sulfate proteoglycan.

In my own research on the molecular impact of inflammation, one of my students, April Reimers, induced inflammation in cultures of cartilage secreting cells, chondrocytes, using a glucan (similar to the glucan elicitor that I used thirty years earlier to stimulate disease resistance in plants.) She used real time reverse transcriptase PCR to follow the expression of the genes involved. The inflammation transcription factor, NFkB, was turned on (as judged by a compensating induction of its inhibitor IkB) and proteins and enzymes involved in the expression of heparan sulfate proteoglycans were shut down. Our interpretation was that inflammation reduces some heparan sulfate proteoglycans. Since there are at least a dozen different HSPGs with different patterns of expression, we would expect there to be some exceptions from the general suppression caused by inflammation.

Inflammation caused by many different diseases opens up capillaries to provide access of the immune system to damaged tissue. Breakdown of the integrity of an endothelial barrier with an inflammatory decrease in HSPG in kidneys is observed as proteinuria (loss of protein into the urine) due to high blood sugar of diabetes. Breakdown of the capillaries also disrupts blood flow to extremities and is responsible for amputations in diabetics with poor control of blood sugar. Inflammation of intestinal blood vessels and HSPG reduction occurs in protein lossing enteropathy (loss of blood protein into the lumen of the intestines) as a result of heart reconstruction to repair congenital structural defects. The same thing happens to the BBB when vessels of the brain experience inflammation.

Breaching of the BBB is an essential initiating event in many diseases of the brain. Subsequent flareups of symptoms may signal repeat compromise of the barrier between the blood and the brain. The reduction of the barrier, including a decrease in HSPG, permits transit of antibodies, other proteins, e.g. the beta amyloid peptides of Alzheimer’s disease, and lymphocytes from the blood into the brain. The region of the brain that is inflamed and the nature of the interaction between the blood components and the brain determine the symptoms. Examples would be the autoimmune attack on the myelin sheath of nerves in multiple sclerosis and the aggregation of fibers of beta amyloid peptides with heparan sulfate on the surface of neurons in Alzheimer’s disease.

Shutting the blood brain barrier would seem to be the first step in disease treatment, but in some cases, the BBB must remain breached for medication to reach the brain -- the BBB is also a barrier to drugs. If the BBB is to be repaired, then inflammation must be stopped. In most cases, this is accomplished by anti-inflammatory drugs. In some cases, such as protein lossing enteropathy, heparin therapy can reverse symptoms. A very effective supplement to reduce chronic or in some cases acute inflammation is an anti-inflammatory diet. The components of these diets are discussed elsewhere on this blog. These diets have been used effectively in the treatment of MS, Alzheimer’s and many other degenerative and autoimmune diseases.

Amyloids and Heparin

Amyloid proteins form fibers as they systematically stack in the presence of heparin. Examples of amyloids are the beta-amyloid and tau of Alzheimer’s disease, prions, and perhaps even sickle cell anemia fibers.

Amyloids are proteins that stack into fibers and can be detected microscopically by binding particular stains, e.g. Congo Red. All of the amyloid proteins are also heparin binding, e.g. Alzheimer’s amyloid is 50% heparin sulfate. I surmise that the Congo Red is binding to the heparin-binding domains, because Congo Red also binds to cellulose and a particular lipopolysaccharide from Shigella flexneri. Antibodies and enzymes that bind to polysaccharides, bind through tryptophan residues that are flat, hydrophobic plates with regular spacing. The amyloid fibers presumably bind the broadly spaced hydrophobic surfaces of Congo Red by providing corresponding complementary hydrophobic regions of the basic amino acids that are grouped on the stacked amyloids.

The interactions between carbohydrates, even highly charged polysaccharides such as heparin, and hydrophobic conjugated ring systems of aromatic molecules, such as dyes (Congo Red, Berberine) or aromatic amino acids (phenylalanine, tyrosine or tryptophan) may seem counterintuitive. The ring structures of sugars are actually hydrophobic on the relatively flat surfaces, surrounded by the hydrophobic edges of the hydroxyl groups. Tryptophans are amino acid-two ring systems that are the typical binding surfaces for sugars. The large, negatively charged sulfate groups on heparin sugar units limit the binding of aromatics, such as the dyes berberin or alcian blue to just one side of the units. Proteins bind to heparin by a combination of the positive charged at the ends and the hydrophobic chain of the basic amino acids, arginine and lysine. Thus, binding of a protein to heparin is a two step process of initial charge interactions followed by stronger hydrophobic interactions. Binding of the basic amino acids to aromatic amino acids can be observed in arginine/tryptophan ladders evident in F-spondin.

The cooperative of action of heparin in the formation of beta-amyloid, and many other experimental protein fibers has been thoroughly studied. Tau protein also forms intraneuronal fibers in Alzheimer’s, but a facilitating polysaccharide has not yet been identified. I have examined the protein sequence of the human tau protein and identified numerous heparin-binding domains. Also the X-ray crystallographic structure of tau has not be determined, because tau is considered an intrinsically unstructured protein. Many proteins with multiple heparin-binding domains are unstructured, because these proteins only reach a stable structure when they are bound to heparin. It is not likely that heparin is intracellular in healthy neurons, so either the integrity of neurons with tau fibers has been corrupted and heparin has entered, or the fiber enhancing molecule is a nucleic acid, e.g. RNA. There aren’t many other choices.

Knowledge of the rapid cycling properties of heparin may explain the toxic properties of amyloids. Amyloids are toxic when they are in contact with the cytoplasmic membrane surface. The contact toxicity is consistent with the amyloid fibers interacting with the heparan sulfate proteoglycans (HSPGs) embedded in the cytoplasmic membrane. It is known that HSPGs rapidly cycle between secretion and uptake. Both heparin and proteins with strong heparin-binding domains, e.g. protamine, HIV tat protein, are rapidly internalized. It is possible that amyloids bind to the uptake system, but the large size of the fibers blocks their internalization and paralyzes the uptake system. Paralysis of the membrane functions may lead to death of the neuron. Prions also appear to form amyloids with heparin and may be toxic for the same reason.

An analogous amyloid process leads to the death of the insulin-secreting beta cells of the pancreas, leading to type I diabetes. Insulin is made by chopping the middle and ends off of a pre-protein. The middle section that is excised has a strong heparin binding, internalization domain, so if insulin processing is blocked, is this the basis for autoimmunity in the presence of inflammation? Antibodies against the beta-cells keep killing them and maintaining the requirement for supplemental insulin. I think that aggressive anti-inflammatory treatment and blocking of the offending antibodies (or induction of tolerance) could permit the beta-cells to recover.

Structures displayed were captured from NCBI MMDB using CN3D.

Vitamin B12 and Disease

Vitamin B12 and the associated amino acids methionine and cysteine are essential to avoid specific symptoms of anemia and the deleterious effects of inflammation.

Vitamin B12, cobalamin, cannot be made by the human body and requires a protein secreted by stomach cells, intrinsic protein, for uptake from the diet or from gut bacteria. In the absence of dietary B12, individuals can develop pernicious anemia. Since vitamin B12 is used by enzymes involved in nucleic acid synthesis, the rapidly dividing cell of the blood show the first symptoms. Another feature of pernicious anemia is the production of autoimmune antibodies against gastric intrinsic protein. This means that even if the diet is supplemented with vitamin B12, the anemic patient would show no improvement. Historically, the cure was beef liver juice containing B12 already bound to intrinsic factor.

I enjoyed learning about the autoimmune aspect of pernicious anemia, because when I examined the sequence of the human intrinsic factor, I found the expected pairs of basic amino acids that I associate with strong heparin binding, uptake and presentation of allergens.  I would predict that pernicious anemia results when intrinsic factor is presented to the immune system as a result of inflammation of the stomach or intestines. Subsequently, anti-intrinsic protein antibodies block B12 uptake. Pernicious anemia is commonly associated with pathogen attack on stomach tissue by Helicobacter pylori, the stomach ulcer and gastric cancer bacterium. Treatment for B12 deficiency has to bypass the required binding to intrinsic factor and uses injection or inhalation of B12 supplements.

There is also a group of proteins that bind to B12 in the human body and apparently block uptake of B12 by pathogens. These proteins are called “R binder” proteins and are present in the body in the same pattern as lactoferrin, an antibacterial protein that binds iron, another critical limited nutrient needed by pathogens.

B12 is used by enzymes to hold methyl groups as the groups are moved from one place to another on a substrate molecule. So B12 is needed to donate a methyl group to homocysteine to regenerate the amino acid methionine. In the absence of B12, homocysteine accumulates in the blood and begins to react with the cysteines and lysines of proteins. It is particularly reactive with enzyme active sites and inactivates lysyl oxidase, which cross-links collagen and elastin that are needed for the integrity of heart and smooth muscle.

Inability to regenerate methionine also eliminates the essential functions of its derivative S-adenosylmethionine, SAM, which is involved in polyamine synthesis and cysteine synthesis. Cyseine is an essential amino acid that is one of the three amino acids in glutathione, the major antioxidant of cells. Thus, a methionine deficiency can result in severe oxidative stress and inflammation.

Disruption of normal nutrition, gut flora and uptake can result in deficiencies of vitamin B12, methionine and cysteine, with a subsequent cascade of oxidative events leading to inflammation, autoimmunity and degenerative diseases. It seems likely that a similar scenario could be associated with loss of physical activity and muscle mass (sarcopenia) of aging. As the older person’s energy requirements decrease, less food will be required, but the composition will need to be adjusted carefully to maintain a healthy gut flora and avoid vitamin and amino acid deficiencies. I would not be surprised if the diets of most older people are grossly inadequate to avoid deleterious chronic inflammation. Poorly managed inflammation could account for most of the symptoms of aging and its associated degenerative diseases.

On a closing note, alcohol consumption has be associated with both hangovers and decreased risk of cardiovascular disease. The hangovers are due to alcohol conversion to acetaldehyde. Reaction of acetaldehyde with cysteine may be both a cure for hangovers and a partial explanation for increased longevity associated with moderate consumption of alcohol. Moderate and consistent alcohol consumption may cause an increase in cysteine storage as a compensation for losses due to alcohol intake. If the alcohol adapted person has higher stores of cysteine, there may be a simultaneous increase in cellular glutathione with a corresponding decrease in oxidative stress and inflammation. Decrease in inflammation is associated with increased longevity. Supplementation with cysteine prior to alcohol consumption eliminates a hangover and may also make you live longer!

Toxic Myco-stories

Mycotoxins are among the most toxic natural products. They are fungal adaptations to enhance competition with bacteria and insects, but they can be very effective in injuring the nervous system and organs of humans and may result in persistent debilitating syndromes.

In retrospect, I must admit that I have spent much of my academic life working with lower life forms. When I entered graduate school in molecular biology, I was willing to work on anything, but fungi and plants. At that time, James Watson had claimed that only nucleic acids and proteins were worthy of study by molecular biologists, because that was the scope of The Central Dogma, DNA to RNA to protein. So of course, I ended up studying the carbohydrates (glucan elicitors) of fungal walls that are detected by plants to signal an infection. It was part of the molecular biology of host-pathogen interactions and of plant disease resistance. People now buy fungal/yeast glucans as supplements to enhance (read cause inflammation in) their immune systems.

Turning on plant disease resistant reactions by pathogens or my glucan elicitor points out one of the most misunderstood aspects of plants -- they are dangerous! Afterall, most of the drugs and nasty compounds that are used come from plants. If you want to really make sure that a greenhouse is dead, dead, dead -- no insects at all, you can bring out the heavy guns and spray with nicotine. Nicotine is so nasty and deadly, that full body protection is needed for the sprayer. Just recall all of the nasty plant extracts that are used to kill people in murder mysteries. What could be more deadly that ricin from castor beans? Plants are not safe to touch, let alone eat -- they are full of compounds that kill or maim potential plant eaters and should never be trusted. Plants are more sensitive to my glucan elicitor than they are to their own plant hormones and their reaction is a slew of more toxins -- sick potatoes are a significant source of nasty compounds that cause birth defects. So, be careful eating sick plants, especially if you are pregnant and be even more careful feeding plants to unsuspecting pets.

While I am on the subject of pregnant women, we should spend a moment on morning sickness. The smell of cooking plants makes pregnant women, especially in the first trimester, evacuate their stomach, i.e. vomit. The point is that women have an important reflex to block them from eating food that will injure their developing fetus. During the first trimester, fetuses are very sensitive to plant compounds that cause disruption of development, teratogens. The pregnant woman’s sensory system associates smelling plant compounds with eating plants and eliminates potential teratogens in the stomach. Pregnant women would be well advised to avoid their veggies, especially early in pregnancy. Plants are not always healthfoods -- women store fat for a pregnancy to help them feed their fetus without the need to eat potentially dangerous food.

Fungi including those forming fruiting structures, such as mushrooms, eat by secreting enzymes and absorbing the digested products. They have to compete with bacteria for the fruits of their secretory labors, so they poison the competition with the cheapest toxins possible, i.e. small molecules with high toxicity. When I was gathering mushrooms in the verdant moss strewn forests on the islands of Stockholm’s archipelago, I picked only the ones that were friends and avoided their lethal brethren. The toxins are legion, including the “brain lesioning agent,” ibotenic acid of Amanita muscaria, that can also be used to kill flies, phalloidin from A. phalloides, that locks up the cytoskeleton and all of the recreational shrooms that bring California trippers to the microclimates of the Idaho hop yards. The sensory and nervous system of animals are high value targets for fungal toxins.

While in Missouri, studying bacterial plant pathogens, I befriended Art Karr, who was studying the most lethal of the mycotoxins, aflatoxin. He was using the fluorescence of the aflatoxins to permit their detection after thin layer chromatography. Subsequently, after I had moved to Harvard, I collaborated with Art to design a fluorescent immunological method using antibodies produced by Gerald Wogen, another collaborator from MIT, for the quantitation of aflatoxin in corn. The aflatoxin detection kits became the first product of our company, Vital Technologies, Inc. Measuring aflatoxin in corn was a big deal, because this mycotoxin was what stopped corn production in southern states of the US. and blocked many international shipments of corn. Aflatoxins also have been implicated in many peanut-based diseases/cancers in Africa and even in kwashiorkor, a wasting disease normally associated with malnutrition.

Sick building syndrome has been attributed in some cases to the house mold, Stachybotrys chartarum and its toxins. These macrocyclic trichothecene toxins cause diverse acute and chronic neurological symptoms and may contribute to such elusive diseases as chronic fatigue syndrome (CFS), multiple chemical sensitivity (MCS), fibromyalgia (FM) and post-traumatic stress disorder (PTSD). In each syndrome, an acute, stessful experience compromises the nervous and immune system, and subsequently a chronic phase persists.

Multiple Chemical Sensitivity -- Severe Chronic Inflammation

Multiple Chemical Sensitivity is a cryptic disease that may be multiple individualized manifestations of severe chronic inflammation. Sufferers respond to anti-inflammatory treatments.

A disease by any other name is still inflammatory. Wikipedia:
“MCS has also been termed toxic injury (TI), chemical sensitivity (CS), chemical injury syndrome (CI),[3] 20th Century Syndrome, environmental illness (EI), sick building syndrome, idiopathic environmental intolerance (IEI), and toxicant-induced loss of tolerance (TILT).”

All of these different names attest to the numerous symptoms and clinical presentations of this syndrome, i.e. multiple symptoms for the same cause. Since I see inflammation and heparin as integral to most diseases, it will come as no surprise that I looked for inflammation as the basis for MCS. A search of the biomedical literature brought me to work by Martin Pall at the School of Molecular Biosciences at WSU. Since he lives just north of me and I think of myself as a molecular bioscientist, I was receptive to his ideas, the nitric oxide/peroxynitirite theory.

The nitiric oxide/peroxynitirite theory basically says that numerous types of chemical injuries can give rise to an inflammatory response that generates both nitric oxide and reactive oxygen species (ROS), which in combination produce peroxynitrite. The peroxynitrite causes oxidation damage to mitochondria, which causes release of more ROS and depletion of ATP. Depletion of ATP in neurons of the brain, particularly in the hippocampus can result in increased sensitivity to chemicals. All of these components can produce cycles of nitric oxide/peroxynitirite production and the maintenance of a high level of chronic inflammation. This mechanism for the cause and maintenance of MCS also provides suggestions about treatment.

Important insights from Dr. Pall’s web site are that MCS, chronic fatigue syndrome, fibromyalgia and post traumatic shock all share related symptoms, underlying biochemistry and treatment. A major feature of the treatment is the use of dietary supplements, e.g. turmeric (curcumin), vitamin C, omega-3 fatty acids, that are anti-inflammatory. A potential mistake in therapy for a syndrome with so many triggering chemicals is to simplify the diet to just starch and protein, in an attempt to avoid triggering plant chemicals. Too much starch or any other diet that causes steep increases in blood sugar are inflammatory and pose potential problems. Small meals and exercise may be helpful.

It is not surprising that numerous dietary and environmental compounds may cause extreme symptoms when such a high level of inflammation is present. This high level of inflammation will suppress immunological tolerance, so that all molecules in the environment become potential allergens. A peculiarity of this disease, is that the allergies should keep changing, because the population of antibody-producing lymphocytes for any allergen are eliminated by constant attrition as they are displaced by lymphocytes responding to new allergens. It also seems unusual that autoimmune diseases are not commonly associated with MCS. It would appear that the high inflammatory nature of the disease is inhibiting the apoptosis that produces the cellular antigens needed for autoimmunity.

Dietary treatment of MCS should be very successful with the caveat that very high levels of anti-inflammatory materials, e.g. omega-3 oils, vitamin C, glucosamine, may be needed, because of very high rates of consumption. Normally 1000 milligrams (one gram) of vitamin C might be effective, but with MCS, the level might be very much higher. One of the unusual feature of MCS may be the depletion of all plant antioxidants in a very simplified diet. Since normal body performance may be based on small amounts of ubiquitous plant compounds, the consequences of complete depletion may be similar to unpredicted vitamin deficiencies.

Gut flora have not been discussed in MCS. It is expected that the unusual diets of MCS sufferers may lead to very peculiar gut flora that may reinforce the symptoms of the disease. Normalization of the diet and gut flora may be very important in reducing the symptoms of the disease.

Pre- and Probiotics

The bacteria in your bowels determine your health. Your gut flora can make you chronically inflamed, obese, cancerous or healthy, and your diet can determine your gut flora. We return to the basic wisdom that you are what you eat, but you can change (or exchange?) your gut flora.

A major follow up to the human genome project is an even more ambitious project to identify and characterize all of the microorganisms that live in or on humans. The justification for this “Human Microbiome Project” is the increasing realization that the bacteria of our guts, our “gut flora”, determine our health status.

Let’s get to the crux of the issue with a simple experiment from the biomedical literature. Two groups of people were identified based on whether they were obese or lean. Their bowel contents were removed, their intestines were flushed and their bowels were recolonized by insertion of either the gut flora from a lean person or an obese person. They were then told to resume their normal diets and were monitored. Obese with obese flora and lean with lean flora had the same weight pattern as corresponding untreated obese or lean. Obese with lean flora lost weight and lean with obese flora gained weight -- the gut flora determined weight gain or loss!

Another interesting observation comes from ambitious transplants of whole digestive tracts from stomach to rectum. Patients requiring this type of transplant are clearly compromised both by the disease conditions that required the removal of their intestines, etc. and by the immunosuppressants required for the transplant. The first approach was to purge the replacement intestines of their bacteria before the transplant. This produced a paradoxical low success. Better results, healthier transplanted organs and less need for immunosuppressants, were obtained when more of the gut flora were retained in the transplanted intestines. This was explained by the need for the healthy gut flora to support the transplanted intestines through the shock of the surgery. The dominance of the new flora also helped to suppress a return to the pattern of gut flora that may have contributed to the original need for a transplant.

The bacteria in your surroundings influence your gut flora. Clostridium difficile is a common spore forming bacterium of hospitals that can colonize patients treated with antibiotics. The longer that a patient is in a hospital, the higher their colonization with C. difficile. This also holds true for newborns -- their gut level of this nasty bug increases with the length of their hospital stay, even if they are exclusively breastfed. Medical representatives don’t want to discuss the source of the C. difficile, but it seems obvious that the hospital staff may be a major source. Perhaps their gut flora should be periodically cleared and replaced with healthy flora. In that way, the health care practitioners may be contributing to the cure of their patients by setting a good example.

The good gut flora that support health can be call “probiotics” and food or supplements that support the probiotics in your gut are called “prebiotics.” Typical examples of probiotics are fermenting bacteria, e.g. lactobacilli, commonly found in the living cultures of yogurts. It is not surprising that the bacteria in the guts of newborns that are exclusively breastfed are essentially just one species, Lactobacillus bifidus. That is why the diapers of breastfed babies just smell yogurty -- the typical feces smell (or stink) comes from compounds, e.g. skatole (also added to cigarettes), produced by adult gut flora. Even a single bottle of formula fed to an otherwise exclusively breastfed baby will cause a complete change in the gut flora and a substantial increase in health risks. That change can be observed in a lifelong increase in the risk of Helicobacter pylori that causes ulcers and stomach cancer.

Formula lacks the antimicrobial, immunological (antibodies and lymphocytes), gut maturational and other protective compounds of breast milk and causes inflammation in newborn guts. The inflammation, ironically, may be what keeps formula fed babies from dying (since it stimulates the immature immune system) without the immune support of breast milk. Formula causes a shift to an adult gut flora that results in a 10-100 fold increase in respiratory and digestive tract illnesses and a six point reduction in IQ compared to exclusively breastfed babies. The protective inflammation caused by formula may be analogous to the protective nicotine that prevents smokers from immediately succumbing to the acute inflammation or cancer initiation of tobacco smoke.

Mother’s milk provides special molecules, the bifidus factor, that support the growth of anti-inflammatory Lactobacillus bifidus. The bifidus factor is actually a group of related molecules that have chains of sugars, oligosaccharides, with a galactose on the end. These galacto-oligosaccharides are potent prebiotics. Not only does breast milk encourage the development of healthy probiotics in the newborn’s gut, but a recent study shows that even synthetic galacto-oligosaccharides with or without added probiotics result in an increase in anti-inflammatory gut flora when fed to school children.

Another indication of the potential power of probiotics and prebiotics is the increase in availability of probiotic yogurts and prebiotic supplements in supermarkets. Some of these may actually be beneficial in reducing chronic inflammation. We may even see probiotic fecal transplants offered at weight reduction and health spas.

Multiple Sclerosis and Inflammation

Multiple Sclerosis (MS) is an inflammatory autoimmune disease of the myelinated sheaths of neurons of the brain and nervous system. Anti-inflammatory dietary and lifestyle changes have a significant impact on prevention and treatment of MS.

A common criticism of the biomedical literature is the focus on therapy and cure rather than cause and prevention. This is evident with multiple sclerosis. This disease is dominated by an immunological attack on the insulating myelin sheaths of the nervous system. The symptoms increase in waves of increasing severity over a number of years. MS patients produce antibodies against a myelin basic protein. Risk factors include smoking, prior infection with Epstein-Barr virus (mononucleosis) and low vitamin D/sun exposure. Treatment focuses on the neurological symptoms of the disease. My interest is what caused the disease and an exploration of whether the subsequent removal of the initiating events can provide more effective therapy. Here is what I think happens, based in part on observations from other inflammatory autoimmune diseases.

The early events in many inflammation-based diseases include a disruption of the expression of heparan sulfate proteoglycans (HSPGs). One HSPG is a component of the extracellular matrix of the cells that line blood vessels and provides a barrier to prohibit cells and proteins from leaking from the blood serum into the brain, i.e. the blood/brain barrier. Inflammation blocks normal heparin synthesis by blood vessels, attacks the BBB and facilitates the recruitment of white blood cells, lymphocytes that can mount an immune attack on the brain.

Since HSPGs interact intimately with most proteins on the surface of cells and these binding interactions are the basis for signaling and internalization, changes in the HSPGs can disrupt the maintenance of immunological tolerance -- inflammation also leads to inappropriate immunological recognition, particularly of proteins with triplets of basic amino acids (strong heparin-binding domains common on nuclear proteins.) This is evident in diabetes, lupus and inflammatory bowel diseases (and also dozens of allergens.) In the case of MS the self-antigen is the myelin basic protein. The antibody binding portions of the myelin basic protein are similar to many nuclear proteins that would be expected by comparison to other autoimmune diseases and allergies to stimulate antibody production if exposed to the immune system during inflammation. Thus an immune attack on myelin in MS could be caused by trauma or viral infections in the brain that release cellular debris and cause inflammation. Subsequent flareups could be initiated by systemic inflammation that attacks the BBB and permits lymphocytes to leave the blood stream and attack the myelin.

The MS risk with smoking may be the unusual combination of potent carcinogens and inflammatory compounds with nicotine. These may have had an impact on the integrity of the BBB via chronic inflammation. Epstein-Barr virus exposure may also have affected the BBB and contributed to chronic inflammation. Vitamin D acts similarly to the steroid hormones in many ways and may have some powerful effects as an anti-inflammatory. It may act similarly to estrogen, which is a potent inhibitor of brain inflammation in response to trauma.

Omega-3 fatty acids of fish oil are now being tested as a treatment for MS. An anti-inflammatory diet and lifestyle may be a powerful deterrent to MS and may also be effective in reducing or reversing the progression of the disease.

Miscarriage and Inflammation

Miscarriage occurs when inflammation of a pregnant uterus occurs too soon and induces labor. Aspirin, heparin and omega-3 fatty acids lower inflammation and decrease the risk of premature birth.

Pregnancy requires that a woman’s body resist the normal immunological response to foreign, non-self antigens, i.e. the fetus that has half of its genes from the father. This means that conception requires suppression of inflammation and if the woman’s reproductive system has too high a level of chronic inflammation, she may be infertile. In these cases of infertility, suppression of inflammation with aspirin and heparin can permit the woman to become pregnant and sustain a pregnancy to full term. Other approaches, e.g. omega-3 fatty acids and an anti-inflammatory diet and lifestyle, should also be helpful.

Labor and birth are a result of a peak of inflammation as the baby reaches full term. A single aspirin at the first stages of labor can suppress labor. Premature labor can be suppressed with aspirin and heparin, another anti-inflammatory drug more commonly associated with one of its other effects, clot prevention. A recent study showed that omega-3 fatty acids taken as a fish oil supplement were just as effective as aspirin in suppressing premature labor. It may be considered likely that the high incidence of chronic inflammation, prominently recognized in the metabolic syndrome associated with obesity, may significantly contribute to increasing rates of infertility, miscarriage and permature births.

Diabetes and Inflammation

Drugs that lower blood sugar in diabetics may reduce some inflammation, but enhance other inflammatory cellular markers that are risk factors for cardiovascular disease.

A recent research article show that two popular drugs for lowering diabetic blood sugar, metformin and rosiglitazone, are effective in reducing hyperglycemia, but differ in their impact on oxidative stress. High blood sugar can lead to accumulation of advanced glycation endproducts (AGE), that result from spontaneous chemical reactions between high concentrations of blood glucose and the amino groups of proteins. AGE are inflammatory and some of the symptoms of diabetes are associated with the hyperglycemia. What the recent research shows, is that metformin can effectively lower blood sugar, some forms of inflammation resulting from residual oxidative stress persist. Rosiglitazone lowers glucose, as well as lowering oxidative stress and thereby reduces chronic inflammation more effectively.

Metformin is an interesting diguanide that mimics the overall properties of many plant alkaloids and is transported into cells using the same organic cation transporters (OCTs). My students and I have studied how alkaloids, sugars and metformin interact with protein enzymes and transporters. Amazingly all of these small molecules, ligands, bind with differing affinities to similar structures in the proteins -- a flat platform of aromatic amino acids exposed to surface water. We have even observed that a common enzyme, beta-galactosidase, that hydrolyzes the terminal galactose sugar from lactose, milk sugar, binds all of these molecules. Thus, the enzyme is inhibited by metformin binding in the active site in place of lactose. This underscores the broad generalization that all of these molecules, carbohydrates, alkaloids and many drugs have a mixture of both hydrophilic and hydrophobic properties, i.e. they act like detergents. It should not be surprising that the molecule that dominates the extracellular signaling environment, heparin sulfate, has detergent-like properties and binds to basic amino acids that have similar detergent-like properties with opposite charges, i.e. cationic and anionic detergents.

Cancer Stem Cells Require Inflammation

Spread of cancer requires stem cells and inflammation. A tumor provides support for development of cancer stem cells and the inflammation transcription factor, NFkB is needed for the expression of the stem cell phenotype.

A recent research article shows that inflammation is the basis for prostate cancer and in particular for the division of cancer stem cells, i.e. proliferation. The stem cells of prostate cancer are a rare form of epithelial (surface layer) cells that produce a particular protein on the surface of their cell membrane that is different from the other cells in a prostate tumor. The researchers examined the genes that were expressed in the presumptive prostate cancer stem cells using DNA array technology that measures gene expression by the amount of mRNA transcribed from each gene. Thus, the total mRNA from the cell sample is extracted, attached to fluorescent dyes, hybridized to each of the 20,000 human genes displayed in tiny drops on a microscope slide and examined by a fluorescence microscope. The amount of fluorescent mRNA attached to each gene is a measure of the expression of that gene. Control cell mRNA extracts can be used for comparison.

The major finding of the research was that the suite of inflammatory genes controlled by the inflammatory transcription factor NFkB were characteristic of the prostate cancer stem cells. If NFkB was inhibited by the active ingredient in feverfew, parthenolide, the cancer stem cells died by programmed cell death, apoptosis. Parthenolide is used to treat inflammation, e.g. in arthritis. NFkB is also inhibited by aspirin and curcumin, the active component in turmeric. These and other anti-inflammatory plant products have been implicated in reducing cancer.

Inflammation and Longevity

An inflammatory life style and diet will kill you four times faster or the equivalent of being 14 years older. A large, new study shows the combined impact of four lifestyle/diet choices (all of which have inflammatory consequences): smoking, sedentary, low fruits and vegetables and low alcohol. The effects were additive and deadly.

It does not surprise most people that eating, exercising and smoking can impact their lives. A recent, long term study measured the impact. Twenty thousand men and women were categorized by lifestyle (smoking, exercise) and diet (fruits and vegetables [serum vitamin C], alcohol) traits and followed for ten years to see which became ill or died. The four factors that increased the rate of death were smoking, a sedentary lifestyle, low fruits and vegetables (low serum vitamin C) and low alcohol consumption. Each independently increased the death rate with combinations of one or two having less impact than three or four. A combination of all four, the couch potato who smokes, eats just chips and drinks soda, yields a death rate, with cardiovascular morbidity prominent, four times higher than the non-smoking, casual drinker who exercises regularly and eats lots of fruits and vegetable (high serum vitamin C). The increased death rate is equal to aging by fourteen years.

Smoking results in the introduction of a toxic brew of inflammatory compounds. Many of the compounds are so toxic that they cannot be handled in typical biological laboratories without special permits. They damage cells of the lungs and produce chronic inflammation. The odd thing about smoking is that smoke also contains nicotine, which has some anti-inflammatory properties. Without the nicotine, I think that no one could tolerate the smoke. Of course without the nicotine there would be no addiction to promote the smoking habit. The fact that smoking kills, is no longer a surprise. It may be less well known that smoking also increases inflammation and thereby increases the cardiovascular (and other) death rate.

Exercise is healthy, but it is also anti-inflammatory. Intense exercise may produce a quick spike of inflammation, but the persistent impact is anti-inflammatory and reduces inflammation-related diseases and death. Increased muscle mass is anti-inflammatory and increased fat is inflammatory. Trading fat for muscle, the typical pattern with advancing age, also produces increasing chronic inflammation and the typical maladies attributed to aging.

Fruits and vegetables are high in vitamin C, but they are also high in a large number of plant compounds that are anti-inflammatory. The compounds can be anti-inflammatory because they are anti-oxidants, because they block inflammatory pathways or because they promote the growth of anti-inflammatory bacteria in the gut. The bottom line is that you can get your nutrients and complex carbohydrates (not starch) from fruits and vegetables, and lower your inflammation as you live longer, or you can each starch from grains, stimulate inflammation and die young (though you appear old.)

The observation of alcohol consumption as anti-inflammatory requires some explanation. Modest alcohol consumption causes some damage to the digestive tract as it passes through and is absorbed into the blood. The surface cells suffer the greatest impact and some are damaged and die. This localized trauma produces a signal to the body that results in a regional suppression of inflammation. There appears to be an adaptation for the body to tolerate and minimize the damage associated with the productive action of eating food. It appears that alcohol is good medicine.

Glucosamine Pain/Inflammation Relief

Glucosamine supplements work through effects on the gut that contribute to general anti-inflammation. It is thought that glucosamine interacts with tissue transglutaminase and blocks production of signaling molecules for chronic infammation. It should also be effective against inflammatory diseases of the intestines, joints and elsewhere.


Cartilage and other connective tissues are made up of collagen protein fibers embedded in a matrix of polysaccharide, chondroitin sulfate. The tightly packed, twisted proteins of the collagen in the negatively charged, acidic polysaccharide act like a fiber reinforced, dense gel that provides impact resistance for long bone joint surfaces, as well as the stretch-resistance and elasticity for tendon and ligaments. Collagen in cartilage is comparable to cellulose (crystallin neutral polysaccharide fibers) embedded in the acidic polysaccharide (pectin) of plant cell walls.

The connective tissue polysaccharides are glycosamino glycans, GAGs, the most intensely negatively charged molecules in the body. They are more negatively charged than the nucleic acids, and the proteins that bind to the negative charge of the phosphates of nucleic acids will preferentially bind to GAGs if they get a chance. An example of this is the removal of small, highly positively charged peptides, protamines, used to pack the chromosomes of sperm. These small proteins bind strongly and can only be removed from sperm chromosomes in fertilized eggs, by the brief introduction of GAGs. Heparin is the most negatively charged, most highly sulfated of the GAGs and protamine, is used medically to neutralize excess heparin that has been injected to block blood clotting.

GAGs are composed of alternating amino sugars, e.g. glucosamine, and uronic acids, e.g. glucuronic acid. These are among the most primitive of polymers and may have evolutionarily predated the nucleic acids. It was initially suspected that glucosamine supplements contributed directly to the production of the glucosamine-rich GAGs of the cartilage of joints and thereby helped to reduce joint pain. This is unlikely, however, because the level of glucosamine in the blood is normally vanishingly low and glucosamine supplements increase the level only a tiny amount. Further reflection on the negligible pain relief of meat that contains huge quantities of glucosamine in its connective tissue, confirms that supplemental glucosamine could not impact joint pain by its impact on GAG synthesis.

The usefulness of glucosamine in reducing inflammation and joint pain is based on its impact on the small intestine. I came to this conclusion through analysis of recent studies of inflammatory diseases of the intestines, e.g. celiac and inflammatory bowel disease. These diseases are autoimmune diseases in which the body produces antibodies to inappropriate proteins in the diet or of intestinal cells. The first step in the diseases is the shift to an inflammatory physiology of the intestines. The intestines normally respond to cellular antigens and food antigens with specific anti-inflammatory signals that actively suppress antibody production and lymphocyte activation, i.e. immunological tolerance. Infection of the intestines by a pathogen triggers an inflammatory physiology that is intolerant and immunologically aggressive. Antigens in this environment are presented to the immune system and antibodies are produced. Proteins with heparin-binding domains, e.g. nuclear proteins that would otherwise bind to nucleic acids or signaling proteins that bind to heparan sulfate proteoglycans, or allergens that have strong heparin-binding domains for unknown reasons, are strong immunogens in this context. An enzyme that is normally bound to the surface of the intestines, but is also brought into cells for additional signaling purposes, is tissue transglutaminase, tTG. In the gluten allergy disorder, celiac, antibodies are produced against the body’s tTG. This disorder is poorly understood, but I will try to use what is known to explain the use of glucosamine in pain relief.

Our focus in glucosamine pain relief is on the structure of glucosamine and how this molecular structure is integral to inflammation through the action of the enzyme tTG. Glucosamine is just a glucose sugar molecule with a nitrogen, amino group, added to one of the carbons. In a similar way, glutamine is just a glutamic acid with an amino group added to one end. Tissue transglutaminase is an enzyme that transfers the glutamic acid of glutamine from one molecule to another. As tTG transfers these glutamic groups, it forms intermediate structures with its own amino acids forming bonds to the glutamic group. Thus, wheat gluten, or gluten fragments with long stretches of glutamines, binds directly to make a tTG-gluten complex. Because tTG has strong heparin-binding domains, there is a tendency for the tTG-gluten complexes to be brought into intestinal cells and in an inflammatory physiological state, to be presented to the immune system for antibody production. As a result, antibodies to both tTG and gluten are produced as a consequence of certain forms of intestinal inflammation.

Another key observation was that many of the proteins involved in inflammatory signaling of the intestines are glycoproteins with unusual modifications of their attached sugar chains. Antibodies can be made and used to identify these unusual modifications. The modifications turned out to be attachment of glutamic acid through its carboxyl group to the amino group of the glucosamine of the glycoprotein sugar chains. It was this linkage that drew my attention, because it is similar to the way that tTG forms intermediates with the glutamines of proteins. It seemed obvious that tTG was the enzyme that was adding the glutamic acids (carboxylation) to the glucosamines of the intestinal glycoproteins! A search of the scientific literature revealed that glucosamine is in fact an inhibitor of tTG, showing that glucosamine binds to the enzyme, probably forming an intermediate as it does with glutamine.

The central piece of this complex puzzle was the observation that antibodies used to identify the carboxylated glycans (the sugar chains of the glycoproteins that have glutamic acid added) can block inflammation in inflammatory bowel disease. This suggests that some form of the glutamic acid/glucosamine conjugate to which the antibody binds is involved in inflammatory signaling. AGE receptors were implicated, because these receptors trigger inflammation and they are glycoproteins on the surface of the intestinal cells that are observed to by carboxylated with glutamic acids. AGE is an acronym for advanced glycation endproducts, the molecules formed at high blood sugar levels in diabetics, as glucose chemically interacts with the amino groups (frequently the lysines of heparin-binding domains) of proteins on the surface of the vascular system.

I think that glucosamine in supplements lowers inflammation and decreases pain by forming conjugates with glutamine through the enzymatic action of tTG. This action and the inactivation of tTG by the formation of tTG/glucosamine conjugates lowers inflammatory signaling and could perhaps block the formation of the toxic gluten/tTG/antibody amyloids characteristic of celiac. Glucosamine/glutamic acid conjugates may also be substrates for direct interactions with the AGE receptor.

Molecular Inflammation

Much of the physiology of inflammation is the response of tissue to injury. At the cellular level, inflamed tissues express a particular set of five dozen inflammatory genes. Several different inputs can trigger inflammation, but a single transcription factor, NFkB, turns on the inflammatory gene program.

Each tissue has unique structures and functions as a result of different proteins, yet essentially all cells have the same genes. The differences are due to the expression of subsets of the total 20,000 or so genes -- each different type of cell produces different amounts of protein from each of the available genes. The history of each cell, its cellular neighbors and the biochemical signals that it has received, determines how genes are expressed by the presence or absence of the master molecular controls, the transcription factors. These control proteins interact by sticking to the control sequences at the end of the DNA sequences that are the genes along the very long DNA molecules called chromosomes. Humans have 64 chromosomes -- 23 pairs and on average each chromosome has about a thousand genes interspersed among long stretches of other DNA. The point here is that a particular protein can bind to part of a group of genes, the control region, and the designated genes will be turned on to produce the proteins of inflammation.

The transcription factor that controls inflammation is nuclear factor kappa B, NFkB. Its name reflects its initial characterization in B lymphocytes that were stimulated to produce the heavy chain of immunoglobulins, the kappa chain. NFkB was ultimately implicated in the control of the expression of many genes integral to immunity and that included the suite of genes characteristic of inflammation.

Transcription factors, such as NFkB, physically stick to particular sequences of the four DNA bases, ATG or C, and this means that the NFkB protein has positively charged (basic, i.e. arginine or lysine) amino acids that hydrogen bond with the negatively charged phosphate groups of the DNA. This general interaction is similar to the binding of extracellular proteins to heparin. Moreover, the proteins that interact with DNA or RNA are all found in the nucleus, so it is not surprising that all of the nuclear proteins have a nuclear localization signal, NLS, that in most cases consists of groups of basic amino acids (BBBB or BBxxxx...xxxBB) that are also classes of heparin binding domains. Proteins with these NLSs exposed will be transported from the cytoplasm to the nucleus and if present extracellularly, they will be endocytosed by binding to heparan sulfate proteoglycans and then transported to the nucleus. I think that these unusual properties are the basis for why extreme autoimmune diseases, such as lupus, result in the production of antibodies against nuclear proteins.

The genes turned on for expression by NFkB when inflammation is triggered yield the inflammatory cytokines, IL-1, IL-6 and TNF; the enzyme that makes omega-3 and omega-6 fatty acids into the corresponding anti-inflammatory and inflammatory prostaglandins, COX-2; and the enzyme producing nitric oxide, iNOS. You may notice that many of these protein products can in turn also stimulate inflammation and they can in fact cause the spread of inflammation in a radiating wave from the point of initiation. As this wave encounters the dendrites of neurons, pain can result, but additional signals are transmitted to the brain and the response down the vagus nerve produces the regional production of anti-inflammatory cytokines that control the continued spread of inflammation.

Many plant products bind to proteins involved in the signaling of inflammation. Aspirin for example binds to COX-2 and stops the production of prostaglandins. The inflammatory prostaglandins are needed for the proper development of stomach lining, so aspirin in high local concentrations can damage the gut. Since COX-2 is needed for anti-inflammatory prostaglanding production it also has this negative result. Aspirin also binds directly to NFkB and in so doing blocks inflammation directly. The turmeric compound, curcumin, is even more effective at blocking inflammation by interacting with NFkB. Unfortunately curcumin is “detoxified” by enzymes in the intestines and its inflammatory actions are thereby diminished. The cultural practice of including black pepper with turmeric compensates for the gut effects, because the piperine in black pepper inactivates the defensive enzymes and temporarily permits serum levels of curcumin to become therapeutic.

Hot/Cold and Inflammation

Are you supposed to apply hot or cold to an inflamed injury? The answer is both and oddly enough I think that this answer involves the science behind acupunture.

Inflammation is a cell and tissue response to insult and injury -- damaged cells release chemical signals that recruit help from neighboring and also from distant parts of the body. The signals and response result in dilation of local blood vessels, swelling, accumulation of white blood cells, warming of the injured tissue and pain, i.e. inflammation. This is a dramatic and potentially dangerous response for the body as a whole. If too much tissue is involved, the blood pressure plummets and lethal shock results. Some inflammation is needed for the body to protect itself and heal following an injury, but the response must be limited.

Inflammation spreads and is self-perpetuating -- inflamed cells tend to stimulate neighboring cells to participate in the inflammation. The body blocks this spreading cycle by a regional anti-inflammatory response. In essence local inflammation occurs in a region of tissue that is becoming less responsive to inflammatory signals -- sensations of pain as well as hot and cold that are interpreted in the brain as a result of nerve signals from distant tissues result in responses through the vagus nerve complex that result in the regional production of anti-inflammatory cytokines.


Application of hot or cold to an injured area results in a decrease in pain and inflammation. Initial application of cold is thought to slow the initial inflammation, but subsequently either hot or cold is anti-inflammatory. This appears to be a skin response that effects deeper tissues. Hot pepper products containing capsaicin (left) or cooling menthol (right) ointments work to reduce inflammation by stimulating the same receptor proteins for hot and cold. A convenient source of fast acting menthol is Vick's VapoRub. Oddly the unusual fatty acids (below) in castor oil also work by binding to the hot receptor.[figures from Wikipedia]

You can test the impact of castor oil by chewing a hot pepper, rinsing and drying your hot mouth and then rubbing your mouth with castor oil. The castor oil is related in structure to olive oil, so it has a similar taste. In about 30 seconds the castor oil penetrates the lining of your mouth and reaches the hot receptors already triggered by the hot pepper caspsaicin and the hot sensation dissipates.

You can also get pain relief by applying castor oil to hands and feet experiencing arthritic pain or tendonitis. Interestingly, with the regional response of the anti-inflammatory system, pain in a finger can be relieved by application of castor oil or menthol to the base of the finger or even around the wrist. Sweat bands or cotton gloves or socks can be used to prolong the treatment overnight and you will awaken with very soft skin [Most of the oil will be absorbed and the remainder is like a mild soap, so it is easily washed off the surface.] and relief from inflammation. Results may vary, but it can be very dramatic. Castor oil treatments seem to be very effective in reducing pain and inflammation. If it is helpful to you please let me know.

It is my observation that the placement of the castor oil can be used to mimic acupunture. If any readers have results to support or contradict this observation, please comment.

Inflammatory Proteins Bind Heparin

Particular amino acid sequences mark a protein for secretion, binding to heparin, uptake and internalization into the nucleus.

You can tell a lot about a protein from the sequence of its amino acids. Basic amino acids (arginine and lysine) arranged in groups, for example, usually mean (if it is an extracellular protein) that a protein binds to heparan sulfate proteoglycans.

It seemed strange to me that heparin-binding was so simple when I tried to determine the rules for heparin-binding by looking at the structures of several hundred proteins known to bind to heparin. Since heparin is heavily sulfated and the sulfates are negatively charged, at first I just color-coded the positively-charged , basic amino acids (blue) to look for oppositely charged heparin-binding sites on the surface of the proteins. Obvious blue patches were found on the surfaces of all of the proteins that bound to heparin and scattered blue spots were on the surfaces of other proteins. Moreover, similarly color-coded amino acid sequences showed that the blue patchs almost always had pairs of basic amino acids flanked within six amino acids by a third basic amino acid, i.e. BBxxxxB, where B is either arginine (R) or lysine (K) and x is a hydrophobic amino acid. It was surprisingly simple.

I was shocked at the simplicity, because most binding sites are made up of parts of regular secondary structures of helices or pleated sheets. If there were basic amino acids on these structures, which bound to heparin on one side, then the R/K would be repeated at specific intervals. For a helix, for example, the repeat would be BxxBxxB, because it takes three amino acids to return to the same side as the amino acids wind around in the helix. For the pleated sheet, the amino acids alternate on each side of the sheet, so the pattern is BxBxB. I found these kinds of heparin-binding domains also. The hardest patterns to find from sequences are groups formed as R/K’s on neighboring helices or sheets are brought together in the final folding of the protein.

One of the reasons that the simple pair plus one (BBxxxxB) was found so easily, is because the sequence is typically found on coils that only take shape in the presence of heparin. Thus the rigid binding of the domains to heparin is a result of the shape of the protein induced by the heparin. A related example of this phenomenon is the facilitation of the formation of amyloid fibers in the presence of heparin. The beta amyloid of Alzheimer’s disease for example, consists of a stack of small amyloid peptides with basic amino acids that line up and bind heparin along the length of the stack. Heparin is also an essential component in the amyloids of diabetes. Prions also seem to involve heparin. It is assumed that the cytoplasmic tau fibers of Alzheimer’s disease also have a similar facilitating polyanion (if not heparin), but it has not been identified.

Because of the essential nature of HSPG recycling, it is interesting that amyloid formation is toxic when the amyloid is in contact with cells. Perhaps the amyloid paralyzes HSPG recycling and thereby kills the cells. Treatments that disrupt amyloid binding to heparin, e.g. methylene blue, spare the neurons. This would also suggest the utility of berberine, a fluorescent dye for heparin, which is also a common herbal cure for arthritis, in treatment of many amyloid diseases.

The pair plus one is the minimal grouping of R/K’s that binds heparin, but larger groups bind more strongly and increase the complexity of the interaction between proteins and a cell. A triplet of R/K’s results in a protein binding to the heparan sulfate proteoglycans (HSPGs) on the surface of a cell, but as the HSPGs are recycled by being brought into vesicles within the ce)ll, the bound proteins are also internalized. These internalized proteins are then fused with lysosomes and the proteins are at least partially degraded by proteases. The proteins were released from the HSPGs by the degradation of the heparan. The modified proteins have a variety of fates. Some return to the Golgi for secretion, e.g.HSPGs and heparanase, whereas others are degraded in proteosomes and presented as potential antigen fragments on surface receptors, and still others are are transported to the nucleus. Those proteins transported into the nucleus have four R/K’s or to neighboring pairs of R/K’s, e.g. HIV-TAT, heparanase and transglutaminase 2 (?) Heparanase is intimately involved in cancer proliferation and transglutaminase is involved in Celiac and inflammation.

I have reproduced below the sequences of several human proteins from the National Center for Biotechnology Information. For simplicity, I have deleted the “uninteresting” amino acids between the heparin-binding domains. You will also see an occasional negatively charged amino acids (D/E) within the R/K groups and their hydrophobic neighbors. These amino acids bind to the amino sugars of the heparin.

transglutaminase 2
M---REKLVVRR---KFLKNAGRDCSRR---RRWK---KIRILGEPKQKRK

heparanase
M---REHYQKKFKNSTYSR---KLLRKSTFKNAK---RRKTAKMLKSFLK---RPGKK---KKLVGTK---KRRKLR

Tat [Human immunodeficiency virus 1]
M---KCYCKK---RKKRKHRRGTPQSSK---KEQKKTVASKAER

Chain A, Interleukin- 1 Beta
A---KKKMEKRFVFNK

lactoferrin
M---RRRR---RNMRKVR---RRAR---KGKK---KRKPVTEAR

Honest Nutrition: Omega-3 fats, depression and mood disorders

Here is a review:

Honest Nutrition: Omega-3 fats, depression and mood disorders

 by Neil Levin (http://honestnutrition.blogspot.com) of several research articles on the impact of omega-3 fish oils on mental disorders.  Most of the studies failed to control the omega-6 intake of the patients and thus the impact of the omega-3 oils would be expected to be minimized or confused by the uncontrolled nature of the experimental population.  The bottom line observed over and over again, is that even without controlling for the level of initial inflammation, omega-3 oils lower chronic inflammation and as a consequence disease symptoms are reduced -- many mental disorders are the symptoms of chronic inflammation.  This is reminiscent of Linus Pauling demonstrating that many mental disorders were symptoms of vitamin deficiencies.

Omega-3 and Heart Failure

A large Italian study of heart failure prevention shows that even small additions of omega-3 fatty acid-rich fish oil decreases heart failure more than statins.

The addition of one gram of fish oil omega-3 fatty acids to diets of patients after a heart attack was found (GISSI study) to reduce second attacks by 20%. A subsequent series of studies showed that the progressive degeneration of the heart muscle that results in heart failure was reversed by omega-3 fatty acids. A relatively minor supplement of omega-3 fatty acids was superior to statins in a parallel study. The improvement seen with the omega-3 supplements was in addition to other standard treatments to reduce risk and the morbidity in the Italian population was lower than would be expected in the US population.

This study used a diverse population without measures of the level of chronic inflammation in each patient. There was also no effort to determine the dose dependency -- would twice the dose have given twice the benefit? In the US, the consensus is that two grams of EPA/DHA per day is needed to see significant reduction of inflammatory symptoms within a week.

Eating Rules -- Omega-3

Fish oils are anti-inflammatory and are most effective when other vegetable oils are avoided, but are eaten in a meal in which other fats stimulate bile production.

Omega-3 fatty acid rules:

• Avoid vegetable oils in general -- corn is very bad, soy is bad and canola is not too bad
• Only olive oil is acceptable
• Flax oil is too short and still has omega-6 fatty acids -- most labeling is misleading
• Saturated fat in butter and eggs is ok and safer than vegetable oil
• More symptoms of inflammation means more fish oil supplements are needed
• Take fish oil supplements with meals and preferably fatty foods to stimulate bile

Explanation: The omega-3 fatty acids that count are those that are essential, i.e. the body can’t make them, EPA (C20) or DHA (C22), or that can be produced from ALA (C18). EPA and DHA can be converted into anti-inflammatory prostaglandins by COX, the enzyme that is blocked by aspirin. COX also converts omega-6 fatty acids into inflammatory prostaglandins. Unfortunately the corresponding short omega-6 fatty acids block the elongation of the short omega-3 ALA. For this reason, supplementing most vegetable oils, that are rich in omega-6 oils, with even high levels of omega-3 fatty acids, will still leave the vegetable oils inflammatory. In most cases the only alternatives are eating more fatty fish than you would normally eat or fish oil supplements.

Most people have found that without any symptoms of inflammation two gram capsules per day of combined EPA-DHA fish oil meet requirements for health. Two more capsules should be added per day for obesity and two more for other symptoms of inflammation, e.g. arthritis, allergy, etc. Spread the supplements over multiple meals. Eating the fish oil with other fat-rich food will improve absorbance in the gut by stimulating the release of bile -- capsules on their own will just slip on past. I would recommend an empirical approach -- start with two capsules a day and see if your symptoms lessen within a week. If not, increase by two more capsules a day and monitor your symptoms. The severity of your inflammatory inputs will determine how much fish oil is required. Other sources of omega-6 oils will sabotage the anti-inflammatory benefits of the omega-3 fatty acids supplements. Saturated fats and cholesterol are not as much of a problem as the omega-6 fatty acids (and of course trans fats.)

Obesity is a particular problem, because the fat is a source of omega-6 fatty acids that were eaten when your diet was worse. You will continue to pay for previous dietary errors. For this reason, a diet rich in olive oil is helpful, because fat stored from this oil will be low in omega-6 fatty acids that could be troublesome in the future. This may be a significant component of the benefit of the Mediterranean diet.

Swine Song

Some parasites and pathogens suppress inflammation and we may learn from them how to block the cycle of conflagration fundamental to many diseases.

“My bowels are alive with the swine Trichuris!”

This may not be a common refrain yet, but it may signal the success of new treatments for destructive inflammation of the intestines, e.g. inflammatory bowel disease, Crohn’s disease and perhaps even Celiac. In these diseases the intestines continue to rachet-up their immune defenses in response to common gut bacteria, food or self-antigens. These inflammatory diseases don’t occur in developing countries with compromised water and sewage systems, and with their related parasites and chronic infectious diseases. This correspondence of diminished exposure to parasites and pathogens, with increased occurrence of inflammatory diseases is the basis for the hygiene hypothesis that associates lack of childhood exposure to subsequent chronic diseases.

Trichuris is a genus of whipworms that live in the large bowel of mammals. These parasites attach to the lining of the bowel and release eggs that carry the infection to other individuals. Different species of Trichuris are adapted to each host species, so that eggs of the swine whipworm will produce only a transient infestation in humans. Consistent with the hygiene hypothesis, people given swine whipworm eggs only temporarily harbor the parasites, but this exposure to the parasite also provides relief from patients with Crohn’s disease.

The intestines are a major organ of the immune system. They are home to most of the lymphocytes and developmentally most of the immune organs start as branches of the digestive tract. The intestines must distinguish between food, friendly microbes and pathogens. Simplistically, that means that it must declare friend or foe, self or non-self, for each molecule, generally protein or carbohydrate, that is presented on its surface. Inflammation causes an alarming shift in the threshold toward foe and in the throes of intense inflammation, many mistakes are made and friendly cells are sacrificed. This is inflammatory bowel disease.

Parasites living in the bowels are adapted to suppress inflammation and thus, it is not surprising that infesting inflamed bowels with parasites might result in a spillover of the suppression into pathologically inflamed tissue. This may even be transmitted to the whole patient. It would make sense that whipworm treatments would benefit a variety of inflammatory diseases including allergies/asthma, arthritis and perhaps some cancers. Generalized suppression of inflammation may even have an impact on heart disease and autoimmune diseases such as MS, Alzheimer’s and diabetes.

There is no opposite to inflammation. There is only the absence of inflammation or anti-inflammation. Perhaps a closer approximation of the opposite would be tolerance. The healthy intestine does not react to itself, i.e. it is self-tolerant, because cells of the immune system that had the ability to recognize and bind to self molecules were killed early in their development (clonal deletion.) Food is a different problem, because it is not present as the lymphocytes develop. The intestine has an active system to suppress immune action against components of food and beneficial bacteria of the gut. In the absence of inflammation, the intestines teach tolerance that spreads throughout the body. Whipworms (and some bacteria) evoke a tolerant state in bowels by suppressing inflammation and researchers are actively trying to figure out how they do it.

Why Guinea Pigs?

Guinea pigs, people and primates can’t make vitamin C, salmon runs make women fertile and seed crops provide resistance to infectious diseases.

What’s the point of vitamin C? How is a college athlete sweating in the sun like a speed freak? Are women (or men for that matter) more fertile after a sirloin or a salmon steak? The questions are a bit tricky and they obviously tie into inflammation, so they will need some explanation.

Most plants and animals synthesize their own ascorbic acid, vitamin C, in four steps from glucose. Humans, primates and guinea pigs do not. Vitamin C is used in the conversion of proline into hydroxyproline. Proline is an amino acid that normally punctuates the regular structures of amino acids in proteins so that helices start and stop at the correct points as the linear necklace of a eight hundred amino acids of a protein twists and turns to make a compact, three dimensional enzyme. With the help of vitamin C, collagen proteins made of repeated amino acid triplets containing proline, have a proline in each triplet converted into hydroxyproline. The result of this substitution is that the collagens can now wind around other collagens to form very strong twisted threads that are the foundation of connective tissues. In the absence of vitamin C, the threads form weakly and the resulting connective tissue literally fails to hang together and the result is the symptoms of scruvy: blotchy skin, swollen gums, loose teeth and depression.

I noticed cases of what looked like modern day scurvy in the graphic anti-amphetamine ads on television. It turns out that speed users end up with symptoms that look to me like the defects in connective tissue that I associate with vitamin C deficiency. I think that the speed addicts are suffering from inflammatory depletion of their vitamin C reserves. Amphetamines are rather nasty, because if you take just one dose, your body goes through withdrawal and responds with a sustained burst of the inflammatory cytokine TNF. If you keep taking amphetamines, your body responds with a sustained burst of TNF. Either way you end up wasted.

TNF was originally called cachexin from cachexia, which is the term for the loss of appetite and wasting associated with terminal stages of diseases such as cancer. So TNF is involved in inflammation, which at the cellular level results in the production of enzymes that produce reactive oxygen species (ROS) that would otherwise be useful in protection from pathogens. In a TNF storm all the ROS do is deplete vitamin C. I would expect that someone showing the symptoms of cachexia or amphetamine-induce scurvy might use up ten grams or more of vitamin C a day, if they could get it.

So what about the sun-drenched college athlete? In this case, I suspect that he has a genetic defect in his mitochondria that also results in a depletion of vitamin C. It is not as drastic as the meth-abuser, but still leads to weakened cartilage and sensitivity to the sun. He fails to use sunglasses and the intense blue light on the field further disrupts the mitochondria in the corneas of his eyes. The damaged mitochondria produce ROS and attack the cornea proteins, making them weak and disrupting the optical properties of the surface of the eye. The sun always causes skin damage that can be partially offset by vitamin C supplements. In this case, the daily need for supplements may be several grams of vitamin C per day.

Ascorbic acid is called a vitamin, because it must be present in the diet or a deficiency disease develops. With the increasing prevalence of inflammatory diets and inflammatory lifestyles, production and accumulation of ROS is also increasing. In previous decades connective tissue destruction associated with scurvy was avoided with a couple of hundred milligrams of dietary vitamin C. An anti-inflammatory dose that could also reduce ROS was probably about a gram of vitamin C per day. Nowadays, I think that to have an impact on the chronic inflammation of our obese population the amount of vitamin C may have to be more than doubled and will vary dramatically from person to person.

I haven’t mentioned antioxidants out of simplicity. The ROS are produced by many different processes associated with inflammation and the presence of ROS can also cause inflammation. There are cellular defenses against ROS, i.e. enzymes such as superoxide dismutase and these enzymes are obviously important, because disruption of their functions causes severe diseases, e.g. ALS, and in animal studies increased expression of these enzymes increases longevity. You can also eat your veggies and many of the unusual plant products, e.g. polyphenols, will calm down the reactive oxygen species, i.e. they are antioxidants. But don’t be fooled by the simplicity of the declaration that a plant molecule is an antioxidant. These “antioxidants” interact with dozens of different signaling pathways. That is why all drugs, many of which started out as plant products, have “side effects”.

I finally turn to fertility. Pregnancy requires that the natural inflammation in response to foreign molecules be compromised. In the absence of heightened body-wide inflammation, due to infection, disease, or physical stress, a fertilized egg can locally suppress inflammation and implant. The embryo can continue to suppress inflammation until it is full term, at which time inflammation is enhanced and a baby is born. Chronic inflammation can lead to infertility that is further reinforced by an inappropriate immune reaction to implantation. One treatment for this condition is heparin plus aspirin, both anti-inflammatory.

The introduction of agriculture to human culture led to grain-based diets. This was a dramatic shift from leaves to seeds, which is also from high to low vitamin C, from low to high starch and from high to low omega-3 to omega-6 fat ratios. In essence, this is from an anti-inflammatory to an inflammatory diet. Just as an added interest, this social structure also led to more consistent use of cooking and the production of sugar-amino acid conjugated compounds call advanced glycation endproducts or AGE, which are also highly inflammatory. Does it make sense that agriculture would lead to chronic inflammation? The answer may come from consideration of the impact of inflammation on communicable diseases. Agriculture leads to dramatic increases in population densities and the potential for epidemics. Chronic inflammation inhibits epidemics by enhancing immunity, so human responses of inflammation to the dietary consequences of agriculture are highly advantageous. Chronic inflammation, however, trades resistance to epidemics for increased aging and degenerative diseases.

And finally, what about the salmon? It is provocative to think that agriculture may have reduced the fertility of women until the annual salmon harvest, which brought with it a dramatic increase in the omega-3 fatty acid composition of the diet, a suppression of chronic inflammation and a window of enhanced fertility. Contact with the sea may have been essential for the fecundity of a civilization.