Toxins, BPA, Estrogens

Toxins and estrogen mimics are potential threats to our health, but diet-induced inflammation is a major contributor to disease and makes us susceptible to environmental toxins.

The world is full of toxins. More industrial toxins are produced each year and many escape. The drugs and bioactive molecules that people consume seeking health end up down the sewer and into waterways via inadequate sewage treatment. Estrogens from birth control pills end up in our waterways in sufficiently high concentrations to alter the fertility of some fish species and impact ecosystems. The big question is, “Are these molecules toxic to us at very low environmental concentrations?”

Can we argue that if some people are sensitive enough to suffer from a millionth of a peanut, that vanishingly low concentrations of a molecule, e.g. bisphenol A that interferes with the binding of estrogen to its receptor, can cause severe development problems and disease? This may be a suitable comparison, because in both the case of peanut allergy and an acute sensitivity to a particular chemical, the sensitivity may be the key issue. Most people may suffer no problems, because a healthy body processes toxic materials and keeps functioning. Those people with defective immune systems, in the case of allergies or unusual sensitivities may be the canaries in the mine -- warning the rest of us of accumulating toxins. Alternatively, the unusual sensitivity may be similar to the allergies and provide a dangerously aggressive response to an otherwise innocuous environmental material, such as a peanut.

How do we know if the toxins in our midsts are real threats? Lets treat the easy cases first. Cigarettes are not too dangerous until they are burned, but then the smoke and the filters or unused butts are quite dangerous. I don’t think that most biochemistry research laboratories could get permission to bring the hazardous materials from a burned cigarette into their laboratories. Special handling, including air purification would be needed. Clearly this type of toxic material that can cause damage at very low concentrations must not leach into our environment. Cigarette butts should be handled as hazardous waste and should be never left unattended. It should certainly be illegal to discard this material where children could come in contact. Radioactive materials similarly should also be handled with great care.

Our bodies can handle relatively large amounts of some toxic materials -- detoxifying enzymes are present in the walls of our intestines and in the liver to convert toxic dietary molecules into other molecular forms that are less toxic and more readily excreted in our urine. Thus, we can eat some plants that would kill our pets, because dogs and cats lack the ability to handle common plant toxins. Some manmade toxins may be similarly handled by functional detoxifying systems in healthy people. A classic argument in this context is whether organic vegetables are healthier than those produced by chemical farming. Organic vegetables have higher levels of naturally occurring, but nonetheless potentially hazardous toxins produced by opportunistic fungi, i.e. mycotoxins, and the plants themselves, e.g. phytoalexins. Chemical farming produces veggies higher in manmade toxins. Which is safer? I would choose the organically grown, but that is probably just my personal prejudice, because the research is fairly clear that organically grown veggies don’t actually make people healthier. It is healthier to eat more veggies from any source. Unfortunately, just adding vitamin supplements also doesn’t improve health. That is why I recommend an anti-inflammatory diet supported by an anti-inflammatory lifestyle.

But what about the bisphenol A that is in the news. As the name implies, this molecule has two ring structures, phenols, that are connected and this combined structure vaguely compares to the structure of steroid hormones. The bisphenols were originally synthesized as potential birth control drugs. They aren’t very effective in competing for binding to estrogen receptors or to the enzymes that are involved in the synthesis of steroid hormones. I don’t know how quickly the bisphenols would be excreted from the body, but I doubt if they accumulate. So BPA should be excluded as an added ingredient in shampoo, for example, but I personally would not worry about the small amounts that leach from polycarbonate bottles.

There is one worrisome observation that I heard in a news report, and that is that BPA may disrupt the release of adiponectin from fat tissues. Adiponectin is anti-inflammatory, so this got my attention. I guess I will keep drinking my water from a glass.

I will leave with a final anecdote. My wife encouraged me to contribute to a listserve that was discussing the dangers of soy baby formula, based on the use of genetically modified soybeans and glyphosate herbicide. Since I had told my wife that glyphosate, which readily breaks down into an amino acid and phosphate in soil, is one of the few safe and environmentally friendly herbicides, I agreed to present my views that genetically modified soybeans were probably a better source for the baby formula than traditionally grown soybeans. (This doesn’t suggest that I think that any formula is safe for babies -- I am only considering the relative safety here.) So after I went out on the limb here and supported glyphosate, I read that this herbicide modifies the metabolism of the soybean plants and increases the level of estrogenic compounds! I had recommended a treatment that potentially exposed this group of soy formula-fed babies to higher levels of phytoestrogens. So, beware of unknown consequences and stick to mother’s milk.

In summary, my personal feeling is that the toxic molecules in our environment are a minor impact on our health. Environmental carcinogens, for example, are a rare source of mutations in our cells compared to the natural mutation frequency due to replications errors of our cellular machinery. Unexposed to any environmental insults our cells would produce at least one hundred times as many potential cancer cells as result from common environmental carcinogens (smoking accepted). I think that we should focus our attention on reducing the big problem of diet-induced chronic inflammation, thereby develop a higher general level of health that would reduce degenerative and autoimmune diseases by at least 90% and then consider the smaller problem of environmental toxins. Most healthy people would resist the minor impact of toxins, as well as their personal genetic predispositions, to disease.

Rattlesnake Venom

Plants attack pathogens, pests and herbivores with toxic chemicals, whereas snakes attack other animals with heparin-binding protein toxins. The rattlesnake toxin, crotamine is an example of a small peptide cross-linked by disulfide bonds, which attach to cells via heparin-binding domains.

I had a sabbatical in Singapore, at the National University. I was seated on the patio of the university canteen eating one of my typical lunches: curried mutton with hot lentils, rice and a durian milkshake, served on a banana leaf. I struck up a conversation with a biochemist who studied the structure and function of snake venom proteins. He was systematically analyzing the proteins in various venoms looking for proteins that interact with essential features of cells. This was a potential initial step for the design of new drugs. I remember asking him how he knew which part of the venom proteins was important and which parts just served as a rigid platform to display the active parts. He said that it was simple, the water-binding, hydrophilic amino acids that formed amorphous loops bordered by sulfhydryl-bonding cysteines were his targets. Ten years layer, I observed that these loops also have the basic amino acids (K, lysine and R, arginine) that form heparin-binding domains.

His conversation came back to me a couple of days ago when I ran across the structure and function of the crotamine toxin from the venom of the South American Rattlesnake, Crotalus durissus terrificus.

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG

The heparin-binding domains were evident in both the amino acid sequence as well as the protein structure. As is true of many small proteins, or peptides, they are held in their functional shape by -SS-, disulfide bonds, between cysteines (C). The presence of two well-defined heparin-binding domains (blue) also predicts that the toxin would be anti-bacterial and that it would bind to phospholipids, i.e. membranes. The observed toxic quality of the toxin is its ability to disrupt ion transport through membranes and it has a shape similar to the mammalian anti-bacterial peptides, defensins. Most venom toxins bind to the heparan sulfate proteoglycans (HSPGs) of their victim's cells and then as the HSPGs are brought close to the cell surface during recycling, the toxins attack the membrane proteins and kill the cells.

I am tangentially interested in snake venom, because the proteins that mediate its toxic effects are related to the hormones that mediate inflammation.

It is interesting that a simple discussion over curried mutton ten years ago would be so consistent with a major shift in my research interests to study inflammation.

Simple Remedies

Simple is usually best. Traditional herbal cures are tried and true. I learn a lot about biology by studying what works in herbal medicine. Some simple plant products, such as Vicks Vaporub, are very potent cures for what typically ails you. Castor oil is an excellent topical pain killer.

Herbs and spices excite our senses and dominate cuisines. Cultures are identified by their food, but the use of particular plant materials to food is not a random act of history, nor is it limited to the regions where the herbs and spices first appeared. Research by Paul Sherman at Cornell, and others has shown that herbs and spices that are used in a culture are also the most effective at inhibiting pathogens and parasites where that food is traditionally served.

I must talk about some related experiences that touch on the same subject, but are simply fun explanations of cultural practices. Milk is used in some interesting cultlural practices, because it has very potent anti-viral, anti-bacterial and anti-fungal components -- milk keep newborns well nurished, but also safe from nasty germs, etc. while the immune system the baby matures. Two astoundingly disparate applications of milk come to mind: pruning fruit trees and walking hot coals. I have observed both. I previously worked with plant pathologists and I watched pruners sanitize their shears in milk between trees. The milk stopped the spread of viruses and bacterial pathogens. I also observed firewalking in the Sri Mariamman temple in Singapore. The firewalkers stepped from the coals into a pool of milk to stop infections of their singed feet. In both examples, milk provided an abundance of anti-microbial molecules that were retasked from protecting babies to protecting trees or adult feet.

Herbs and spices are plant products that are toxic to plant pathogens or herbivores, which are retasked to protect people. Some of these, such as the curcumin in turmeric, are potentially more effective that commercial drugs. I want to point out some of the common plant materials that are very useful in our diets and to remedy common infections, aches and pains.

My current champion cure-all is Vicks Vaporub. This thick ointment has the pleasant scent of its ingredients, menthol, eukalyptol, camphor and terpentine. I associate the smell with childhood treatment for congestion. I also remember that my father used to rub it on his arthritic hands to loosen them up before a day’s work. There is a solid physiological basis for the action of Vicks. Many of the ingredients are powerful antibiotics effective against a variety of bacteria and fungi. Vicks is one of the most effective topical treatments for athlete’s foot and ringworm fungal infections. The menthol is cooling, because it binds to the cold sensing receptors and it is an effective analgesic and anti-inflammatory, because it triggers acupuncture like responses through the vagus nerve. I would also try Vicks on autoimmune conditions of the skin, because of both the anti-inflammatory and anti-bacterial properties. I have even seen Vicks recommended for the same reasons for the treatment of acne. TMJ pain and inflammation apparently responds to the menthol.

Castor oil binds to heat detecting receptors of the skin and works similarly to hot pepper capsaicin. Castor oil can be used to stop many aches and pains in arms and legs by topical applications. Since most of these plant products act through the pain sensors in the skin, they don’t actually penetrate to the joints involved, but rather they trigger release of neurotransmitters from nerves that do penetrate to the sites of interest. I also think that the use of castor oil packs applied to the skin of the abdomen, may have systemwide anti-inflammatory impact.

Garlic is the most anti-bacterial of the herbs, but most of the common herbs added to food probably affect the gut flora and shift it to a more anti-inflammatory composition. Many herbs and spices are used as topical cures for acne, because of their combined antimicrobial and anti-inflammatory qualities. Plant materials are very potent. They contain many compounds that are highly effective at low concentrations in neutralizing plant pathogens and herbivores, and so they are also very potent in their impact on the bacteria of our gut and potential on our own systems. Plants are powerful, but just because they are natural does not mean that they are safe. Plants are also rich sources of poisons. Domesticated plants are safer, because we have selected for variants that have lower levels of the compounds that the plants need to otherwise protect themselves. This also means that the compromised varieties need to be sprayed with antibiotics, e.g. apple trees sprayed with streptomycin, fungicides and herbicides. We have traded one group of plant toxins for manmade toxins. All this aside, plants are necessary for our health, but it is better to browse over many different plants than eat a lot of just one. Grains are a relatively recent addition as large components of the human diet, and should also be limited because of their high starch and inflammatory omega-6 oil content.

Turmeric, red pepper and black pepper are commonly ground together and used to enhance many dishes in a variety of different cuisines. It turns out that the curcumin in turmeric and the capsaicin in red pepper are very potent anti-inflammatory agents, but they are enzymatically modified as they are absorbed through the intestines. The black pepper piperine inactivates the enzymes of the intestines and enhances the effectiveness of the other two chemicals. Thus, there has been a lot of trial and error optimization in the use of spices. It makes a lot of sense to eat the way that locals eat when traveling.

Chronic Disease and Bacteria

There is increasing evidence that many, if not all, chronic degenerative and autoimmune diseases have a bacterial component and that antibiotics may be much more broadly useful.

Antibiotics are fed to children with ear infections, fed to cattle to fatten them, sprayed on apple orchards to stop fireblight and used to treat AIDS patients with nuisance to nasty infections. Bacteria are in many ways better adapted for exploiting nutrients than plants or animals, but antibiotics level the playing field.

Fungi have the same problems with bacteria stealing their food and they have adapted their biochemistry to kill off competing bacteria with their antibiotics, while they digest the world around them with enzymes. Thus, the classic antibiotic, penicillin, is made by a green mold and this antibiotic kills bacteria by mimicking one of the ingredients used to make bacterial walls -- the unsuspecting bacterium incorporates the penicillin into its wall and the compromised wall stretches apart. Bacteria grow themselves to death in the presence of penicillin.

Antibiotics discriminate between bacteria and us, because the bacteria use different molecular machinery, e.g. ribosomes, cell walls, than we do. So antibiotics kill bacteria and our cells are spared. Some antibiotics are somewhat specific and can be used to kill just certain types of bacteria, while other antibiotics are broad spectrum and rather indiscriminate.

Use of antibiotics is problematical, because most of our bacteria are necessary for our health. Antibiotics kill a substantial fraction of the beneficial bacteria that live in and on us, and leave us exposed to opportunistic pathogens and parasites. Everyone is familiar with yeast infections following a course of antibiotics and the rapid acquisition of hospital strains of antibiotic resistant bacteria.

Another problem with antibiotics is that they fail to reach every nook and cranny of the body. Sinuses, for example, provide vast distances that separate bacteria from the blood stream. Similarly, pieces of metal that pierce cartilage can produce bacterial infections that can’t be reached by antibiotics, because cartilage inhibits capillary production, so their is no blood circulation to carry the antibiotics to the bacteria. In the same way, the depletion of capillaries in the extremities of diabetics with poorly controlled blood sugar, makes infections difficult to treat with antibiotics.

Even under the best of conditions it is difficult for antibiotics to reach lethal concentrations for resident bacteria and biofilms make survival of bacteria certain. Bacteria have adapted to the hostile environment of human tissue by making structured colonies. The outer layers of bacteria are sacrificed to spare the inner, proliferating bacteria. Bacterial biofilms are highly resistant to antibiotics and to attack by the cellular defenses of the immune system. Unfortunately the bacteria in biofilms, and in other forms adapted to human tissue and have a different metabolism and stain differently from the same bacteria grown on laboratory agar plates. Thus, it is quite possible for pathogenic bacteria to be undetectable and fail to be associated with a particular disease.

Bacteria may avoid scrutiny by pathologists, but they cannot fail to release cell wall fragments and debris, which triggers inflammation and other responses from the surrounding tissue. Numerous researchers have implicated mycobacteria, chlamydia and many other bacteria as causal agents of degenerative and autoimmune diseases -- there is a distinct correspondence between inflammatory diseases and these bacterially-associated diseases. There are also recent patents supported by provocative experimental data, that claim that aggressive antibiotic treatments (usually supported by anti-inflammatory lifestyles and diets) can cure these diseases.

I expect that antibiotic cures for many degenerative and chronic diseases will be demonstrated with a huge accompanying upheaval of traditional medicine. There will be, and there has been for the last century, substantial resistance to antibacterial approaches. Even though Helicobacter pylori was implicated as the causal agent of ulcers, antacids and blockers of acid production persist as the predominant treatment. The role of bacteria in chronic disease will be substantiated, but for me the lingering question will be, “where are the bacteria that are being treated?” I still think that the best candidate is the gut. Afterall, the gut is the body’s major interface with the outside world and there is a compelling research literature on the impact of gut flora on health and disease.

Migraine, Heparin, Glucosamine

Is it possible to repair the damage that leads to migraine headaches? If chronic inflammation is a risk factor, then depletion of brain heparan sulfate proteoglycans could be repaired temporarily with heparin or gradually with glucosamine.

Mast cells, the secreters of histamine in allergic reactions, also secrete heparin at the same time. In fact, cattle and swine intestines are the sources of commercial heparin. The crude source of heparin became dangerously apparent in recent scandals over the adulteration of Chinese heparin raw materials with hypersulfated chondroitin sulfate. But why is heparin secreted along with histamine in mast cells and what does this have to do with migraine headaches and glucosamine?

A basic observation is that migraineurs who receive heparin or glucosamine treatments for other symptoms, see relief from their headaches. Heparin treatments can produce dramatic effects that may be shortlived. Glucosamine may require substantial amounts taken orally for four to six weeks before migraines abate. Heparin and glucosamine are both effective, but how do they work?

Heparin and glucosamine therapies are both awkward. Heparin is rapidly internalized and degraded by cells. Glucosamine is also rapidly taken up and processed. In both cases, ingested heparin or glucosamine do not enter the blood stream. I think it is highly unlikely that either taken orally will have their effects directly in the brain or joints, simply because they are commonly metabolized molecules. Glucosamine may be a building block for heparin and other glycosaminoglycans, but it just passes directly into glucose metabolism, so that logical connection is unimportant in joint pain or migraines. I covered glucosamine in more detail as an anti-inflammatory molecule in another article, so here I will only emphasize that glucosamine is very effective for prophylaxis of migraine headaches.

Heparin can have an impact orally for some bowel diseases, but for headaches it must be administered IV or by inhaling. The major point here is that heparan sulfate proteoglycans (proteins with long heparan polysaccharides) mediate most of the hormonal signaling by mediating the binding of hormones to their receptors. Heparan fragments, called heparin, are a mixture of molecules that may interfere with or augment signaling, dependent on the quality of the heparinoids and the signal pathways under consideration. In most cases, there are so many different hormones and receptors involved, that it is more straightforward to try heparin to see if it works, rather than attempt to sort out all of the side reactions. If heparin does work, it should also be noticed that the amount of heparin required for an anticoagulant effect is on the high side, reflecting the depletion of circulating heparin.

Mast cells and histamine release have been implicated in migraine, but we have to return to the question of why heparin is normally released at the same time. From my own experiments with chondrocytes, the cartilage secreting, developmentally related sisters of the arterial endothelial cells, I would expect that inflammation inhibits heparin synthesis. Prevailing inflammation may reduce the production of heparin by mast cells and neuronal cells. As a result, triggering mast cells may release histamine into heparin-depleted brain tissue. If heparin normally serves to control the spread of the inflammatory signal from mast cells, then the absence of adequate heparin may lead to a spreading inflammation, a kind of neurological shock and awe. Addition of circulating heparin may temporarily repair the blood brain barrier, just as it does the lining of the bladder with interstitial cystitis or the kidneys in diabetes or the intestines in protein lossing enteropathy.

It appears that migraines are based on chronic inflammation of the brain and an associated compromise of the blood brain barrier. The source of the chronic inflammation may be shared with other degenerative and autoimmune diseases that lead to migraine attacks. Underlying infections may be hard to identify. The solution is to reestablish the blood brain barrier by eliminating chronic inflammation. Separate reports indicate that glucosamine, omega-3 fatty acids and anti-oxidants are all effective in reducing migraines. It seems obvious that the first step would be to begin an anti-inflammatory lifestyle (including exercise, dental hygiene, etc.) supported by an appropriate diet.

NO Migraine

Migraine attacks are based on inflammation and nitric oxide production in the capillaries and nervous tissues of the brain. Drugs that impact NO have a major impact on migraine attacks.

What do angina/nitroglycerine, impotency/Viagra, and migraine/L-NMMA have in common? The answer is nitric oxide, NO, a potent vasodilator and messenger molecule derived enzymatically from the basic amino acid arginine. NO is ubiquitous, short-lived and associated with inflammation. One of the NO synthetases (iNOS) that make NO is inducible as part of the suite of inflammation genes under the control of NFkB. Other NOS enzymes are controlled by cellular calcium levels and since we will be talking about neurons, it is important to mention that neurons ultimately release neurotransmitters when an action potential reaches the synapse and causes a sharp increase in calcium.

NO acts on a cell by activating cellular GMP cyclase to produce cyclic GMP. cGMP in turn can activate MAP kinase or NFkB. Viagra, by the way, enhances the vasodilation effects of NO by inhibiting the breakdown of cGMP and thereby causing more dilation with a limited amount of NO. Nitroglycerine/glyceryl trinitrate (GTN) increase the amount of NO available to endothelial cells and relax coronary arteries.

People who suffer from migraines, migraineurs, are sensitive to NO donors such as GTN and have a migraine attack after a delay of 4-6 hours. The headaches are not usually preceded by the typical visual auras. Non-migraineurs have no response. These results suggest the critical role of NO in the early stages of the migraine attack. Inhibition of NOS by L-NMMA reduces symptoms in most migraineurs after a spontaneous attack starts. Metabolites of NO also increase during migraine attacks.

Viagra, which blocks breakdown of cGMP, also induces migraine attacks in migraineurs and it does not cause dilation of the middle cerebral artery, suggesting that vasodilation may be only associated with migraine attacks, but not integral. Histamine can also cause attacks, but requires the NO pathway.

NO appears to act by increasing the release of the neuropeptide called CGRP, because NO initiated attacks can be stopped by drugs that block the CGRP receptor.

Inflammation is both a precondition for migraine attacks and a response to administration of NO donors such as GTN. The inflammatory transcription factor NFkB is activated by GTN and elevated levels of inflammatory cytokines, IL-1, IL-6, are released. Pretreatment with parthenolide, the active ingredient in feverfew, which is used as a migraine prophylaxis, reduced activation of NFkB by GTN. This observation reinforces the view that migraine pain results from NO induced inflammation.

The most effective and selective treatment for migraine headaches are drugs that block the action of the 5-hydroxytryptamine (5-HT) receptors, e.g. triptans, such as pizotifen and methysergide. The 5-HT receptor stimulation can result in NO production and blocking these receptors, blocks subsequent NO production, inflammation and pain. Prophylactic administration of NOS inhibitors, such as L-NMMA or NO scavengers, such as hydroxocobalamin, can reduce attacks.

There is substantial evidence that anti-inflammatory lifestyle and diets have a significant impact on the frequency and intensity of migraine attacks, because they minimize the participation of inflammation and its product, NO.

Neeb L, Reuter U. 2007. Nitric oxide in migraine. CNS Neurol Disord Drug Targets. 6(4):258-64.

Toulouse-Lautrec

Cathepsin K and Heparin

The power of simple sequence pattern analysis in predicting protein behavior is illustrated in the case of Cathepsin K, a papain-like cysteine protease, involved in many degenerative diseases, bone development and Pycnodysostosis (Toulouse-Lautrec syndrome.) Triplets of basic amino acids are typical of heparin binding proteins that are internalized.

I admit that I am obsessed with inflammation and heparin. My daughters automatically yell out “Give him heparin!” when a patient on ER has a severe migraine attack. They think it is a good joke until the savvy doc reveals the latest approach and actually injects heparin with satisfying results. Heparin and inflammation are intimately involved and I predict that blood tests that determine the quality and quantity of protein-bound heparin, will ultimately be used as measures of chronic inflammation, as well as revealing a variety of diseases.

I have a habit of examining the molecular basis of diseases that I encounter on TV, in newspapers or in books. Wikipedia is my first source, followed by the National Center for Biotechnology Information (NCBI). As soon as I find the genes/proteins involved, I check to see if the structures has been determined by X-ray crystallography or NMR, and then I look at the amino acid sequence. I check for pairs or triplets of basic amino acids. Invariably the pairs are matched with a neighboring basic amino acid, and that is a putative heparin-binding domain. Triplets almost always indicate secreted proteins that are brought back into cells dependent on strong affinity for recycled heparan sulfate proteoglycans. Within minutes of hearing about a new disease, I usually know something about the molecules involved and particularly whether or not inflammation is going to be a major factor.

I was just reading Outlander by Diana Gabaldon and one of her characters has the short stature and disablity of Toulouse-Lautrec syndrome. I literally ran to my computer, because I am particularly interested in diseases of cartilage and bone. Since TLS is a genetic disease, I checked the NCBI Online Mendelian Inheritance in Man (OMIM) site and found that the genetic defect is in the cathepsin K gene. Cathepsin K is a protease similar to papain, which is intimately involved in many different facets of development, as well as cartilage and bone production. The cells that degrade cartilage to remodel bone, osteoclasts, use cathepsin K to degrade collagen.

I found a structure for cathepsin K bound to chondroitin sulfate. The structure looked all wrong, based on my prejudices -- the sugars of the polysaccharide should have been bound to the basic amino acids or to surface aromatic amino acids. The accompanying amino acid sequence told the whole story:

---DYRKKGYVTPVKNQGQCGSCWAFSSVGALEGQLKKKT---

There were two triplets of basic amino acids (R, arginine or K, lysine), indicative of internalization and strong heparin binding. I performed a quick literature search for heparin binding and internalization and found a reference that confirmed my hunches (note the title):

Nascimento FD, Rizzi CC, Nantes IL, Stefe I, Turk B, Carmona AK, Nader HB, Juliano L, Tersariol IL. Cathepsin K binds to cell surface heparan sulfate proteoglycans. Arch Biochem Biophys. 2005 Apr 15;436(2):323-32.

The article demonstrated that cathepsin K bound only to the surface of cells that produced heparin sulfate and was internalized only by heparin-producing cells. Moreover, cathepsin K changed shape as it bound to heparin, but not to chondroitin sulfate.

This story underscores the predictive power of simple generalizations derived from the dominating interactions between heparin and proteins. Heparin-binding domains, because of their positive charges, stay on the surface of the protein, don’t tend to fold well into helices or other secondary structures and are readily recognized in amino acid sequences of proteins. Stronger heparin-binding domains involved in internalization or transport into nuclei are even more stereotyped as triplets or quadruplets, respectively, of basic amino acids.

There are some complicating special cases involving basic amino acids, since these amino acids are also involved in glycosylation, nucleic acid binding, inositol phosphate interactions, phospholipid interactions, protein folding/chaperone binding, and protease action, but the generalizations outlined here provide a starting point for exploring the exciting area of heparin binding.

Knowing just a few typical patterns, you can just look at a protein sequence and amaze people by telling them that they should be able to purify their protein on heparin Sepharose! You can also point and gasp at the fact that in the early 1990’s in China the bird flu hemagglutinin picked up a new sequence with a quartet of four basic amino acids. When I saw that I called the CDC and explained the new cell receptor! It still has me scared -- am I the first one to notice the potential for a pandemic much more severe than the 1918 Spanish flu?

Inflammation Protects

Inflammation in response to infection mobilizes the immune system and serves a vital purpose in defense against disease and parasites. Too much or too little inflammation is unhealthy.

A recent report (ref. below) provides a note of caution in the use of anti-inflammatory dietary supplements. Modern diets are inflammatory and provide a major predisposition to degenerative and autoimmune diseases, but the inflammation has some qualified advantages in providing protection to infectious diseases. Densely packed humans survive against the constant threat of epidemics, in part because of heightened dietary inflammation.

The paper listed below shows that habitual use of the potent anti-inflammatory compound in turmeric, curcumin, can effectively lower chronic inflammation. Inhibition of inflammation is not a good thing, however, if the source of inflammation is a chronic parasitic infection of Leishmania. While this article indicates that suppression of inflammation may not be uniformly advised, it does demonstrate the effectiveness of natural products, such as curcumin.

A different study of tuberculosis comparing  land-locked versus genetically related, fish-fed populations, found that the fish eating group was more susceptible to the disease. The interpretation of the results was that the high omega-3 fatty acids of the fish diet reduced chronic inflammation in the coastal community and thereby increased the susceptiblity to TB.

The bottom line for me is that a healthy, anti-inflammatory diet is much safer than the typical modern inflammatory diet of high carbs, high vegetable oils (omega-6), low fruits and vegetables (few antioxidants, low in vitamins C, D and A) and harsh for supportive gut flora. It does leave open the possibility that during an unusual infection a return to fast food may shift your body into a state of high alert.

Adapala N, Chan MM. Long-term use of an anti-inflammatory, curcumin, suppressed type 1 immunity and exacerbated visceral leishmaniasis in a chronic experimental model. Lab Invest. 2008 Sep 15. [Epub ahead of print]

Steroid Hormones

Numerous cholesterol-derived hormones (testosterone, estrogen, corticosterone, vitamin D, vitamin A, retinoic acid) influence gene expression by directly binding to cytoplasmic transcription factors. These hormones control inflammation, as well as development and numerous examples of cellular differentiation.

Hormones are a form of molecular communication between cells in different locations in the body connected by the blood stream. Cells, for example in the pituitary of the brain, receive a nerve or chemical signal and respond by the secretion of a steroid hormone that changes the behavior of cells in other parts of the body. Steroid hormones are produced enzymatically in mitochondria by the enzymes (P450) related to those used to detoxify dietary molecules.

Steroid hormones are useful for transmitting messages, because when they stick to the surface of a protein, they can make it change shape. The shape change is the result of the hybrid structure of steroids -- they are large flat molecules that don’t bind to water, i.e. hydrophobic, on their faces, but form bonds with water, i.e. hydrophilic, on the edges. This dual, amphipathic, nature means that the steroids will bind to hydrophobic pits in proteins and as the steroid squeezes into the pits, it will change the shape and ultimately the activity of the protein. Sugars, on the other hand, have small rings of carbon atoms that are hydrophobic, but their small size means that they bind to small aromatic amino acid residues in shallow surface pits that don’t usually change the shape of the proteins. Hence, there aren’t sugar-responsive transcription factors, but large, highly charged polysaccharides, such as heparan sulfate proteoglycans, can dominate the structures taken by proteins, e.g. most protein hormones and their receptors.

Steroid hormone receptors are cytoplasmic, whereas receptors for proteins hormones, such as insulin, are found on the surface of cytoplasmic membranes. It is commonly assumed that steroid hormones, because of their amphipathic characteristics, can diffuse through cytoplasmic membrane and move directly from blood to the cytoplasm of cells. I doubt the evidence and think that diffusion is too slow to account for the action of steroid hormones. It is more likely that steroid hormones bind to carrier proteins in the blood, and general transporters transfer the steroids either directly from their carriers to intracellular receptors (testosterone, estrogen, corticosteroids) or the carriers are transported all the way to the nucleus for transfer of the steroids to nuclear receptors (vitamin D, vitamin A, retinoic acid). Binding of the steroids to cytoplasmic receptors displaces chaperones (heat shock proteins) and expose nuclear localization receptors (groups of basic amino acids that would function as heparin-binding domains, if the proteins ever found themselves in the extracellular environment) that result in transport into the nucleus.

Receptors with bound steroids act as transcription factors in the nucleus -- they bind to unique DNA sequences and control gene expression. Thus, anti-inflammatory steroids can block the signaling via NFkB from a bacterial infection (LPS triggering the TLR) by directly interfering with transcription (binding to inhibitory control elements) of inflammatory genes. The presence of estrogen receptors in a wide variety of tissues provides an overall decrease in inflammation in the presence of estrogen. Thus, women going through menopause and reduction in estrogen, may experience a sudden increase in inflammation-based symptoms that were previously suppressed by anti-inflammatory estrogen.

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.