Cooling Inflammation

Baldness Cure: Low Dose Naltrexone?

2011-08-29T16:04:00.000-06:00

Naltrexone can be used to block opioids and provide the basis for treatment for drug abuse, but in low doses (LDN) it provides a paradoxical increase in natural endorphins that reverses inflammation and provides an effective treatment for autoimmune diseases, e.g. MS.

Receptors
A recent anonymous post brought the role of cannabinoid and opioid receptors in baldness to my attention. The relationship between these receptors, inflammation and autoimmunity is very complex. The heat and cold sensors, which also bind capsaicin and menthol, appear to be mediated by endorphins. Acupuncture also seems to function by similar mechanisms and is inhibited by high dose Naltrexone.

Low Dose Naltrexone
A side effect of high dose Naltrexone (e.g. 50 mg/d) is hair loss. Low dose Naltrexone (e.g. 1 mg/day, taken at night) appears to stimulate hair production and it may reverse the effects of Finasteride, since LDN improves libido.

Endorphin-Suppressed Inflammation
I would expect hair loss to be prevented/reversed by topical treatments that block inflammation and autoimmune attack on hair follicles. Curcumin, from turmeric, blocks NFkB and appears to help hair loss. Capsaicin can block inflammation via endorphin production and also helps hair loss. I would also expect that topical menthol and castor oil would reduce hair loss.

Anti-Inflammatory Diet
The anti-inflammatory diet that I recommend, may not be sufficient to block hair loss, but it may provide a good foundation for other anti-inflammatory treatments. In fact, other topical treatments may not be effective unless chronic, diet-based inflammation is eliminated. It may also be important to reduce oxidative stress by optimizing glutathione and vitamin C.

I would appreciate comments by others who may have experience with LDN and balding.

Baldness/Prostatitis Treatment, Impotence, Inflammation

2011-07-19T19:35:00.003-06:00

Male pattern baldness appears to result from the interaction between enzyme-modified male sex hormones (DHT) and receptors in some hair follicles of the scalp. Inhibition of the enzyme by topical (Rogaine) or oral administration (Propecia) of an inhibitor, e.g Finasteride, can stop hair loss. One of the significant side effects of oral use of Finasteride is loss of all sexual functions, which can be temporary and reversible after the drug is stopped, or permanent. I think that inflammation may play a critical role in both hair loss (and prostatitis) and loss of sexual functions in response to Finasteride and an anti-inflammatory approach my be helpful.

Testosterone is Converted to Dihydrotestosterone (DHT) by 5-Alpha Reductase

The male hormone, testosterone, is produced in the testes and travels to hair follicle or to other parts of the body via the blood in either a free state, or bound to a carrier protein. Only the free form interacts with the 5-alpha reductase enzyme in the scalp to produce the DHT that diminishes hair follicles. Application of the Reductase inhibitor, Finasteride, directly to the scalp stops the production of DHT in the scalp. Taking oral Finasteride blocks DHT production throughout the body, and unlike topical application, can also result in apparent changes in the brain, which can explain loss of sexual behavior.

DHT Role in Hair Loss and Brain-Based Sexual Behavior is Poorly Understood

Surprisingly, the molecular biology of male pattern baldness (MPB) is not known, even though this is one of the classic examples of a dominant, sex-associated phenotype, i.e. a single copy of the baldness allele in males produces baldness. Of course, this is not a sex linked gene, since baldness is not inherited from mothers with their X chromosome, and in some cases as many as 80% of the males in a single family exhibit male pattern baldness. It appears to me that baldness is also likely to have an environmental, e.g. gut flora, heritability similar to obesity. In fact, metabolic syndrome and type 2 diabetes are substantial risk factors for male pattern baldness.

There is also an association between MPB and polycystic ovary syndrome (PCOS) in females of the same family. I would also expect that MPB is related to prostatitis, since the prostate is a major processor of testosterone to DHT via 5-alpha reductase and prostatitis can be treated with Finasteride. PCOS is also treated with Finasteride. PCOS is also associated with obesity and metabolic syndrome. Prostatitis, PCOS, obesity and metabolic syndrome can all be treated as inflammatory diseases with significant contribution of dysfunction of gut flora.

PMB, Testosterone, 5-Alpha Reductase and Aromatase

In PMB, testosterone levels are lower and 5-alpha reductase is higher. This suggests that testosterone has been converted into DHT in scalp hair follicles. In order for DHT levels to make a difference, the hair follicles have to have specific receptors for DHT. Testosterone/DHT receptors, like all steroid hormone receptors, are proteins in the cytoplasm of cells, which bind the hormone and become activated as transcription factors that migrate to the nucleus and control the expression of particular genes.

Testosterone can also be converted by another enzyme, aromatase, into estrogen. DHT cannot be converted enzymatically into estrogen. Estrogen has a separate receptor and controls a different set of genes. Thus, enhanced conversion of testosterone into DHT in MPB follicles, may shift the balance away from estrogenic in favor of androgenic effects. Women exposed to aromatase inhibitors, stop converting their limited testosterone into estrogen and more is converted into DHT, resulting in rapid signs of baldness. All brain estrogen is produced from testosterone via aromatase in the brain and aromatase inhibitors can reduce libido in women.

DHT Activates Inflammatory NFkB (and Block Nerve Apoptosis?)

Prolonged exposure of cerebral blood vessels to DHT has been shown to activate the inflammatory transcription factor NFkB. Conversion of testosterone to DHT by 5-alpha reductase may amplify the inflammatory impact of testosterone by virtue of the stronger activation of the androgen receptor by DHT. Activation of NFkB also suppresses apoptosis and may be necessary to maintain some neural cells. Reducing DHT production by 5-alpha reductase inhibitors, may reduce NFkB activation in the brain and expose androgen-sensitive parts of the brain to apoptosis. This loss of brain cells may result in loss of sexual behavior.

Vitamin D is also Steroid Hormone/Co-Transcription Factor

It should be remembered that vitamin D also has a cytoplasmic receptor that acts as a transcription factor and that vitamin D deficiency can result in hair loss. If fact, vitamin D is required for the normal hair growth cycle, as well as intestinal villi development (defensin production). The vitamin D receptor can also inhibit the inflammatory transcription factor NFkB.

Thus, multiple steroid hormone receptors are involved in hair development, prostate function and brain sexual behavior. Modification of the conversion of testosterone into DHT or estrogen can have diverse consequences directly or indirectly by modification of inflammation/development signaling. This is also true of fertility/menstrual cycles, mammary tissue and perhaps intestinal epithelial villi or skin/follicle development in the case of rosacea. All of these processes are affected by enzymatic interconversion of steroid hormones and interaction of hormone/receptor and NFkB transcription factors.

Questions

The questions that I have about hair loss (or prostatitis), treatment with 5-alpha reductase inhibitors, and subsequent loss of sexual function are:

Why are the genetics of MPB so unusual/non-Mendelian? There are too many males with MPB in the same family. This points to some hereditary predisposition, but with a major environmental component, e.g. “inherited gut flora.”
Why are only a few of the people treated with 5-alpha reductase inhibitors rendered permanently impotent?
There is anecdotal evidence that dexamethasone (or prostate message and antibiotics) can reverse some impotence. Does this indicate that inflammation is involved in hair loss and/or impotence? Where do the antibiotics act and is their action to kill bacteria?

Treatment for Finasteride-Induced Impotence

Impotence is a severe side effect of a few men using Finasteride to treat baldness or prostatitis. I doubt that those with induced impotence are genetically predisposed, but rather these individuals probably had an altered immune system. I suspect two types of alterations: a compromised blood/brain barrier and a compromised suppressive immune system. Diet-based chronic inflammation is a typical path to a leaky blood/brain barrier that facilitates the penetration of Finasteride into the brain to alter 5-alpha reductase in the DHT-responsive regions responsible for sexual function. I presume that the subsequent reduction of DHT also results in inflammation that contributes to loss of function. Dexamethasone and some antibiotics could attenuate the inflammation and return normal function.

Suppression of attack of normal tissues by the immune system is mediated by development of the suppressive immune system in the gut in response to specific bacteria of the gut flora. A history of antibiotic treatment can yield a dysfunctional gut flora and a compromised immune system that results in allergies and autoimmunity. Prostatitis may have an autoimmune component and may result from compromised gut flora.

All of the symptoms discussed from hair loss to prostatitis to impotence should be improved by normal function of the gut and immune system by my anti-inflammatory diet and normal gut flora. Use of antibiotics will always lead to further side effects by perturbing and limiting the function of gut flora and the immune system that is dependent on the gut flora.

I particularly suspect that vitamin D deficiency is a significant contributor. Most “anti-inflammatory diets” will lead to chronic inflammation, because they are just high carb diets with a few vegetables. All of the complex phytochemicals produced by plants will be “antioxidants”. Adding these antioxidants to an inflammatory diet has no impact. Look at my anti-inflammatory diet and note that it requires attention to serum vitamin D levels, gets most calories from saturated fat and not carbs (low carb/high saturated fat), no vegetable oils and high omega-3 (EPA, DHA) to 6 ratio. That means meat/fish/eggs/dairy and lots of fresh vegetables for new gut bacteria.

Podcast on Jimmy Moore's Low Carb Show

2011-07-02T18:40:00.000-06:00

Jimmy Moore

Jimmy Moore invited me to speak on his Livin' La Vida Low Carb show. So I recorded a conversation with him about low carb diets, inflammation and disease. It was great fun to talk about fixing gut flora and the benefits of an anti-inflammatory diet that is based on low carbs and high saturated fats.

Here is the link for show # 476, June 12, 2011.

Soluble Fiber: Food for Gut Flora

2011-06-29T16:34:00.000-06:00

The human body only produces enzymes to digest proteins, fats, starch and a few simple sugars. The remaining components of food either pass through the intestines undigested (insoluble fiber) or are digested by bacteria and fungi in the colon (soluble fiber.) Soluble fiber feeds gut flora. Insoluble fiber is usually minimized by traditional food preparation, for example grains, because it contains unhealthy materials, such as phytic acid. Soluble fiber is healthy and required for normal development of the gut/immune system, whereas insoluble fiber should be avoided.

Soluble Fibers in Vegetables are Carbohydrates/Polysaccharides

Plant cells are surrounded by cell walls composed of long chains of sugars, polysaccharides. These wall polysaccharides, e.g. pectin, arabinogalactans, xyloglucans, and storage glucans and fructans, are highly complex in structure and can only be digested down to simple sugars by the action of dozens of different enzymes produced by dozens of different bacterial species in the colon. Many plants (as well as fungi and bacteria) also produce unique polysaccharides that are only susceptible to additional unique bacteria enzymes. Thus, digestion of diverse vegetables requires hundreds of different species of bacteria in the gut. Healthy gut flora consists of more than 150 different species of bacteria, which were eaten with food and adapt to the gut environment.

Food Intolerances/Most Food "Allergies"/Constipation Reveal Missing Enzymes

Enzymatic treatment of complex polysaccharides in the gut is a complex process that also yields many intermediate products that can influence both gut flora and the gut itself. A well adapted gut flora can systematically digest most of the food molecules that pass into the colon and produce only short chain fatty acids (CFAs) that feed the colon and pass through the liver to the rest of the body.

Antibiotics or a history of limited food choices and excessive hygiene can result in a simplified gut flora that only partially digests soluble fiber and results in accumulation of unusual byproducts that irritate the gut, and cause bloating and gas. Adverse reactions are called food intolerances or food allergies. Since bowel stools are composed predominantly of loosely packed gut flora, inability to fully digest and convert soluble fiber into more gut flora, also results in constipation.

Soluble Fiber in Meat is also Polysaccharide

Meat is made of fibers of protein connected to bone by polysaccharides. The tendons, gristle and other chewy parts of meat are made of chondroitin sulfate and other glycosaminoglycans (GAGs). Heparin is another GAG, which is released onto the surface of the intestines to block the adhesion of viruses and pathogens to the gut, and is subsequently digested by colon bacteria. Other components of meat (and vegetables), such as nucleic acids and some fats are also digested by enzymes of the gut flora. The versatility of gut flora to adapt to a huge variety of foods permits people to live on very diverse diets, ranging from vegan to paleo.

Modern Diets Starve and Simplify Gut Flora

Modern diets consist of processed foods that are made of fat, protein and starch, all of which are digested and absorbed before reaching the colon. These simplified foods produce a simplified gut flora that may also produce more CFAs rather than stool forming gut bacteria. In other words, eating larger amounts of simpler foods can result in more of the nutrients being absorbed and making it easier to gain weight on less food with a tendency toward constipation. These diets may also select for bacteria that maintain the simplified, "efficient" gut flora community and provide the potential for the spread of obesity through a population. Having friends and relatives who are obese and presumably have gut bacteria that favor obesity, increases the risk of obesity. It seems likely that obesity is contagious.

Simplified Gut Flora also Means a Compromised Immune System

Complexity in the gut flora is also needed to produce a healthy immune system, because different species of bacteria in the gut stimulate the development of different parts of the immune system, which develop in the lining of the gut. Soluble fiber is the normal food for the colon bacteria that control the part of the immune system that regulates autoimmunity and allergy, for example. Obesity is also associated with increased risk of degenerative and autoimmune diseases, which is consistent with defects in the gut flora that reside in the colon. Thus, the modern high carb diet contributes to the symptoms of obesity by elevating blood sugar, blood CFAs, as well as compromising the gut flora needed for normal functioning of the immune system.

Healthy Gut Flora = Anti-Inflammatory Diet + Eating New Bacteria

A damaged or simplified gut flora can be fixed by eating foods that supply nutrients for the body as well as feeding the gut flora, e.g. plenty of different types of soluble fiber. It is also necessary to eat the missing bacteria. Just adding a few probiotics with yogurt will not fix the problem and cooking kills all of the good bacteria. Fermented foods, especially those based on bacteria from your own home and garden, are good sources of health-providing bacteria. Raw vegetables will also provide bacteria that may be useful in your gut flora, as long as the vegetables are not too thoroughly washed. Sterilizing and cooking vegetables may avoid rare pathogens, but will certainly prevent contributions to a healthy gut flora.

Food Poisoning and Manmade E. coli

2011-06-14T15:26:00.000-06:00

Bacteria on food is a problem for diet-compromised people.

Gut Flora are Required for a Healthy Immune System

Healthy people don't get sick from food poisoning, because their gut flora provide protection. Gut bacteria control the development of the human immune system by producing interesting compounds, including short chain fatty acids and vitamins. In response to the gut bacteria, the healthy immune system produces white blood cells that can effectively attack bacteria, and also control this aggressive behavior to spare human cells and avoid unnecessary attacks on beneficial bacteria.

Disrupted Gut Flora Lead to Susceptibility to Disease/Infection

Gut flora can be compromised by what we eat and antibiotics. Those normally affected by food poisoning are the very young (on formula), the old (constipated) and those treated with antibiotics. Each of these groups have abnormal gut flora. Food poisoning is rarely observed in exclusively breastfed babies being introduced to foods, because human milk contains potent antimicrobial polysaccharides (human milk oligosaccharides) that only permit the growth of a few species of Bifidobacteria. Formula (in any amount) disrupts the normal development of the gut and immune system by stimulating an inflammatory growth of adult gut bacteria, making these babies more susceptible to intestinal and respiratory diseases, including food poisoning.

Constipation, which is more common in older people, reflects a disruption of the gut flora and decreases the effectiveness of the immune system in these individuals. In most cases the compromised gut flora results from a long history of a restricted diet and reduced access to environmental sources of bacteria.

Antibiotics are usually ignored as major corruptors of the immune system, even though they are known to produce diarrhea and constipation. Doctors reluctantly suggest that people taking antibiotics should just eat some yogurt. This is a silly oversight that severely compromises future health, because probiotics supply only a tiny fraction of the 150 different species of bacteria needed for a healthy body and immune system.

Pathogenic E. coli is Made by Antibiotic Use in Cattle

E. coli is a common and essential resident of the human gut and the best studied bacterium. This bacterium is not normally resistant to antibiotics nor does it produce deadly toxins. Antibiotic resistance and toxin production results from treating cattle with antibiotics to increase fat production prior to butchering.

Antibiotics Select for E. coli that Stick to Rectal Surface of Cattle

Pathogenic E. coli are not found throughout cattle fecal material, but rather they are only in the outermost surface layer. This outer layer of material contains bacteria from the surface of the rectum just as the cow pies are deposited. E. coli does not normally stick to this surface, because it lacks a protein, such as a hemagglutinin capable of binding to the surface polysaccharides, heparan sulfate. Antibiotics kill off the bacteria normally residing on the surface. As a member of the intestinal biofilm community, E. coli continually exchanges DNA/genes with other bacteria in the gut and picks up three useful genes, to become a pathogen:

Antibiotic resistance
Hemagglutinin for sticking to surfaces
Toxin to release nutrients from the intestinal walls.

E. coli with these three genes can colonize the rectal tissue of cattle in feed lots.

Pathogenic E. coli Can be Easily Avoided

We have to work hard as a society to have problems with E. coli. Pathogenic E. coli results from absurd use of huge quantities of antibiotics just to disrupt the normal gut flora of cattle so that they become unhealthy and store fat in their tissues, i.e. prime beef. The same effect can also be achieved just by feeding the cattle some short chain fatty acids, or better still avoiding this step by feeding exclusively on grass. It would also be easy to treat the few cattle that have pathogenic E. coli, so that it doesn't become a problem. Proper treatment of manure and meat processing would also block transmission of pathogenic E. coli to agricultural crops or meat. Finally, an Anti-inflammatory Diet and Lifestyle would provide a healthy gut flora and immune system that would make people less susceptible to the pathogen.

Udder Nonsense

2011-06-09T17:12:00.002-06:00

Recent articles in the popular press have heralded the genetic engineering of cows with some human milk proteins. Milk produced by these transgenic cows is advertised as being similar or the same as human breast milk.

This is like claiming that the udder in the picture is an all natural, low BPA container for fortified water.

The breakthrough in humanized cow's milk, announced by Chinese researchers in PLoS One, actually documents replacement of cow lysozyme with the corresponding human enzyme. That does not make the milk human anymore than adding egg white lysozyme would turn the cows into chickens. If it moos like a cow...

Cow's milk-based formula harms infants, because the carbohydrates it contains do not support the normal development of infant gut flora. The result is gut inflammation, and not normal gut and immune system development. Even human proteins produced in cows will have characteristic cow sugars attached. It is these cow sugars on milk proteins that are associated with colic. The chains of sugars (milk oligosaccharides) free and/or associated with milk proteins are different in cows and humans, and cow carbs are a problem in formula.

I think that it is silly to support humanizing cow's milk formula, when the sensible solution is to support breast feeding and licensed human milk banks. The natural approach is much cheaper and far healthier. Only human milk and human milk-derived fortifiers should be used for infants (especially preterm) in hospitals. It is time for the healthcare industry to realize that disruption of gut flora by antibiotics or artificial formula is a health risk. The data are clear -- cow's milk (including transgenic cow’s milk) in the hospital may be profitable, but it is unhealthy, e. g. contributes to Clostridium difficile and necrotizing enterocolitis infections, and contributes to long term health problems, such as inflammatory and autoimmune diseases.

References:

China Genetically Modifying Cows To Produce Human Breast Milk

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

Contagious Health

2011-06-01T12:36:00.001-06:00

Healthy gut flora: bacteria from family, friends, Fido and food provide the foundation for the complex microbial community of the intestines, which controls the immune system. Antibiotics and hygiene are detrimental to gut flora and health.

Gut Flora Are Complex

Recent studies of the gut flora, e.g. the human gut biome, show that each individual maintains more than 150 different species of bacteria. Worldwide, that means that about a thousand different bacterial species are common residents of the human gut and together those gut bacteria use more than 1 million different genes. Many of those genes code for the enzymes used by gut bacteria to digest plant polysaccharides, i.e. soluble fiber.

Hygiene Isolates People from Healthy Sources of Gut Flora

Every time we speak, we release a mist of bacteria from our lungs, mouth and GI tract. These bacteria are on our skin, clothes and personal items, and provide a source of the bacteria that make us healthy. Parents and older siblings pass these bacteria on to younger children. These donated bacteria are essential for the development of a healthy immune system and children growing up with healthy relatives and exposed to soil bacteria via pets, farm animals, etc. are healthier than children who are more isolated.

In this sense, hygiene is unhealthy, because an individual is isolated from new sources of bacteria that could replace those lost by limited diets, antibiotics, etc. Otherwise, health is contagious, since gut bacteria from healthy individuals can spread among the population. Washing hands and food is unnatural and unhealthy.

Few Bacteria Make You Sick, but Many Are Essential for Good Health

Food intolerance can result from “good” family hygiene, limited diets and exposure to antibiotics. A common intolerance results from the absence of bacteria that produce an enzyme to digest dairy lactose, i.e. lactose intolerance. Lactose intolerance can be readily cured by eating a dairy product, such as yogurt, that contains both lactose and live bacteria (probiotics) that can digest the lactose. Simply eating moderate amounts of live yogurt daily for a couple of weeks resupplies the gut flora with bacteria that can digest lactose, and the intolerance is gone.

Soluble Fibers Are Plant Polysaccharides that Are Digestible by Bacterial Enzymes

Humans only produce enzymes to digest one polysaccharide, starch. All of the other hundreds of polysaccharides present in plants are only digestible by bacterial (and fungal) enzymes of the gut flora. If the bacteria and enzymes needed to fully digest a particular food polysaccharide are absent, then digestive problems ensue and the polysaccharide can act as a laxative. Continual eating of the problem food with a new source of diverse bacteria, e.g. lightly rinsed vegetables right from the garden, then the gut flora will incorporate new bacteria that can digest the problem polysaccharide and the gut is happy.

Soluble fiber feeds the gut bacteria that convert it into short chain fatty acids that nourish the colon. Constipation results from the absence of the bacteria needed to digest dietary fiber and to produce the large volume of bacteria that make up well hydrated stools.

Gut Bacteria Are Needed for Healthy Immunity

Cells of the human immune system are stored predominantly in the lining of the intestines. Intensive study of the interaction of the gut bacteria with the gut has revealed that both the aggressive half of the immune system that attacks pathogens and the suppressive half that protects the body itself from attack, develop in the gut in response to particular types of bacteria. Thus, the absence of one type of bacteria can cripple responses to infection, while other bacteria are needed to block autoimmune diseases and allergies. Most diseases are caused by disruption of the normal interactions between gut bacteria and the immune cells developing in the gut.

Antibiotics Lead to Autoimmunity

Antibiotics have dramatic and lasting impact on gut flora. Cattle treated with antibiotics and a high carbohydrate diet have an altered metabolism (obesity) that leads to rapid fat accumulation in their tissues. This is good for making tasty beef, but the same approach in people produces the suite of diseases in affluent societies.

Children treated with an antibiotic for a simple ear infection, are much more likely to return to pediatricians for treatments of subsequent obesity, infections and diseases. Compromised gut flora can take years to return to normal function after antibiotic treatment. Loss of the appendix, which is the normal source of bacteria to replenish gut flora after diarrhea, results in an increased risk of abnormal gut flora and numerous autoimmune diseases. It is likely that most autoimmune diseases are preceded by prior treatment with antibiotics that disrupted normal gut flora and permanently altered the immune system.

Interventions to Treat Disease: the Anti-Inflammatory Diet and Fecal Transplants

It should be obvious that a disrupted or unhealthy gut flora will compromise the immune system and contribute to disease. Treatment of diseases is complicated by the use of drugs that also impact the gut flora and produce additional side effects. An alternative approach would be to support the healthy gut flora and normal development of the gut immune system. As always, the answer is a supportive diet and a source of gut bacteria. The diet is obviously the Anti-Inflammatory Diet that provides support for almost anything that ails you. Probiotics are not retained in the gut, but they can contribute a few of the genes needed for a healthy gut flora. The source of bacteria for a healthy gut flora may range from minimally washed garden vegetables, to the more aggressive total replacement of gut flora with a fecal transplant from a healthy donor.

Honey, Hydrophobicity and Biofilms

2010-12-31T13:21:00.000-07:00

A reader (Jay Bryant) recently pointed out a PNAS article on the structure of a bacterial enzyme that uses sucrose to make the glucan matrix of dental biofilms. This article released a cascade of associations in my mind and illustrated why honey does not contribute to dental plaques, but is antimicrobial and aids wound healing. People forget that sugars combine both hydrophilic and hydrophobic properties, and thereby act as soaps.

The starting point of the chemical versatility of carbohydrates is the inability of the central portion of a sugar ring structure to form hydrogen bonds. Each sugar is made of a linear chain of carbon atoms with each carbon linked also to a hydrogen and a hydroxyl. Only the hydroxyl can participate in hydrogen bonds, so each carbon has a hydrophilic side (bonds with water to make hydrogen bonds) and a hydrophobic side (that makes van der Waals bonds with other hydrophobic molecules.) The sugars circularize and the rings have faces that are predominantly hydrophobic and perimeters with hydroxyls that are hydrophilic. Polysaccharides (long chains of sugars), such as cellulose, can sometimes form long fibers that form a hydrophobic context for hydrogen bonds between the hydroxyls of adjacent polymers. These cellulose fibers are very resistant to chemical or biological attack and accumulate as the most abundant biological molecules on Earth.

The PNAS article provides another example of how protein enzymes interact with carbohydrates, in this case sucrose and a polymer of glucose. Typical weak bonds between the amino acid residues of proteins and other molecules are hydrogen, ionic or van der Waals bonds with energies of a couple of kcals/mol. In contrast, the bonding of the hydrophobic face of a sugar to the hydrophobic face of an hydrophobic amino acid, e.g. tryptophan, phenylalanine, histidine, lysine or arginine, releases more than ten kcals/mol of energy. Thus, the structure of the bacterial enzyme that makes biofilm glucan chains from dietary sucrose, the sucrose is bound to the enzyme on the face of a prominent tryptophan. Examination of enzymes that bind to polysaccharides will show a series of tryptophans arrayed across the surface of the enzymes with spacing appropriate to bind to the individual sugars of the polysaccharide.

Biofilms are communities of multiple species of bacteria held together by a polysaccharide matrix. In the case of dental plaque, the polysaccharide is made of glucose links, whereas many other matrix polysaccharides are negatively charged and held together by positively charged metal ions. The bacteria bind to the polysaccharides using protein receptors that exploit the display of hydrophobic binding sites of the polysaccharides. It takes energy to make polysaccharides and the dental plaque bacteria use the energy already expended in the formation of sucrose to produce a polymer of glucose, an alpha-glucan, and free fructose. Thus, sucrose is essential in forming this type of biofilm and without this sugar, the dental plaque cannot form. Milk lactose, or glucose would be a more appropriate sweetener. Unfortunately, high fructose corn syrup would be a poor substitute, because of the high liver toxicity of the fructose (it causes fatty liver, just like alcohol) and very high activity in forming advanced glycation end products (AGEs), which contribute to the symptoms of diabetics.

Honey seems to be magical, because at low concentrations the sugars present in honey (mostly glucose and fructose, and not sucrose) are nutrients for bacteria, but at high concentrations honey is anti-bacterial and useful as a wound treatment. I think that the explanation for its antimicrobial activity is that sugars are amphipathic, that is they have both hydrophilic and hydrophobic properties, just like soap, and at high concentrations they kill bacteria, just as soaps at high concentrations kill bacteria. In fact, the gentle soaplike properties of sugars are exploited experimentally to dissolve proteins that are normally embedded in cellular membranes. This explanation predicts that corn syrup, which can also be used to form very stable soap bubbles, should also be useful in wound healing.

Why Discuss Mother’s Milk on an Inflammation Blog?

2010-08-03T15:02:00.000-06:00

Milk Is Perfectly Adapted for Infant Nutrition/Development

I think that I should explain, if it is not obvious, why I keep bringing up breastfeeding/infant nutrition on this blog about diet, inflammation and disease. The starting point is that infants need nutrition, protection from disease and continued normal development or they won’t be able to reproduce. That means that milk is the focal point of a lot of natural selection and absence of the natural functions of milk, e.g. use of formula, would be expected to lead to inflammation and disease, which it does. Analysis of milk and how formula makes infants susceptible to disease and alters normal development, provides an extreme example of the interactions of diet, gut flora and the immune system.

Since this is World Breastfeeding Week and my wife is an Internationally Board Certified Lactation Consultant, I think that writing a few articles on milk is good for my health.

Milk Provides Complete Nutrition, Controls Gut Flora, Promotes Gut Development -- Formula Doesn’t

I find it amazing that supplements are encouraged for exclusively breastfeeding infants. It doesn’t make sense to suspect that breast milk is inadequate and why is formula fortified with ingredients at ten to a hundred times the levels in breast milk? The answer is in the infant gut flora and perhaps in poor maternal nutrition/gut flora.

Mother’s Milk Promotes Normal Infant Gut Flora -- Bifidobacteria

The normal infant gut flora is very simple, Bifidobacteria. Adults have gut flora composed of hundreds of different species of bacteria and infants have just Bifidobacteria. The name of the infant bacterium should be familiar if you read labels on yogurt. Bifidobacteria are common probiotics. That also explains why the diapers of exclusively breast fed babies smell like yogurt.

Formula Promotes Adult Gut Flora

One bottle of formula kills the Bifidobacteria and replaces it with adult gut bacteria. The diapers are forever changed, because the adult gut flora is very persistent. The presence of the adult gut flora also explains why formula has the high levels of supplements. The adult gut flora consumes the supplements and leaves only tiny amounts for the infant. All of the breast milk nutrients go to the baby if only Bifidobacteria is present, but after the adult gut flora are established much higher levels are needed and the infant may still be deficient. Adult, formula-supported gut flora also digest antibodies and other protective factors in mother’s milk.

Supplement Mothers to Improve Infant Nutrition

Modern mothers are also eating modern diets that produce deficiencies. Chronic diet-based inflammation leads to a compromised ability to produce vitamin D in sunlight. Nursing mothers with poor diets may not be transferring enough vitamin D to their nursing babies. Since formula and the adult gut flora that it stimulates, causes gut inflammation, I suspect that formula fed infants are also compromised in their ability to produce their own vitamin D in sunlight. It makes more sense to supplement mothers than babies.

Formula Is not Breast Milk, It’s Not Even Close

Formula is cheap to produce, but expensive to buy. Formula is promoted as the next best substitute for milk from a baby’s own mother, but that isn’t true either. The next best alternative is mother’s milk from a certified human milk bank. It is available at a reasonable cost. Hospitals should know better and provide the only appropriate alternative. Early formula use dramatically increases healthcare costs.

Formula Lacks the Oligosaccharides Needed to Support only Bifidobacter

Fructose oligosaccharides (FOS) and other short chains of sugars are being promoted to support the growth of beneficial gut flora. These oligosaccharides do promote the growth of adult gut flora, but not just infant Bifidobacteria. Formula plus FOS and/or other prebiotic oligosaccharides other than those present in human milk support the growth of bacteria that rob nutrients from and degrade the protective components of breast milk. If the diaper smells like adult gut flora, then the baby was not fed mother’s milk.

Development of Gut and Brain

The newborn gut and brain are only partially developed. Mother’s milk is needed to supply growth factors to close and differentiate the gut epithelium, and long chain omega-3 fatty acids for brain growth. Formula may eventually be supplemented with the needed fatty acids, but the growth factors/hormones present in mother’s milk will not be provided in formula. Recent studies have shown that hundreds of different genes are activated in gut cells from infants fed either breast milk or formula. Formula leaves the gut leaky and fails to stimulate the development of the immune system that is dependent on interaction with normal infant gut flora. These dysfunctions partially explain the increased (10X to 100X) gut and respiratory infections resulting from formula use. The reduced brain development with formula explains the five point reduction in IQ of formula fed infants.

It’s worldwide breastfeeding week. Support healthy, non-inflammatory gut flora (infant Bifidobacteria) around the globe!

Autoimmune Diseases, Bacteria and GALT (Gut Associated Immune System)

2010-07-30T14:15:00.002-06:00

Celiac, Oxidative Stress, Peroxiredoxin, Alopecia

Grain/gluten intolerance, celiac is an immunological attack on the small intestines with increased risk for numerous autoimmune diseases. Hashimoto’s thyroiditis is a common sequela of celiac and the two diseases share the same autoantigen, tissue transglutaminase (tTG). Thus, the development of celiac and the production of antibodies to the tTG produced in the intestines, results in a subsequent immunological attack on other tissues that produce lots of tTG, e.g. the thyroid. Gluten intolerance, because of its attack on the intestines and the proximity of a major part of the immune system (GALT), may play a major role as the foundation for autoimmune diseases.

Gluten Intolerance First Step in Autoimmune Diseases

Celiac may also lead to herpatic lesions of the skin, dermatitis herpetiformis and loss of hair, alopecia. In these cases, the autoantigen is peroxiredoxin, an enzyme that eliminates hydrogen peroxide produced as a result of accumulation of reactive oxygen species, e.g. superoxide, associated with inflammation. Peroxiredoxin is also implicated as an autoantigen in periodontal disease, suggesting that celiac may also contribute to dental gum inflammation.

Peroxiredoxin 5 Gene Associated with Alopecia Risk

A recent study (see ref. below) of genes associated with alopecia identified genes involved in Treg and Th-17 development, as well as peroxiredoxin 5 as contributors. As expected, several genes involved in antigen presentation (HLA-DRA, HLA-DQA) were also identified. Th-17 lymphocytes are involved in immune attacks on self tissue, i.e. autoimmune diseases, such as alopecia, in which hair follicles are attacked by the immune system. Tregs control immune attacks on self tissues. Peroxiredoxin is an autoantigen and is produced in elevated amounts around hair follicles attacked in alopecia.

Basic Amino Acids of Peroxiredoxin as Expected for Autoantigen

I checked the amino acid sequence of human peroxiredoxin 5 and found an alternative (-nrrlkrfsmv-) to the triplet of basic amino acids that I expect for an autoantigen. In this case there are two adjacent pairs of basic amino acids (blue rr and kr) that I think precipitate immunological presentation of peroxiredoxin. Peroxiredoxins are produced in response to oxidative stress at sites of inflammation and the presence of celiac compromises the gut associated immune system (GALT) that provides Tregs to restrict autoimmunity, so celiac sets the stage for peroxiredoxin presentation to the immune system and for subsequent production of anti-peroxiredoxin antibodies, autoimmunity and destruction of hair follicles, alopecia.

Anti-Inflammatory Diet Should Avoid and Treat Autoimmunity

Control of autoimmune diseases mediated by peroxiredoxin should benefit from a reduction in the conditions that spawned the diseases:

Th-17 elevation -- celiac inflammation stimulated by grain/gluten
Treg loss -- GALT inactivation due to inflammatory diet and inappropriate gut flora
Autoantigen (basic amino acid concentration) presentation -- oxidative stress stimulation of peroxiredoxin

Treatment would be supported by dietary changes:

anti-inflammatory diet to control gut inflammation and minimize celiac symptoms (vitamin D, low carb/high saturated fat, high omega-3 to -6 fatty acid ratio, no grains)
probiotics and soluble fiber (e.g. pectin, inulin) to re-establish gut flora (cure dysbiotic constipation) and GALT function, and development of Tregs
supplements to compensate for depletion of vitamin C and glutathione by oxidative stress, e.g. vitamin C and acetylcysteine (NAC)

Th-17 and Tregs in HIV Infections

Th-17 cells are also reduced by HIV infection, producing susceptibility to infection, but this infection should also reduce autoimmune disease. The reduction in Th-17 also may be a consequence of problems in the GALT. Therapy for HIV infection should also include diet considerations to increase Th-17 and also Tregs to reduce autoimmune diseases due to unbalanced Th-17.

ref.

Petukhova L, Duvic M, Hordinsky M, Norris D, Price V, Shimomura Y, Kim H, Singh P, Lee A, Chen WV, Meyer KC, Paus R, Jahoda CA, Amos CI, Gregersen PK, Christiano AM. 2010. Genome-wide association study in alopecia areata implicates both innate and adaptive immunity. Nature. 466(7302):113-7.

Infant Milk Allergy, Colic and Sialic Acid

2010-06-17T00:44:00.001-06:00

Speculation on the cause of infant reactions to cow’s milk in formula or transmitted into mother’s milk. Are mother’s priming their newborns in utero with antibodies to react to non-human sugars (Neu5Gc)?

Sialic Acids Mark the Surface of Human Cells

Human cells are covered with a forest of long and short carbohydrates, polysaccharides and oligosaccharides resp., which control the interaction of the cells with the outside world. The sugars exposed on the ends of these sugar chains are sialic acids. It is not surprising that pathogenic viruses and bacteria target sialic acids as the first step in attacking human cells and that policing immune cells avoid attacking their own cells by recognizing the sialic acids. The surprise is that essentially all other mammals have the same two sialic acids, Neu5Ac and Neu5Gc, but humans have only Neu5Ac. Meat and cow’s milk have both. Babies and mother’s milk should have only Neu5Ac.

Evolution to Lose Neu5Gc to Avoid Pathogens

Surviving defective remnants of genes needed to make Neu5Gc suggest that loss of Neu5Gc was an adaptation to avoid general mammalian pathogens and to facilitate brain development. One of the limitations of using other mammals as models of human diseases is the differences in sialic acids that are commonly used for initial docking of pathogens on human cells. Other mammals have different forms of malaria than humans and we are well aware that influenza adapted to birds and pigs does not infect humans without adjusting to the lack of Neu5Gc.

Antibodies Against Neu5Gc

Humans make antibodies to Neu5Gc when injected with blood products from other mammals. A sudden change from a long term vegan diet to a meat diet can also lead to the production of anti-Neu5Gc antibodies. These types of antibodies may contribute to some types of non-lactose milk intolerance/allergies.

Neu5Gc from Cow’s Milk Gangliosides to Mother’s Milk

A significant problem in infant health is the reaction of the infant with abdominal distress after eating cow’s milk-based formula or in some cases from breastfeeding after the mother has eaten milk or other dairy products. Milk oligosaccharides, proteins and lipids have Neu5Gc. It is unlikely that cow’s milk proteins or oligosaccharides can move from the mother’s intestines to her breast milk, but it is possible that Neu5Gc attached to fatty acids in the form of gangliosides may be transferred to breast milk.

Mother’s Anti-Neu5Gc in Infants Gut Reacts with Cow Neu5Gc

If cow’s milk gangliosides are the source of Neu5Gc in breastmilk, then how do the infants develop antibodies to these relatively rare antigens? Babies receive all of their antibodies from their mother until their immune systems start to develop at about six months of age. The answer is hinted at by the observation of a mother whose exclusively breastfed infant developed sensitivity to breast milk after the mother ate dairy products. The mother reported that she shifted from a long term vegan diet to a meat diet to improve her nutrition during her pregnancy. It is also likely that she produced IgE antibodies to Neu5Gc, which were then transferred to her baby across the placenta during gestation.

Anti-Neu5Gc Antibodies May Explain Infant Milk Intolerance and Colic

Infants with anti-Neu5Gc antibodies obtained from their mother during gestation in utero, will have mast cells in the lining of their gut that are primed to react to Neu5Gc in cow’s milk present as components in formula or in trace amounts transferred into breast milk. Infants may respond to these immunological reactions with a variety of symptoms, including those observed as rejection of formula or breast milk after the mother has eaten dairy products or as colic.

reference:

Varki A. 2010 Colloquium paper: uniquely human evolution of sialic acid genetics and biology. Proc Natl Acad Sci U S A. 107 Suppl 2:8939-46.

Necrotising Enterocolitis, Low Birth Weight and Formula

2010-05-28T16:10:00.001-06:00

Human Milk and Milk Supplements Protect Newborns

Babies born prematurely are at risk of a serious bacterial infection of the intestines, necrotizing enterocolitis (NEC), that can be prevented if formula based on cow’s milk products is avoided and human milk is used for all feedings.

Human Milk Protects Against Formula Based NEC

Feeding low birth weight, premature babies formula made from cow’s milk increases their risk of NEC ten fold. Replacement of some of the cow’s milk formula with human milk from a milk bank reduces NEC.

Human Milk and Cow’s Milk Are Very Different

What is present in human milk that protects against NEC? The major components in milk are proteins, lactose, fats and oligosaccharides (short to medium length chains of sugars.) Human milk and cow’s milk have the same amount of fat (35 grams/liter) and about the same amount of lactose (65 vs. 45 g/l) and protein (10 vs. 35 g/l). The big difference is in the amount (5-10 vs. 0.05 g/l) and quality of oligosaccharides. Human milk has more than 100 times the amount of oligosaccharides as cow’s milk. That also means that about 10% of the carbohydrates in human milk are non-nutritive human milk oligosaccharides (HMOs).

HMOs Are Not FOS

Human milk oligosaccharides are complex, with over 200 different structures identified so far. Essentially they are made up of a lactose (a disaccharide consisting of galactose linked to glucose) extended by different numbers of N-acetyllactosamine (lactose with a modified glucose) and a few other sugars attached a various locations. A different enzyme is used for each modification and the synthesis of these oligosaccharides has not yet been figured out in detail. More than a dozen different genes are devoted to the synthesis of these oligosaccharides. These oligosaccharides are not structurally or functionally related to the frucose oligosaccharides used as prebiotics.

HMOs Are Prebiotic and Stop NEC

Human milk oligosaccharides have been tested both for their ability to act as prebiotics to encourage the development of normal baby gut flora and to suppress NEC. The HMOs were found to be the elusive bifidus factor that stops the development of adult gut flora and facilitates only the development of the Biﬁdobacterium biﬁdum monoculture found in exclusively breastfed babies. HMOs also reduce NEC in the same way as whole human milk. Another interesting aspect of HMOs is that they modify the oligosaccharides produced on the surface of baby intestinal cells. Babies fed human milk also secrete HMOs in their urine, indicating that ingested HMOs are absorbed in the intestines and reach the blood stream.

Neonatal Nurseries Should Use Only Human Milk

Human milk is now available to neonatal intensive care nurseries through milk banks and purified components of human milk are also available to supplement feedings for very low birth weight premature babies. Nursing is still best for baby and mother, but those mothers who choose not to nurse need not compromise the health and development of their babies by using cow’s milk-based formula or supplements. Every dollar spent on pasteurized donor milk ($3/oz.) reduces costs in neonatal intensive care units by more than ten dollars. It seems to be time to eliminate the added risks of formula use in hospitals and provide every baby with a healthy start and normal gut flora by only using human milk products in hospitals.

ref.

Lars Bode. 2009. Human milk oligosaccharides: prebiotics and beyond

Nutrition Reviews® Vol. 67(Suppl. 2):S183–S191

Healthcare: DNA Testing vs. Diet and Exercise

2010-05-15T17:07:00.000-06:00

Contributions of genetic alleles to disease are useful for understanding, but not in predicting disease. Diet and lifestyle are the major determinants of disease and not genes for most common diseases.

OTC Genetic Screening Kits

A recent headline touted the availability of a kit at Walgreens to screen for “predisposition” to a hundred common diseases. A few months earlier, scientists admitted that after lengthy examination of a dozen major diseases, the genetic contribution was negligible. It may now be possible to cheaply (less than $25,000) determine the sequence of the entire genome of an individual or even more cheaply test for the presence of particular genetic alleles, but that information is useless compared to diet for predicting if the person will actually get the disease. The screening kits were pulled before they reached the shelves.

Gut Flora Dominates Gut Genotype

I think that the reason why an individual’s genes don’t dominate health issues, is because the composition of meals dominates the development of the gut flora community and it is the interaction between the gut and its bacteria that dominates health. The genes of the individual are just not that important in determining disease.

You Are What You Ate

For each individual, the meals eaten over the last years have cultivated the existing gut flora, composed of hundreds of different species of bacteria with unique metabolic capabilities to digest unusual meal molecules and modulate the immune system. Molecular communication between gut and the bacteria in intimate contact determine food intolerance, allergies, autoimmunity and many other disease processes. Healthy eating produces a healthy gut flora and bad meal decisions can lead to unhealthy gut flora and the modern litany of inflammatory ailments. Some genes may mitigate or magnify the development of unhealthy gut flora, but it is difficult to be healthy with compromised gut flora.

Antibiotic Disruption of Gut Flora Trumps Good Genes

It doesn’t matter if there are great genes to help avoid disease, if the function of those genes is compromised by gut dysbiosis, a lack of functional gut flora. Many antibiotic treatments, e.g. for acne, act by attacking the gut flora that support a specific portion of the immune system. Deletion of this function causes cosmetic improvement, e.g. relief of skin inflammation, but at the expense of producing a dysfunctional immune system that may lead to other diseases. Presence or absence of healthy genes can be made irrelevant, if the gut flora is dysfunctional.

Aging Gut Flora

2010-04-30T16:44:00.001-06:00

Diet selects for the bacteria that grow in the GI track and control the development of the immune system. Diet-based inflammation produces aging symptoms.

Returning to the Subject of Aging

I want to return to the subject of aging. A year and a half ago I wrote, “You don’t wear out, you flame out.” I still think that is true, but I need to update that idea of inflammation and aging to include diet, gut flora and immune system development. So here is my old article with a new focus on the gut.

Wearing Out Only Happens with Inflammation

I don’t think that aging happens -- most symptoms associated with aging are just medically mismanaged chronic inflammation. The major observations are that older people have more degenerative/autoimmune diseases and they suffer from fewer infectious diseases. The typical explanation is that the bodies of older people have figured out infections with an experienced immune system and that mechanical damage takes its toll over time -- joints wear out. I think that there may be a minor amount of truth in this cultural perspective, but there is something more profound at work, sarcopenia combined with a compromised gut flora.

Replacing Muscle with Visceral Fat Is Inflammatory

Sarcopenia (muscle loss) is the gradual loss of muscle and replacement by fat. Thus, by age fifty most people are physically less active and even if they appear to have the same weight and shape as in their active youth, the muscle of their abdomens and limbs has been partially replaced with fat. This fat, as in those who are obese, releases inflammatory cytokines into the circulation and the body reacts as if it has a low grade infection.

Chronic Inflammation Taxes Immune System

Senior citizens are constantly expending energy and taxing their immune system by chronic inflammation. As a result they get fewer infections, but the chronic inflammation provides the foundation for cancer and autoimmune diseases. Their bodies aren’t mechanically wearing out, but they are wearing out by over use of the immune system.

Aging Symptoms Are Inflammation Symptoms

Those seniors who are physically active and eat an anti-inflammatory diet, do not appear to age as fast as those who are sedentary, obese and display the typical symptoms of chronic inflammation, the metabolic syndrome. Most of the characteristics associated with advancing years are merely symptoms of poorly managed chronic inflammation that can be reversed by an anti-inflammatory diet and exercise.

Diet Determines Gut Flora

Diet also contributes to aging, because diet controls development of gut flora and gut flora control development of the immune system. The gut flora of an individual reflect the bacteria that have entered the GI tract, nutrients available to the bacteria in previous meals, bacterial growth regulators released by the gut, exposure to antibiotics, exposure to phytochemicals and gut transit time.

Gut Flora Is Diverse and Adaptable

Gut flora appears to be amazingly diverse from individual to individual with thousands of bacterial species inhabiting humans worldwide and about 150 species in each individual. The same species remain in an individual for long periods of time regardless of diet. The dominance of particular species depends on recent diet. Major changes can result from antibiotics or gut diseases, e.g. Crohn’s.

Constipation Means Dysfunctional Gut Flora

Bowel stools are made up predominantly of bacteria and not undigested plant parts, i.e. fiber. Fiber is made up of plant polysaccharides that are not digested by salivary, stomach or pancreatic enzymes, e.g. proteases and amylases that degrade proteins and starch. Fiber polysaccharides pass into the colon where they are digested by gut flora. People with constipation usually have disrupted gut flora, e.g. wiped out by antibiotics, and so the minimal volume of remaining undigested fiber is all that passes out in compact, dehydrated lumps. If gut flora have been exposed to a particular type of fiber and bacteria having the needed enzymes have been brought into the gut previously, then the fiber is digested to sugars that feed the gut bacteria. The increased population of bacteria is what makes up normal, hydrated bowel stools.

Gut Flora Changes Slowly to New Foods (Polysaccharides)

Bacteria grow quickly and with ample nutrients gut bacteria can double in number in about an hour. Bacterial species are usually defined by the ability to utilize various carbohydrates or polysaccharides as nutrients. Depending on the food eaten, nutrients favor the growth of particular bacterial species and the gut flora population changes dynamically. New species are incorporated into the gut flora only if they find their way into the gut on food, e.g. riding on fresh, uncooked vegetables, and food provides nutrients that can permit the new bacteria to grow. It will take several meals for new bacteria to reach appreciable numbers. In the mean time the new fiber may be partially degraded and produce chemicals that disrupt other gut flora and cause bloating symptoms of food intolerance. This is not an allergic reaction of the immune system. It just takes time and persistence to permit the gut flora to adapt. Most people systematically make themselves intolerant to particular foods by over-reacting to initial maladaption of their gut flora to the new food. If they persisted with progressive exposure to diverse foods, their gut flora would adapt.

Simplified Aging Gut Flora Contributes to Inflammation

People of increasing age who maintain a diverse, anti-inflammatory diet and maintain muscle mass by weight-bearing exercise, avoid age-related inflammation and disease, i.e. they age more slowly. Conversely, those who simplify their diets by eating processed foods high in starch and vegetable oils, show symptoms normally associated with advanced age, even when young. The aging diet is inflammatory and it also produces a gut flora which is different from the youthful.

Aging Gut Flora Contributes to Disease

Constipation is an extreme example of dysfunctional gut flora and since gut bacteria are needed for the normal development of the immune system that is located in the lining of the small intestines, constipation is also an indicator of a compromised immune system. Aging is frequently accompanied by digestive problems with one extreme being constipation. It should not be surprising that individuals with compromised immune systems also develop numerous degenerative diseases indicative of a lack in the immunological tolerance systems that develop in the gut in response to normal gut flora. Constipation and digestive problems are not normal signs of aging.

Eliminate Symptoms of Aging by Cultivating Gut Flora

A healthy diet, healthy gut flora, and a competent immune system are all tightly connected. The typical symptoms of aging merely reflect an unhealthy diet and lifestyle that leads to chronic inflammation, a compromised immune system and disease. The process of aging can be slowed by attention to the next meal. Most people who fail to be healthy and active well into their 80’s are simply victims of bad choices (or of bad medical advice.)

Antibiotics, Gut Flora, Food Intolerance and Disease

2010-04-22T13:32:00.002-06:00

Cattle Are Finished by Selective Killing of Gut Flora. The Sickened Animals Store Fat that Grills Great. People Get Metabolic Syndrome.

The likening of modern humans to potatoes sacked out on a couch is misleading. The obesity epidemic linked to diets of processed foods more closely resembles the stumbling progression of cattle to abattoir. Antibiotics and diet systematically lead in both feedlot and food court to gut dysbiosis, immune system failure, hormone disruption, rampant fat accumulation, physical inactivity, depression and the modern suite of chronic diseases. Healthcare costs escalate, but vet bills, in contrast, are forestalled by a captive bolt pistol.

Background Observations

Antibiotics kill bacteria and not humans, because the bacteria have different machinery for making proteins, nucleic acids and cell walls.
Antibiotics kill bacterial pathogens and not viruses or fungi.
Antibiotics kill helpful bacteria in the gut (gut flora) even more readily than pathogens.
Antibiotics are used in meat production to alter gut flora to change animal metabolism; e.g. cattle treated with antibiotics gain fat. Protection from disease is secondary.
Simple diet means simple gut flora. Processed foods are simplified foods that simplify gut flora.
Probiotics can replace only a small fraction of the gut flora diversity.
Gut bacteria control the immune system development in the lining of the gut.
Chronic antibiotic use permanently simplifies gut flora and compromises the immune system.
The appendix stores gut bacteria as a reserve to replenish gut flora following diarrhea.
Diseases based on inflammation and immune system intolerance result from gut dysbiosis (inadequate gut bacteria).

Antibiotics Kill Good Bacteria

This is a rant about antibiotics, not about humane actions. Humane actions are not the point here, since I am talking about health care and not treatment of agricultural animals. I am pleading for the rights of gut flora everywhere and antibiotics are the casual killers. Compromised gut flora is collateral damage in attempting to eliminate bacteria characterized as pathogens. Every time the pediatrician treats the mother by acceding to her pleas for an antibiotic prescription to silence a howling ear ache and get a good night’s sleep, or the dermatologist treats teen acne with antibiotics, billions and billions of domesticated bacteria die.

Constipation Is a Sign

Countless hours are wasted waiting, because antibiotic-depleted gut flora cannot hydrate and form normal stools. Probiotics are gulped down, but they supply only a handful of the hundreds of bacterial species that are needed for health. Yeasts and other fungi that are naturally resistant to antibiotics quickly replace the lost beneficial bacteria in the gut, vagina and on other body surfaces. Surcease for simple sorrows leads to lingering and lasting laminations. Don’t mess with mother nurture.

Damage of Antibiotic Use Is Slow

Most of the impact of antibiotic annihilation of bacteria normally present in humans is unobserved, because the deleterious effects lag months behind the initial treatment. After all, cattle treated with antibiotics to restructure their gut flora to induce bovine obesity, appear to thrive as they rapidly gain weight and avoid symptoms of infectious diseases. Humans on antibiotics also display fewer dental and incidental infections. Constipation is not a high price to pay for a better mirror image.

Antibiotics Compromise the Immune System

Unfortunately, allergies, autoimmune diseases, degenerative diseases and cancers are not usually linked to prior use of antibiotics. There is no evidence that gut flora recovers after antibiotic treatment, but constipation as a consequence of chronic antibiotic use is a common indicator of gut dysbiosis, collapse of normal gut flora bacterial communities. The harbingers of inflammatory and degenerative diseases are present, but are usually discounted, because they are a common consequence of the Western diet.

Food Intolerance Reveals Inadequacies in Gut Flora

Food intolerance is a sign of depleted gut flora diversity. Gut flora have hundreds of genes that can break down a huge diversity of polysaccharides derived from plant cell walls. Gut flora of Japanese who routinely consume kelp have specialized enzymes to hydrolyze unusual algal sulfated polysaccharides. Essentially all of the polysaccharides in plant fiber can be consumed by bacteria in the anaerobic environment of the colon. Inability of individuals to digest particular food components usually results from a deficiency of the gut flora and an indication of a history of dietary simplification and antibiotic use. Lactose intolerance, for example, results from depletion of lactose-degrading bacteria from the gut flora and can be remedied by simply eating lactose with probiotics for a couple of weeks. Gut flora can adapt, but they need persistent exposure to diverse, i.e. non-processed, food.

Antibiotic Allergies Are Natural

Allergies develop from a combination of inflammation and compromised immunological tolerance. Inflammation heightens processing of antigens for presentation to the immune systems, whereas loss of immunological tolerance means that aggressive immune responses are inadequately controlled. Thus, innocuous environmental molecules are incorrectly recognized as pathogen components. Allergies to antibiotics, such as penicillin, make sense, because the antibiotic is used to treat inflammatory infections and the antibiotic treatment eliminates the gut bacteria that are needed to develop gut lymphocytes (Tregs) to produce tolerance. Antibiotics lay the foundation for immune system dysfunction that is central to many chronic diseases.

Healthy gut flora and a healthy immune system require:

avoidance of antibiotics
systematic (not simply eating yogurt) rebuilding of gut flora following diarrhea or antibiotic use; lack of an appendix means gut flora reservoir is gone
eating a variety of vegetables; avoiding processed food
using herbs and spices
don’t overdo hygiene; gut flora diversity derives from bacteria that you eat and those that rub off acquaintances
eat seasonally to increase diversity

Lateral Gene Transfer in Gut Flora

2010-04-13T13:04:00.000-06:00

Japanese Gain Ability to Digest Algal Polysaccharides from Marine Bacteria
Gut flora adapt to the food nutrients that are prevalent in different parts of the world. Bacteria able to digest unusual nutrients, such as the sulfated porphyrans found in seaweed eaten in Japanese cuisine, are also consumed along with algae. Formation of bacterial biofilms triggers the exchange of genes among gut bacteria and the acquisition of new polysaccharide-degrading enzyme activities.

Gut Flora Adapts to Diet
The million or so genes of the thousands of bacterial species found in the guts of humans around the world are adapted to the diet of each of those individuals. Each individual gut harbors a couple of hundred different bacterial species and those different types of bacteria increase or decrease in number in response to the composition of each meal.

Diversity of Plant Polysaccharides Provides Digestion Challenge
Plants provide the greatest challenge for digestion, because plants differ the most in their carbohydrate (sugars, oligosaccharides, polysaccharides) composition. Some of those carbs, such as sucrose, starch or the components of the plant cell walls, pectins, arabinogalactans and xyloglucans, are present in all vegetables. Whereas other polysaccharides, such as the sulfated porphyrans from red algae of the same name (Porphyra) are restricted to particular plants. Each different linkage and sugar requires a different digestive enzyme.

Gut Bacteria of Algae Eaters Have Algae-Degrading Enzymes
A recent report (see ref.) traced genes from marine bacteria that digest marine algae/seaweed, to gut bacteria of people who routinely eat seaweed. Researchers studying marine bacteria identified genes coding for new enzymes, porphyranases, that hydrolyzed porphyrans. When they checked gene databases for other porphyranase genes, they found that some gut bacteria had previously unassigned genes that were apparently, based on their nucleotide sequences, porphyranases. Curiously, these genes were only present in gut bacteria isolated from Japanese sources, i.e. from people who traditionally ate seaweed. In some of these bacteria there were more than 260 genes for degrading a huge variety of different plant polysaccharides.

Marine Bacteria on Seaweed Release DNA Incorporated into Gut Bacteria
Bacteria recognize that other bacteria are around by a process called quorum sensing. This signaling system triggers the production of matrix polysaccharides produced by the bacteria to hold the bacteria together in complex communities. Quorum sensing also mobilizes the release of copies of the bacterium’s genes, which is coordinated with uptake of DNA from the surrounding environment. [Note that the proteins that take in foreign DNA have basic amino acids arranged in the same heparin-binding domains that are also used by growth factors and their receptors or the numerous proteins that bind to nucleic acids in the nucleus or in ribosomes.] Thus, biofilm formation is accompanied by enhanced lateral gene exchange that would also enhance the incorporation of porphyranase genes from ingested marine bacteria.

Gut Bacteria Are Made in Guts and Shaped by Diet
Species of gut bacteria are defined in the micro lab by their ability to grow in Petri dishes of agar containing particular combinations of sugar, polysaccharides, etc. The sugars that different bacteria are able to metabolize for growth reflect sugars available as niches in different parts of the gut. Thus species are defined in part by the sugars and polysaccharides they can metabolize, i.e. by the enzymes they can produce.

In each human gut, however, bacteria of the species filling a particular niche will have many other additional genes than those that define the species. These atypical genes are present as a consequence of serendipitous encounters with genes from other bacteria (lateral gene transfer) and may reflect peculiarities of individual diets. Different regional cuisines also shape the regional gut flora. Persistent diet components would be expected to provide selective advantage for bacteria with genes capable of metabolizing new nutrients. Access to a rich diversity of bacterial genes to augment typical gut flora genomes will facilitate adaptation. Food processing to refine and simplify nutrient diversity, and hygiene to eliminate bacterial diversity in food, will reduce diversity in gut flora and minimize adaptation to novel foods. Antibiotics, especially persistent use, can permanently disrupt gut flora. Decreased diversity in gut flora may eliminate species of gut bacteria that are essential for normal physiological functioning of the gut and associated immune system, and may be major contributors to degenerative and autoimmune disease.

Sources of Personal Gut Flora:

Exposure to Mother, Breast Milk
Pathogens from Others
Pets, Farm Animals
Environmental Sources
Ingested with Food
Appendix Reservoir of Gut Flora

Selection Pressures on Gut Flora:

Breast Milk Normalizes Flora Development
Formula Disrupts Flora
Food Nutrients
Food Phytochemicals (herbs and spices)
Antibiotics
Gut Secretions
Secretory Antibodies
Bacteriophages, Bacteriocins
Lateral Gene Transfer

reference:
Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature. 2010 Apr 8;464(7290):908-12.

Helminths, Oligosaccharides and Immunotolerance

2010-04-01T16:22:00.000-06:00

Parasitic worms reverse allergies and autoimmune diseases using oligosaccharides to mimic self and silence immune over-responsiveness.

Helminth therapy, i.e. infection with parasitic intestinal worms to provide remission from allergies, inflammatory bowel and other autoimmune diseases, has been examined as a potential therapeutic model to rehabilitate immunological dysfunction. The surface oligosaccharides of these worms have been found to mimic human oligosaccharides and alter immune responses by binding to carbohydrate-binding, i.e. lectin, receptors.

Immune Tolerance
The essence of allergic and autoimmune diseases is a defect in distinguishing between pathogen, innocuous and self molecules. Heightened immune reactions as a result of inflammation move the immune system toward production of antibody and T cell receptors specific for antigens. Those antigens respond to unique receptors on the surface of each B and T lymphocyte. The lymphocyte population has been previously depleted of cells that can produce receptors that will bind to most self antigens. This depletion makes the lymphocyte population generally non-responsive, or tolerant to self antigens. Thus, the immune system is blind to the body.

Regulatory T Cells and Tolerance
Most of the immune cells of the body are present in the lining of the gut. It is in the gut that various immune cells continue to develop for their various roles, including controlling immune reactions to self antigens and to common food molecules. Immune cells in the gut are exposed to some food molecules and bacteria that leak through the cells of the intestinal villi. Responding to these common antigens by inflammation can lead to inflammatory bowel disease. This pathological over-responsiveness is normally avoided by development of regulatory T cells, Tregs, that suppress immune responses to common food molecules and to surface antigens of common bacteria.

Treg Development Depends on Gut Flora
Gut bacteria are needed for the normal function of the immune system. Oddly, Helicobacter pylori, Hp, the cause of stomach ulcers and cancer, also stimulates the development of Tregs. Thus, the pathology of Hp may result not from its presence, but rather from how it is growing. Since Hp uses hydrogen gas produced by Klebsiella in the lower bowel and hydrogen production is dependent on dietary starch, then it follows that the pathological behavior of Hp may be dependent on dietary starch. A low starch diet may actually result in Treg stimulation from Hp and a reduction in allergies and autoimmune diseases.

Tregs Enhanced by Heliminths
Immunological tolerance is also stimulated by parasitic worms, Helminths. Helminth infestations, therefore, reduce allergies and autoimmune diseases and may contribute to the hygiene hypothesis to explain the prevalence of allergies, autoimmune and other inflammation-based degenerative diseases in modern societies. Examination of worms to find the molecules responsible for inducing immunological tolerance has identified complex surface and secreted oligosaccharides (small sugar chains) as the active molecules. Helminth oligosaccharides mimic human cell surface oligosaccharides and bind to carbohydrate-binding, lectin, receptors on immune cells to stimulate Treg development.

Lectin Receptors Control Tolerance
There are many implications of the modulation of the immune system via oligosaccharides. Note that related oligosaccharides are components of human milk and prepare the gut and develop the immune system. This explains why formula, which lacks these unique oligosaccharides, results in aberrant gut flora, contributes to neonatal necrotizing colitis and supports the development of allergies and autoimmune diseases. In contrast, judicious use of self or Helminth oligosaccharides may provide a means of restoring the function of damaged immune systems and therapy for allergies and autoimmune diseases. Also note that the critical use of lectins, which have oligosaccharide-binding sites rich in aromatic amino acids to bind the hydrophobic faces of the sugars, will also bind and provide entry into immune cells for allergens and autoantigens that have triplets of basic amino acids. The binding sites of lectins should also bind many aromatic phytochemicals. Immunomodulation by phytochemicals may result from interference with or mimicking the binding of oligosaccharides to lectin receptors.

reference:
van Die I, Cummings RD. Glycans modulate immune responses in helminth infections and allergy. Chem Immunol Allergy. 2006;90:91-112.

Rosacea: Alzheimer’s of the Face

2010-03-19T00:00:00.000-06:00

Is Rosacea Caused by Amyloid LL-37, as Alzheimer’s Is Caused by Anti-microbial Abeta?
A recent article in PLoS One (Thanks Daniel!) suggests that the amyloid beta (Abeta) proteins that aggregate to form fibrous plaques in the brain tissue of Alzheimer victims, function as typical defensive anti-microbial peptides (AMPs), similar to the LL-37 cathelicidin implicated in facial tissue in rosacea. The structural and functional similarities of Abeta and LL-37 suggest to me that Alzheimer’s and rosacea may also be similar in initiation and treatment. Let’s compare amyloids and AMPs.

[The figure shows a model protein (from ref.) used to examine stain binding to amyloids. The stains appear to bind to aromatic amino acids spaced evenly between adjacent proteins, but adjacent basic amino acids (blue) are spaced the same way and provide sites for heparin binding.]

Amyloids:

Amyloid proteins/peptides align into stacks and fibers
Stacked beta sheets bind amyloid stains: Congo Red, Thioflavin-T
Fibers form on anionic polymers: heparin, DNA
Short amyloid stacks are toxic to cells
Proteases produce multiple sizes of amyloid peptides

Anti-microbial Peptides:

AMPS typically contain heparin-binding domains -- basic peptides/ plus charge
Some AMPs, e.g. LL-37, form fibers on DNA, heparin (stain with amyloid stains)
Toxic to cell membranes
Kallikrein stimulated by gut flora migrates to face and clips LL-37 to a smaller peptide that binds to host DNA and stimulates the TLR receptor to produce inflammation
Stomach pepsin hydrolyzes dietary proteins into anti-microbial peptides (heparin is secreted by mast cells onto to the intestinal surface to protect from any amyloid-like effects)
Defensins, cathelicidins and other AMPs are under transcriptional control of vitamin D receptor

Abeta Is Anti-microbial Like LL-37

Amyloid beta is the well-known source of the fibrous plaques forming brain lesions in Alzheimer’s disease. The normal function of Abeta has not been firmly established. The recent article shows data to support Abeta as an anti-microbial peptide comparable to LL-37 against several pathogenic bacteria and yeast. Knock-out mice deprived of a gene corresponding to Abeta are susceptible to bacterial infections. The anti-microbial activity present in extracts from Alzheimer’s disease brains was inactivated by anti-Abeta antibodies.

Implications of Abeta as an AMP Like LL-37

The similarities between AMPs and amyloid peptides suggest some implications for both Alzheimer’s disease and rosacea. Vitamin D is a hormone that binds to a cytoplasmic receptor and the vitD/receptor complex then acts as a transcription factor that controls the expression of defensins in the intestines, LL-37 in facial skin and perhaps Abeta in brains.

Amyloids form fibers on a scaffolding of heparan sulfate (HS). There is usually an excess of HS on the surface of cells and the HS is rapidly recycled back into cells. During inflammation, mast cells release heparin, short fragments of HS, that should also inhibit amyloid fiber formation on HS. Chronic inflammation, however, reduces HS production and may set the stage for amyloid fiber formation. HS metabolism of the brain may be vitally important to the development of Alzheimer’s disease, especially since the increasing chronic inflammation of aging people should deplete brain HS.

LL-37 forms complexes with DNA from damaged host cells in rosacea skin. The LL-37/DNA complexes trigger TLRs and inflammation. LL-37 may normally bind to cell surface HS and chronic inflammation of the skin may cause the shift to pathogenic autoinflammation. Topical application or perhaps low dose IV heparin may be effective in disrupting the autoinflammation due to LL-37. Part of the toxicity of LL-37 in the skin may be due to amyloid like structures that could form with inadequate HS and overabundant LL-37 production. Vitamin D metabolism should also be very important, since LL-37 synthesis is controlled by vitamin D. This is consistent with the benefits that some rosaceans observe with high doses of vitamin D3 supplements.

references:
Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD. The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS One. 2010 Mar 3;5(3):e9505.

Abedini A, Tracz SM, Cho JH, Raleigh DP. Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation. Biochemistry. 2006 Aug 1;45(30):9228-37.

Biancalana M, Makabe K, Koide A, Koide S. Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies. J Mol Biol. 2009 Jan 30;385(4):1052-63. Epub 2008 Nov 14.

Heparin, Growth Factors and Rosacea

2010-03-12T19:57:00.003-07:00

Knock-out Mice and FGF Receptor Inhibitors Mimic Rosacea
Heparin Nanofibers Loaded with VEGF and FGF Mimic Stem Cells

In previous articles, I have emphasized the mediation of extracellular signaling by heparan sulfate proteoglycans (HSPGs, polysaccharides attached to proteins) and heparin (HS fragments, oligosaccharides) and the sensitivity of HSPG expression and HS degradation by inflammation. I return to that subject, spurred on by reading two articles that together show both the significance of heparin-mediated growth factors in general and in the specific case of symptom development in rosacea.

FGF Receptor Inhibitors Cause Symptoms Like Rosacea
Fibroblast growth factors stimulate the development of cancers, and antibodies against FGF receptors block cancer growth (see ref.) FGF receptor inhibiting antibodies are now being used to stop cancers. Unfortunately, FGFR antibodies (e.g. cetuximab, panitumumab) also cause symptoms in the skin (telangiectasia, acneiform eruption) similar to the facial inflammation of rosacea. Similarly, in knock-out mice, that lack the ability to produce FGFR, there are related symptoms. It appears that lack of some FGF signaling may produce the symptoms of visible blood vessels and pus-filled (though lacking bacteria) follicles of rosacea.

FGF Mediated by HSPG
FGF binds to the heparan sulfate of membrane bound HSPG in pairs and these FGF dimer/heparan sulfate complexes activate a pair of FGF receptors. The result is activation of protein phosphorylation activity (tyrosine kinase) and normal skin development. HSPG synthesis is modified by inflammation and heparanase activity is increased. This suggests that inflammation will decrease FGF signaling and could lead to symptoms of rosacea.

Growth Factors (VEGF, FGF) Bind to Heparin Nanofibers that Mimic Stem Cells
Stem cells produce lots of different growth factors and when stem cells are introduced into damaged cardiovascular tissue, more healing results (see ref.) To determine if the growth factors produced by the transplanted stem cells was sufficient for the improved healing, fibers made of heparin were dipped into stem cell cultures and the resulting growth factor-coated fibers were injected into damaged tissue. The heparin-binding growth factors were just as effective at enhancing healing as were the stem cells in previous experiments. This demonstrated that heparin-binding growth factors were the key to normal repair/revascularization and function.

Rosacea Results from Inflammation and Aberrant Vascularization
Rosacea is poorly understood and is probably numerous diseases that have related symptoms and complex development. As I indicated in previous articles, neurotransmitters from stimulated facial nerves, enzymes (kallikrein) and cytokines from intestinal interactions with gut flora, mast cell products (heparin, protease) and modified antimicrobial peptides (cathelicidins), as well as cryptic bacteria in facial tissues, may all be involved. Inflammation in the skin of the face and in the intestines is involved. Vitamin D, omega-3 fatty acids and anti-oxidants have a variety of responses (sometimes paradoxical) that differ from individual to individual and at different stages in the development of the disease. Facial inflammation leads to abnormal development of blood vessels (telangiectasia) and in accumulation of lymphocytes and neutrophils (papulopustular rosacea).

Facial Inflammation May Depress HSPG Production and Disrupt FGF Function
One of the key ramifications of persistent facial inflammation may be the depletion of of HSPGs that normally coat cells. HSPGs are continually produced, reabsorbed and degraded. The half-life for HSPGs, even those that surround the cells that produce cartilage in connective tissue, is six hours. HSPGs are also the source of heparin, that is produced as a counter ion bound to histamine and proteases in the secretory granules released by activated mast cells. Thus, inflammation-based depression of HSPG production, which is also accompanied by heparanase activation, will remove the HSPG coating of cells. This HSPG coating is needed for normal growth factor function. Lack of an HSPG matrix on the surface of cells will also result in the migration of growth factors away from where they are normally functional and into adjacent tissue where they may stimulate aberrant development of blood vessels. This may explain telangiectasia.

Is Topical Heparin a Rosacea Treatment?
Topical heparin does penetrate the skin. It would appear to be a logical treatment, if HSPG depletion is contributing to symptom development in rosacea. The length of the heparin fragments may be important. I am unaware if anyone has tried the heparin lotions that are available for treatment of wounds to minimize scarring, on rosacea. Heparin may be useful in combination with vitamin D3 and remediation of gut flora in a general scheme to treat rosacea.

refs:
Segaert S, Van Cutsem E. Clinical signs, pathophysiology and management of skin toxicity during therapy with epidermal growth factor receptor inhibitors. Ann Oncol. 2005 Sep;16(9):1425-33. Epub 2005 Jul 12.

Webber MJ, Han X, Prasanna Murthy SN, Rajangam K, Stupp SI, Lomasney JW. Capturing the stem cell paracrine effect using heparin-presenting nanofibres to treat cardiovascular diseases. J Tissue Eng Regen Med. 2010 Mar 10. [Epub ahead of print]

Human Gut Flora Genes

2010-03-05T12:58:00.004-07:00

Thousands of Species, Millions of Genes

A research paper in this week's Nature shows a major advance in the study of the role of the human gut flora in disease and health. Metagenomics of the Human Intestinal Tract (MetaHIT) Consortium examined the role of gut flora in health by a massive project (ref. below) to determine the DNA base sequence of the majority of the millions of genes in the thousands of species of bacteria that abide in human feces. Gut flora have been linked to many human diseases, as well as the normal function of the human immune system.

Major Findings:

576.7 gigabases sequenced (150 times larger than human genome)
Identified 3.3 million gut flora genes
Represents more than 1,000 bacterial species
Each individual harbors approx. 160 different species
Most prevalent species are identified and shared by everyone

Feces from 124 Europeans

Feces samples (124 individuals total) from a Danish project to determine the role of gut flora in obesity and from a Spanish project to determine the role of gut flora in Crohn’s disease and ulcerative colitis, were extracted for total DNA.

Illumina Multiplex Sequencing of PCR Amplified Fragments

DNA samples from each individual were fragments (<800 bp) and amplified to include indexing information and sequencing primers. The sequences of a dozen of the fragments at a time was determined using a dozen different dyes (Illumina multiplexing). Sample preparation and analysis was fully automated to permit identification of overlapping contiguous sequences and assembly of bacterial genomes (1,000-1,150 most common). The sequence data in this study represents most of the bacterial species of the gut.

Comparison to 89 Existing Human Gut Bacteria Genome Sequences

The new study represented more than 200 times the sequence information than in all previous studies combined. Existing genome sequences could be identified among the bacterial genomes assembled from the new data. The identified and sequenced bacterial species represent approximately a tenth of the common bacteria in the gut.

Polysaccharide/sugar Metabolism Common among Gut Flora

Genes coding for enzymes needed to metabolize pectin, sorbitol, mannose, fructose, cellulose and sucrose, were common among the gut flora. Bacteriophages were also significantly (5%) represented in the total gut metagenome. Most of the genes were assigned recognizable bacterial functions, but many genes, presumably involved in interactions among the bacterial community or in modification of gut function have not been characterized.

Obese, Crohn’s Disease, Ulcerative Colitis, Compared to Healthy

The bacterial gene compostion of individuals diagnosed with Crohn’s Disease and ulcerative colitis were different and distinct from healthy individuals. Individuals with Crohn’s disease had 25% fewer species of gut flora than comparable healthy controls.

This study demonstrates the feasibility of using current techniques to examine in detail the interactions between gut flora and tissues of the gut that are involved in health and disease. This also suggests the risks of antibiotics in altering critical functions of the gut flora, as well as the alteration of gut flora to support health and cure disease.

reference:
Junjie Qin, et al. 2010. A human gut microbial gene catalogue
established by metagenomic sequencing. Nature 464, 59-65.

ABO Oligosaccharides, RBCs, VWF, Endothelial Cells, Megakaryocytes

2010-02-26T14:33:00.000-07:00

Blood Group Antigens Are Synthesized in the Golgi by Glycosyl Transferases A or B

This is another rant about poor teaching and false text book information. I have been trying to summarize some of the key points of genetics and cell biology that I think should shape the minds of young biologists. To make my point, I want to outline where the common ABO blood group antigens are made and displayed.

ABO Antigens Are Oligosaccharides, Not Proteins

Red blood cells that are type A will clump together with antibodies that bind to A antigens. At this point many instructors leap ahead and say that the expression of the A gene results in the production of the A protein, that is displayed on the surface of RBCs. Wrong. The antigens are carbohydrates, short chains of sugars called oligosaccharides, attached to a lipid embedded in the RBC membrane.

Glycosyl Transferases - Enzymes that Assemble ABO Oligosaccharides

Oligosaccharides are synthesized by adding one sugar at a time onto a growing chain. Information-rich oligosaccharides, such as the ABO antigens, are made of several different sugars and are linked in a variety of ways to other sugars, so each sugar is added by a different enzyme. The sugar-adding-enzymes are called glycosyl transferases. Similar to other macromolecular polymerizations, e.g. protein and nucleic acid synthesis, the monomer sugars are first activated by bonding to a nucleotide phosphate (typically UDP) and the sugar is transferred from this activated intermediate to the growing sugar chain, in this case the H antigen.

The glycosyl transferase coded by A allele transfers an N-acetylgalactosamine to the end of the H antigen oligosaccharide, whereas the glycosyl transferase coded by the B allele transfers a galactose residue.

Dominance Means Functional Enzyme, Recessive Means Dysfunctional

The ABO blood group system provides a simple molecular interpretation of genetic dominance. An allele is dominant if it produces a functional enzyme that gives the phenotype, in this case a particular glycolipid attached to an RBC. A or B alleles that have mutated to yield dysfunctional proteins that no longer function as glycosyl transferases are recessive. Recessive alleles don’t impact phenotype. O antigen is nothing more than the original unmodified H oligossacharide.

ABO Is Bizarre because A and B Code for Two Alternative Functional Enzymes - Codominance

Multiple mutations converted an allele that coded for a glycosyl transferse into a second allele that transferred a different sugar. A and B alleles code for two different glycosyl transferases that transfer two different sugars. The result is codominance. AB individual have both enzymes and produce both A and B antigens on their RBCs. This is very unusual and inconsistent with Mendelian genetics.

AB Glycosyl Transferases Are in the Golgi to Produce Oligosaccharides for Export

Part of the peculiarity of the ABO system derives from the action of the A and B glycosyl transferases in the Golgi. Proteins destined for secretion have a hydrophobic group of amino acids, the signal peptide, that is synthesized first as a messenger RNA is translated into protein on a ribosome. The signal peptide orchestrates binding of the ribosome to the endoplasmic reticulum (ER) and the growing polypeptide is extruded into the ER. A series of vesicle budding and fusing events transfers proteins to the lamina of the Golgi apparatus and a final fusion event with the cytoplasmic membrane results in secretion.

Specific enzymes retained in the ER and the Golgi recognize particular amino acid sequences and attach sugars to exposed hydroxyl of amino groups (O or N glycosylations). The ABO antigens are a little unusual in that the oligosaccharide is attached to a lipid anchor, so that the A and B glycosyl transferases localized in the Golgi attach terminal sugars and the glycolipid remains bound to the cytoplasmic membrane and is displayed on the surface of the RBC.

RBCs Don’t Have Nuclei, ER or Golgi, but Normoblasts Do

Mammalian RBC’s don’t have nuclei, because the nuclei were expelled during RBC development. Since the ER is an extension of the outer membrane of the nucleus and the Golgi is derived from the ER, it follows that RBCs don’t have any of these structures. RBCs are just collapsed bags of ribosomes, hemoglobin mRNA, cytoplasmic enzymes, a few mitochondria, lots of hemoglobin and stiffened membranes displaying ABO oligosaccharide antigens. The ABO antigens were assembled and displayed on the membrane before the loss of the nucleus.

ABO Antigens Are Also on von Willebrand Factor of Endothelial Cells and Platelets

I have never heard a good explanation of why a transfusion from a universal, type O donor to recipients with type A, B or AB blood doesn’t cause clumping of RBCs. The galactose and galactosamine sugars that decorate B and A RBCs also commonly decorate the surfaces of bacteria. The immune system is prevented from making antibodies against its own antigens and so it only produces antibodies against A or B antigens found in bacteria, if they are not own self RBCs. Type O individuals lack A and B antigens of their own, so they produce anti-A and Anti-B against bacterial oligosaccharides. So why don’t the anti-A and anti-B antibodies in type O blood serum clump type A or type B blood?

The answer is that a serum protein, von Willebrand Factor (vWF) is also decorated with the ABO oligosaccharides. A type A person has type A vWF and a type B person has type A vWF. Thus, the antibodies against A and B oligosaccharide antigens that are present in type O blood will be inactivated by binding to the A or B oligosaccharides of the recipients vWF.

vWF is synthesized by endothelial cells that line veins and arteries. It is present in the secretory Weibel-Palade bodies of endothelial cells. It is also present for secretion by platelets.

So, the ABO blood group antigens are very strange examples that are not protein, not Mendelian and are not even predominantly found on RBCs. The AB oligosaccharides do provide good examples of the use of activated intermediates in macromolecular synthesis, the origin of organelles, secretion, erythropoiesis and immunology.

Last Week of the Eades Cure

2010-02-16T17:46:00.001-07:00

Week 6 of The 6 Week Cure for the Middle-Aged Middle

It feels like I have established a new set point ten pounds lower than my start. I dropped ten pounds easily in the first two weeks and then bounced around plus or minus two pounds for the next month. The Cure is simple and effective.

The First Weeks of The Cure

The 6 Week Cure for the Middle-Aged Middle was written by Drs. Mary Dan and Michael Eades to efficiently lose abdominal visceral fat and tone the abs. It starts with two weeks of three whey protein/cream/leucine shakes and one high fat/protein-low carb veggie meal per day. This surprisingly tolerable regime (without all but essential medications, no alcohol and no grains) helps to reduce fatty liver and use up visceral fat around the abdominal organs. I noticed the impact immediately and lost about a pound a day. This also eliminated hunger and exposed snacking habits.

The Middle Weeks of The Cure

The second two weeks of The Cure permits occasional alcoholic beverages and three low carb meals per day, but without dairy. That is basically meat/fish/eggs and low carb veggies for each meal. Most calories were from fat rather than carbs. Portion control became a new issue, but hunger was still not a problem. The meals were very satisfying. Energy for exercise returned, but weight loss ebbed. It was harder to stay away from old snacking habits, since meals were back to a more normal pattern.

The Last Weeks of The Cure

During the last weeks of The Cure there is a final turn to what may be for some a new, low carb, higher fat eating style. I chose The Cure, because I already knew that the eating philosophy of the Drs. Eades was consistent with my own anti-inflammatory diet and lifestyle. I did not expect to be surprised by The Cure, but I was. I learned a lot about my own eating habits.

Gut Flora Matter

I started The Cure, because I thought that the progression of dietary components would destabilize my gut flora and simultaneously destabilize my weight set point. I anticipated that my gut flora would reorient, and they did. There were all kinds of changes and some of the weight loss and gain was probably elimination of a pound of gut flora and reestablishment of a new bacterial order. The new order also came with a lower body weight.

Hunger Comes with Carbs

For the first month of the diet, I was only hungry if I went longer than six hours without eating or if I slipped on the diet and introduced some extra carbs. Straight protein early in the morning can cause an insulin rise and a blood sugar dip that leads to a little hypoglycemia, but it produces dullness, rather than hunger. The only problem with the easy weight loss first two weeks, was that there was less energy with the protein shakes.

BMs Are Bacterial Motivated

The noticeable changes in bowel movements during The Cure, should have been expected, but they forced me to contemplate stools. When I first realized the absurdity of eating breakfast cereals, because of their high carb/grain content, I went in search of alternative day-starters in other cultures. At that time, I was still hesitant to embrace saturated fats, so I ran across salsas and stewed tomatoes. The addition of stewed tomatoes to my breakfast (usually stewed tomatoes on a poached egg) made my gut happy and regular.

The point in this context, is that my studies of BMs and constipation brought pectin to my attention again. I had previously considered pectin (poly galacturonic acid) as a competitor for biofilm acidic polysaccharides, but in this context pectin is also recommended to aid the development of probiotic biofilms. Thus, I added apples to The Cure, to help the establishment of a new bacterial order. The rapid result was a return to a happy, regular gut. This was the duh moment. Apples = pectin, tomatoes = pectin, and pectin = happy gut flora. Adding either apples or tomatoes to your diet can make your gut flora happy. An apple (or tomato) a day keeps the antibiotics away.

The Cure Works

The Cure did what I expected and more. My wife was also pleased with the rapid initial weight loss and an ongoing loss of about a pound per week. The continued loss is due to alteration of diet with a further reduction in carbs. The Cure makes the connection between weight retention and a high carb diet. Elimination of grains/starch makes weight loss much easier. Absence of sugar, high fructose corn syrup and other sources of fructose also makes weight loss easier. The inclusion of saturated fats and elimination of omega-6 vegetable oils is anti-inflammatory and provides an improved sense of well being. I recommend The Cure, because it simply works.

Arthritis, Autoimmunity and Arginine Deimidation

2010-02-06T22:52:00.003-07:00

Celiac and Antibody Production Against Tissue Transglutaminase as a Model

Arthritis is an autoimmune disease in which the immune system attacks and degrades the connective tissue of joints. Antibodies against modified amino acids, arginine converted to citrulline, and proteins commonly found in joints, mediate the arthritis disease process. The development of arthritis mimics the development of gluten intolerance, celiac, in which another enzyme, transglutaminase ( tissue transglutaminase, tTG or TG2) modifies the major gluten protein, gliadin, and antibodies are produced against both modified gliadin and TG2 autoantigen.

Arthritis of Joints Is Like Coeliac of Intestines; Autoantibodies to Protein Modifying Enzymes

In other articles, I outlined the pathology of gluten intolerance:

The major protein of wheat gluten, gliadin, contains long stretches of glutamines.
An intestinal enzyme, TG2, converts the glutamines to glutamates by deamination.
As TG2 works it binds to gliadin.
In celiac, the TG2-gliadin complexes are internalized and fragmented to stimulate antibody production against both TG2 and gliadin.
I think that the internalization and processing for antibody stimulation is dependent on the basic triplet found in TG2.

Arthritis Is Mediated by Autoantibodies to Peptidylarginine Deiminase and Citrullinated Proteins

Parallel to the celiac example, in some forms of arthritis, antibodies are produced against an enzyme that modifies proteins. In arthritis, the enzyme involved, peptidylarginine deiminase (PAD) removes the terminal nitrogen from arginine (deimination) to produce citrullinated proteins. Antibodies are produced to both PAD and citrullinated proteins.

PAD Also Has a Triplet of Basic Amino Acids for Internalization

I of course wondered if PAD had the same triplet of basic amino acids, e.g. RRK, that I had found on all other autoantigens and allergens. Examining the sequence of human PAD in the NCBI sequence databases and comparing to other sequences, I found the basic triplet near the carboxy terminus. The same or an alternative basic triplet was found in PADs from other mammals.

Autoantigens and Predicted Basic Triplets of Amino Acids Reveal the Cause of Arthritis

Arthritis is an inflammatory disease. That means that without inflammation, arthritis cannot start and if inflammation is inhibited, arthritis cannot progress. It is likely that arthritis is the result of chronic inflammation plus a precipitating event, such as joint injury or joint infection. Alternatively, in a manner similar to Hashimoto’s thyroiditis, in which celiac produces anti-TG2 antibodies that attack the TG2 also produced in the thyroid gland, arthritis may be produced by autoantibodies stimulated in the inflammation of other tissues and spreading to the joints. Celiac is also a risk factor for arthritis. Trauma-based inflammation of a joint can also result in migration of Clamydia pneumonia (Cpn)-infected macrophages to the site of inflammation. Cpn could contribute to joint inflammation and promote immunological presentation of autoantigens and autoantibody production.

reference:
Stenberg P, Roth B, Wollheim FA. Peptidylarginine deiminases and the pathogenesis of rheumatoid arthritis: a reflection of the involvement of transglutaminase in coeliac disease. Eur J Intern Med. 2009 Dec;20(8):749-55. Epub 2009 Sep 19.

The Eades Cure Week 4

2010-02-02T12:41:00.004-07:00

Meals, Alcohol, Caffeine, but no Dairy

I embarked on The 6 Week Cure for the Middle-Aged Middle to drop ten pounds and reveal my 6-pack. Now in week 4, I am half way there. I lost ten pounds (from 188 to 178 lbs) and the visceral fat under the muscle of my belly is greatly diminished, but my 6-pack is yet to be revealed. My wife dropped a couple of more pounds. Her blood stats also improved: glucose dropped 10, total cholesterol dropped 30, LDL dropped 20 and triglycerides dropped 26.

The Cure Weeks 3&4
The middle third of The 6-Week Cure is more like a normal low carb diet. There are three meals a day, but dairy is excluded. Alcohol, limited to two drinks a week and caffeine (dash of cream, no sugar) are OK. Plenty of no/low carb veggies, but minimal fruit. I enjoyed the eggs and bacon for breakfast and popped a whole chicken in the crock pot, after slipping some herb-saturated butter under the skin of the breast. The second day I pierced the surface of a couple of thick steaks with slivers of garlic and grilled them to produce some lusciously browned edges of fat.

Bad Habits and Portion Control
Alas, I had forgotten that the main reason for going on this gut transition diet, was to destabilize my gut flora, so that I could more easily change my metabolic set point to lose some visceral fat. I apparently succeeded, because after a couple of days my weight had very easily increased again. I was clearly taking in too many calories and there was little resistance to weight loss or gain. During the first two weeks on protein shakes, it was easy to eliminate bad snacking habits and carbs, but the return of normal meals in the 3rd and 4th weeks, made portion control harder. Before the diet, I would have a poached egg with two strips of bacon. The Cure version had crept up to two fried eggs, two strips of bacon and a sausage patty. I was satisfied, but it wasn’t a cure for the middle-aged middle. The harsh lesson, was that portion sizes still matter.

Energy for Exercise
More normal meals also meant a return of energy and an interest in exercise. I had only a few times when I felt that my low carb/ high protein & fat meals, stimulated some insulin and lowered my blood sugar. Common sense prevailed. I limited my portion sizes, enjoyed coffee and occasional drinks. I also started walking three miles a day watching the construction for the new community college along Indian Creek. Another interesting result was my weight training. I started testing my strength and found that for the first time in forty years I could bench press and crunch my weight, and I could squat twice my weight. Hand stands and pull ups were also much easier with less weight and more strength.

Altered Gut Flora
Absent PCR tests of my gut flora rRNA genes, I have to settle for empirical changes. My diet is still predominantly protein and fat, and I haven’t put back as many veggies as The Cure recommends. [It is winter in Idaho and the veggies in the market are not that appealing.] That also means that my gut flora are not getting very large meals either, so my stool volume has been cut about in half, compared to before The Cure. The largest impact is the impression that I can gain or lose weight with each meal. That makes further weight loss to remove the baby fat covering my 6-pack very approachable. Unfortunately, I can just reach out and ... feel it jiggle.

Constipation, Gut Flora and Health

2010-01-28T11:55:00.003-07:00

“If you notice your bowel movements, they are unhealthy.”

Scatological jokes are common. The guide on my Swedish language tour of Moscow in 1976, told a joke about Russians. She described Russian toilets that are dry, with a shelf for stools to drop upon and a lower hole in the back into which the stool is swept with water. This was contrasted to Western toilets that partially concealed the stools as they dropped into water. The joke was that Russians had no art, because they could easily observe their creations each morning.

You Are What You Eat and the Proof Is in the Toilet

Refer to Mr. Monastyrsky for a broad discussion of constipation and stool characteristics. This web site has lots of information about stool types, how to get them and how to change them. His recommendations to avoid constipation center on a healthy diet, like my Anti-Inflammation Diet, plus glutamine to help the gut heal.

Another useful perspective on gut flora is provided by a Nature web site on gut flora genomics. This site describes genomic research to show that changing diet changes relative proportions, but not the types of bacteria in gut flora. Each person has a recognizable, individual composition of gut bacteria.

Feces Is Primarily Bacteria

What you eat and your eating/health history determines your gut flora, and feces is made up of gut flora and some undigested food. Healthy bowel movement stools are made up of more than 50% bacteria and the consistency of the stools is determined by the bacterial content. Less bacteria means drier, harder stools. Bacteria hydrate stools and prevent constipation.

Pathogenic Gut Flora

Why does a total bowel irrigation with PEG, polyethylene glycol, make people with chronic diseases feel as good as if they had an antibiotic treatment (barring die off)? I think that the answer is that both disrupt and change the gut flora and in many cases disease symptoms are supported by an unhealthy gut flora/biofilms. In many cases, the antibiotic cannot have a lasting impact, because it is hard to kill bacteria in biofilms. PEG may actually clean out more of the biofilms, because it should also disrupt the polysaccharide matrix of the biofilms.

Disruption of Gut Flora Leads to Disease

Babies fed breastmilk vs. formula display very large difference in inflammation and susceptibility to disease. Formula causes gut inflammation and susceptibility to intestinal and respiratory diesease. Formula also causes a dramatic shift in gut flora from a simple flora dominated by Bifidobacter to a complex adult gut flora.

Gut Controls Immune System

The impact of the two different gut flora on development of the GI tract and on the newborn immune system is dramatic also. Remember that most of the immune system of the body is located in the lining of the gut and immune organs, such as the tonsils, are outgrowths of the GI tract. The thymus, which is responsible for producing T lymphocytes, is twice as large in breastfed babies. Thus, feedback from the gut of formula fed babies inhibits thymus and immune system development.

Change Your Gut Flora and Change Your Health

Experiments in mice, and I think in humans, have shown that changing the bacteria in the gut changes interactions with food. Exchanging gut bacteria between fat and lean individuals, causes fat people to lose weight and lean people to gain weight. I think that this indicates that gut flora participate in the so called metabolic set point, that determines if it is going to be easier to gain or lose weight. These experiments suggest that a powerful approach may be to eliminate the gut flora of individuals with chronic disease and replace it with healthy gut flora. This healthy gut flora, along with a healthy diet may make a powerful contribution to elimination of chronic diseases. Rosacea, which involves both the face and gut, might best be treated by topical antibiotics and anti-inflammatory agents, after the gut contribution has been eliminated by a fecal transplant with healthy gut flora/diet.

Anti-Inflammatory Diet Should Support Healthy Gut Flora

The efficacy of an anti-inflammatory diet (AID) should be displayed in reversal of inflammatory symptoms and unmemorable bowel movements. A gut and gut flora that resist inflammation as a result an AID, should also produce a healthier immune system and contribute to a reduction in chronic inflammation and disease. The gut may also have an impact on gut flora and a diet that does not contribute to inflammation in the body, e.g. lacks fructose and vegetable oils, may also support an anti-inflammatory gut flora.

Cure for Middle-Aged Middle

2010-01-23T11:26:00.007-07:00

My First Two Weeks with the Drs. Eades Diet

The Drs. Eades (Mary Dan and Michael) see eye to eye with me on the health benefits of a low carb diet for avoiding chronic inflammation, the foundation of most degenerative and autoimmune diseases and cancers. Recently they applied their clinical experience with the diets they developed to combat obesity to produce a book that focused on the protruding gut: The 6-Week Cure for the Middle-Aged Middle: The Simple Plan to Flatten Your Belly Fast! That book seemed to be a great way to introduce most people to a healthy diet based on obtaining calories from fat rather than carbs, so I featured it as the recommended book on my blog for the last several months. Two weeks ago my wife and I decided to practice what I preach and started The Cure.

The Cure Reduces Visceral Abdominal Fat

"The Cure” consists of three, 2-week blocks, that adapt the body metabolism to a new low carb, higher fat diet. The focus is to reduce visceral fat stored primarily in the abdomen. Equally important, from my perspective, are the changes that take place in the gut flora -- The Cure changes the composition of the gut flora to facilitate change in body fat and to restore complete function to the immune system.

Basic Outline of The Cure

The first two weeks of The Cure consist of three protein shakes (low carb flavored whey plus cream) and a very low carb meal each day. Weeks 3 and 4 are no-carb, dairy-free meals of meat/fish/eggs with calories from fat and protein only. The final two weeks are the new low carb, higher fat diet. Research on diet and gut flora would predict that the bacterial species in the gut stay basically the same and provide a recognizable individuality, but the relative quantity of each species in the gut changes to maintain body fat. The efficiency of the total gut flora would increase, if the diet resulted in weight loss and become less efficient, if there was increased fat deposition.

After the First Two Weeks of Protein Shakes

A diet of protein shakes changed my view of food. I was simply not hungry and cravings disappeared. My wife was shocked that she felt satisfied after drinking a shake and did not panic without snacks or seconds. It was relatively easy to eliminate most of the bad eating habits associated with weight gain. There was no problem about portion sizes or eating outside of meals. All of the bad habits became noticeable and I could see when I normally reached for some food out of boredom. There was simply no physiological support for snacking. Hydration is important and bowel movements were irregular as gut flora adapted to the severe change in diet. [Also note that the milk whey protein used in the shakes is high in lactoferrin, a protein found useful in controlling bacterial pathogens, e.g. Clostridium dificil, and intestinal candidiasis.]

Weight Loss Was Effortless

It was easy to drop some weight on the protein shakes. My wife (15 lbs) and I (5 lbs) both lost weight easily. This is significant, since neither of us was able to reduce our weights in the last several years. My weight at the start was 20 lbs over what I weighed as a high school gymnast. I didn’t feel like I had a lot of energy in the first two weeks of The Cure, so I slacked off on walking and weight training. Now that I am into the luxurious second phase with lots of meat, gaining strength by exercising is a pleasure.

Reduced Need for Fish Oil with Altered Gut Flora

Something that I need to highlight and that I attribute to the change in my gut flora, is a lowered need for fish oil. I was taking four fish oil capsules per day, to eliminate minor aches in my fingers. After the first two weeks of The Cure, without vitamin D supplements or fish oil, I still don’t have any finger aches. My wife, also no longer needs fish oil to make our 3-mile hikes along Indian Creek painless for her knees. The Cure seems to have uncovered a remaining source of inflammation in my previous diet. The most likely inflammatory candidate was the small amounts of grain and starch still in my old diet. It will be very easy for me to transition to a new anti-inflammatory diet more consistent with the one I have been advocating on this blog. I think that the anti-inflammatory diet would be a simple, healthy and enjoyable way to avoid most diseases or as an essential part in the treatment of most degenerative and autoimmune diseases and cancers.

My thanks go to the Drs. Eades for creating a smart diet sequence that alters gut flora for loss of visceral abdominal fat and provides a transition to a healthy low carb, increased fat diet. The diet sequence may also be useful in restoring gut flora destroyed by antibiotic use or dysbiosis indicated by constipation.

Rosacea, Brain Cooling and Niacin Flush

2010-01-13T11:20:00.004-07:00

Other players include: Cathelicidins, Prostaglandins, Cryptic Bacteria, Nerves, Gut

What does it take to make your face red? Excessive solar exposure can lead to apoptosis of skin cells overloaded with DNA damage and trigger inflammation: vasodilation, recruitment of neutrophils, swelling, etc. Similarly, a local infection can cause inflammation and the accumulation of neutrophils (see The Inner Life/Extravasation for slide show), lymphocytes, etc., that is observed as pus. These are general responses that occur in skin anywhere, but the face also blushes in response to emotional cues and flushes with exercise. Rosacea seems to involve all of these reactions to produce a variety of symptoms of wide severity. Here I try to provide an overview of the complex physiological interactions involved in rosacea.

Rosacea is Persistent Vasodilation of the Face with Accumulation of Neutrophils

The nervous and circulatory systems of the face are unique and provide numerous triggers for inflammation. Emotional blushing is a common trait among those who progress to rosacea, even though this type of vasodilation is not easily observed with some facial characteristics. Thus, many rosaceans claim to have never flushed before their first outbreak, but tests of skin circulation indicate that these individuals had skin types that prohibited display of the blushing. The face is also adapted to control brain temperature, so changes in body temperature, physical activity, etc. can also trigger flushing.

Facial Blood Circulation to Cool the Brain

The cooling of the blood as it traverses the facial skin is used to cool the brain during extensive exercise or in warm environments. This unique adaptation also means that control of facial vasodilation can potentially be disrupted in disease and cause symptoms of pathology. In rosacea, the brain cooling response is disturbed (see reference below), resulting in persistent vasodilation and suggesting that the unique control of inflammation in the face is why rosacea is limited to the face. The pattern of blood circulation in the face, however, only roughly approximates the inflammation pattern in rosacea.

Nerves to the Face

The face receives sensory branching from the trigeminal nerve. The enervation pattern of the branches matches emotional blushing, but they also appear to approximate the pattern of reddening in rosacea. It makes sense that rosacea involves nerve-triggered dilation of the blood vessels of the face. One contrast between emotional blushing and rosacea is that emotional blushing does not lead to the offloading of lymphocytes, whereas rosacea produces localization of neutrophils that exacerbate and prolong inflammation.

Cathelicidin, Vitamin D Receptor, DNA Complexes, Autoinflammation

A major component of the innate immune system is the group of basic antimicrobial peptides, cathelicidins. Cathelicidins are effective against bacteria and they are produced during inflammation and are partially controlled by the vitamin D receptor acting as a transcription factor. Thus, part of the action of vitamin D in providing protection against disease is by enhancing cathelicidin production. Cathelicidin action in the skin parallels the control of intestinal villi development by defensins, that are also basic antimicrobial peptides under the control of vitamin D. Cathelicidins also form complexes with host DNA from damaged cells. These cathelicin/DNA complexes bind to toll-like receptors (TLRs) and trigger inflammation. This reaction has been associated with psoriasis and may explain how neutrophil damage can perpetuate inflammation in rosacea.

Niacin Flushing Implicates Arrestins

The unique circulatory system of the face also makes it susceptible to flushing with niacin, a.k.a. nicotinic acid or vitamin B3. Niacin is cheaper and much more effective at raising HDL and lowering triglycerides and LDL than statins, but is not fully utilized because it also produces intense facial flushes. A recent article (below) has demonstrated that the lipid benefits can be separated from the flushing and implicated beta-arrestin 1 activation by niacin binding to GPR109A (G-protein-coupled receptor) as the triggering event. Arrestin, which is involved in clathrin-mediated endocytosis, activates phospholipase A2 that in turn releases arachidonic acid (ARA) from phospholipids. The ARA (that got into the phospholipids as the omega-6 fatty acid in vegetable oils) is converted by COX-2 into the inflammatory prostaglandin D2. This prostaglandin is what stimulates vasodilation. It is possible to produce chemicals that will stimulate the lipid metabolism alterations of niacin, without producing the arrestin activation and inflammation. Aspirin can be used to inhibit COX-2 and other parts of NFkB-mediated inflammation and eliminate the niacin flush. It is also interesting that the modified lipid metabolism of schizophrenics also eliminates niacin flushing. Salicylic acid, the same as the acetylsalicylic acid of Aspirin without the acetate, is also used in some topical applications to quiet the symptoms of rosacea. Arrestin activation may be involved in rosacea.

Gut Flora, Biofilms and Cryptic Bacteria

The gut is probably involved in most cases of rosacea and bacteria are also implicated by the modification of rosacea symptoms by antibiotics. This area has not been explored, but I suspect that gut flora controlled by diet, as well as pathogenic biofilms and cryptic bacteria, e.g. Clamydia pneumoneae, in facial tissue are involved in varying degrees in the panoply of pathologies called collectively, rosacea. Since the bacteria in contact with the gut determine the development of the lymphocytes in the lining of the gut, e.g. Tregs vs. T cells that fight infections, pathogenic gut biofilms may disrupt the normal function of the immune system and support rosacea. Die off and release of cell wall endotoxin from cryptic bacteria could explain the paradoxical inflammation in response to many treatments that are normally anti-inflammatory. I have discussed in another article potential approaches to strip off biofilms.

Treatment with Anti-Inflammatory Diet

The Anti-Inflammatory Diet (AID) and Lifestyle that I advocate on this blog would seem to be a natural cure for rosacea. It should eliminate the inflammatory background that supports rosacea and was probably essential for its development. This diet also eliminates acne, which is directly related to the accumulation of lymphocytes to make pus. Inflammation is also needed for the offloading of neutrophils that exacerbate inflammation in rosacea. Vitamin D is instrumental in cathelicidin production to eliminate cryptic bacteria.

In most cases of rosacea, the AID should be helpful. Eliminating dietary sources of inflammation, especially vegetable oils (the source of omega-6 fatty acids that are converted into inflammatory prostaglandins), should reduce rosacea symptoms.

In advanced, severe cases, however, it appears that maintenance of the suppression of the response to cryptic bacteria is required to prevent endotoxin-based inflammation. Thus, most treatments that decrease inflammation, e.g. omega-3 oils, vitamin D3, Vagal maneuvers, can paradoxically produce elevated inflammation. These treatments may also inadvertantly contribute to inflammation by upsetting pathogenic interactions between bacteria and intestinal cells. I have discussed these paradoxical ramifications in another article.

references:
Brinnel H, Friedel J, Caputa M, Cabanac M, Grosshans E. 1989. Rosacea: disturbed defense against brain overheating. Arch Dermatol Res. 281(1):66-72.
Walters RW, Shukla AK, Kovacs JJ, Violin JD, DeWire SM, Lam CM, Chen JR, Muehlbauer MJ, Whalen EJ, Lefkowitz RJ. 2009. Beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice. J Clin Invest. 119(5):1312-21.

2009: What I Learned Last Year

2009-12-30T16:05:00.001-07:00

This year followers of this blog checked in more than 100,000 times to read my 150 articles on diet, inflammation and disease. I learned a lot and I hope that my readers gained some insights into anti-inflammatory food choices that are helpful in pursuing enhanced health. Here is a status report.

What We Eat Contributes More to Disease Risk than Genetics

I started this blog to try to understand how food, exercise, sun exposure, etc., contribute to health and disease, because I was shocked that recent, comprehensive studies demonstrated that genetic defects were only minor contributors. I am trained as a molecular biologist and I search for explanations of disease in terms of the interactions of the proteins coded by the genes in our cells. History of defective genes that code for defective proteins in sickle-cell anemia, Huntington’s disease or ALS, suggested that personal genetic defects might explain personal diseases. Fortunately, it appears that in most cases genetic defects only matter when our actions produce chronic inflammation. What we eat is far more important than our genetics in determining if we are going to suffer from allergies, autoimmune diseases, degenerative diseases, various forms of mental illness or cancer. If we eat to avoid inflammation, in most cases it doesn’t matter how genetically defective we are.

Diet-Based Inflammation Is the Major Risk

Modern diets rich in starch/sugar/fructose and polyunsaturated fats (omega-6 oils), and deficient in saturated fats and omega-3 oils produce the chronic inflammation that forms the foundation of most diseases. Vegetable oils, such as corn, soy or safflower oils are inflammatory and should be eliminated from our kitchens. We should only use olive oil, butter or lard. Saturated fats from meat, dairy and eggs are healthier than polyunsaturated vegetable oils. There was never adequate scientific data to justify the shift from saturated fats to polyunsaturated vegetable oils. That was a tragic, unscientific medical error that contributed significantly to deteriorating health in the developed/developing world.

Anti-Inflammatory Diet and Lifestyle Is the Cure

It came as a surprise to me that simply eliminating inflammatory foods could prevent most diseases. After diseases have developed, it is harder to reverse the process and return to health, but even in that case, diet is of paramount importance.

Back to Basics of a Healthy Diet (the Food Pyramid Is Wrong)

Starch/sugar/fructose are inflammatory. Low carbohydrate is the healthiest diet.
Grains, even whole grains, and especially cereal are a big part of the problem and should be avoided.
Fat and not carbohydrates, should be the major source of dietary calories/energy.
Saturated fats are healthier than vegetable oils -- use olive oil and butter.
Meats/fish (not fed on grains) are healthy. A healthy vegetarian diet is difficult.
Leafy vegetables are a good source of healthful antioxidants.
Fruits and fructose are inflammatory and should be eaten sparingly.
Healthy gut bacteria are important. Eat fermented foods with live bacteria, e.g. yogurt.

Living with Inflammation

Chronic inflammation can lead to many problems that diet and supplements can help to remedy. For example, vitamin D deficiency is an epidemic in America, because chronic dietary inflammation appears to compromise the ability to make vitamin D in the skin with sunlight. Most individuals eating a diet high in polyunsaturated fats, starch and high fructose corn syrup, are deficient in vitamin D and would benefit from a vitamin D3 supplement of at least 2,000 IU per day. Vitamin D deficiency also contributes to inflammation. Fish oil supplements can also help to reduce dietary inflammation and should always be taken with at least equal amounts of saturated fats in the same meal.

Resolve to Eat Your Way to Health

It is easy to avoid most diseases by avoiding dietary inflammation. Since chronic dietary inflammation produces depression, lethargy, obesity and a lack of energy, a healthy anti-inflammatory diet will also lead to weight loss, increased energy and reduced symptoms of aging. Most symptoms of aging and disease are actually poorly managed inflammation that exposes genetic defects. Most people increase in inflammation with age, but proper diet can avoid this risk to health and prolong youthful activity. The healthiest resolution for the new year is to stop eating blatantly inflammatory foods (starch and vegetable oils) and start eating more spicy meats, fish and leafy vegetables.

Humans vs. Naked Mole Rats

2009-12-09T11:27:00.003-07:00

Blood Sugar, Insulin, Superoxide, Couch Potatoes

(Thanks to my loyal readers for the inspiration for this article.)
There is a lot to be learned by sticking one's head in the sand. Mole rats of East African deserts are just as naked as humans, but beyond the lack of hair and complex social structures, we are as different as night and day. These differences explain some of our unusual physiological characteristics. Maybe our health problems are linked to our sweaty skin, predatory nature and our need to run, just as the naked mole rats (NMRs) are adapted to their dark, high carb, climate-controlled burrows.

Mole Rats:

low metabolic rate controlled by eating
live in low oxygen burrows
poor temperature regulation
live in the tubers that they eat -- sweet potatoes with legs
no insulin or superoxide dismutase
vitamin C and D production (in darkness)?
no pain sensors in skin, no stress, no sweat
mostly vegetarian, starch

Humans:

high metabolic rate controlled by physical activity
live in high oxygen
temperature regulation by sweating
hunters, runners, farmers
no vitamin C production, vitamin D via sunlight
insulin used to regulate blood sugar, insulin resistance by superoxide
oxidative stress leads to inflammation and disease
carnivores, fat

Naked Mole Rats Are as Unique as Humans

Naked mole rats and humans are odd compared to most mammals. Those oddities may explain a lot about modern human diseases. The biggest difference between humans and NMRs is the control of blood glucose. It seems that NMRs control their metabolism by their eating. In times of starvation, the NMRs eat less and their metabolic rate lowers. At the cellular level, this must mean that fat stores are converted to blood glucose to modestly regulate blood sugar as it drops, but the lack of insulin does not permit control of high blood sugar. Thus, a rise in blood sugar must lead to cessation of eating. This would make sense, because NMRs husband their resources -- they typically encounter few, very large, starchy, underground tubers/roots, eat into them and continue to live off of them for their lifetimes. They are underground farmers. They do not wolf down their slow moving prey and hunt for more.

NMRs Know When to Stop

Individual cells of NMRs regulate their metabolism without apparent recourse to adjusting their surface glucose transporters, since their blood glucose levels are constant or unmanipulateably low. There is no mechanism for blocking influx of glucose by insulin stimulation when intracellular glucose is too high. It would be expected that intravenous injection of excess glucose could kill NMRs by producing excess intracellular glucose spilling excess high energy electrons of the electron transport chain into superoxide damage. Of course low tissue oxygen levels would provide protection, since the rate of superoxide formation is proportional to oxygen concentration at the mitochondrial surface.

Humans Are Runners

Humans are adapted to running down prey during the heat of the day, which means that they produce high metabolic rates, high demands for cooling, high tissue oxygen levels and high glucose/fat utilization. In a lengthy chase, glycogen is rapidly depleted and fat metabolism ensues. Human brains are adapted for access to lots of oxygen and nutrients. Human tissues are adapted to low serum glucose and high levels of oxygen. Moderate levels of serum glucose lead to increased cellular metabolism via insulin production and increased glucose transport into cells. Low serum glucose leads to lipid mobilization and liver gluconeogenesis.

Humans Kill for Fat

Physical activity regulates human cellular activity. Depletion of celllular ATP leads to an increase in cell surface glucose transporters. Inadequate serum glucose, low intracellular glucose (phosphates) and low ATP lead to lipid utilization. Lipids are all metabolized in mitochondria and require oxygen as the last, low energy electron acceptor in the electron transport chain. Brain evolution in humans was adapted to high metabolism and intelligence is associated with intense brain vascularization, oxygen supply and lipid utilization. It could be argued that glycogen storage is a way for humans to handle excess blood sugar during sleep inactivity, since humans are adapted for handling fats and tolerating carbohydrates.

Sweet Tooth Is Deciduous

Why do humans have a sweet tooth? A group of early humanoids stumbling onto a cache of cookies made by elves, would quickly eat themselves into a stupor as their blood was diverted from brain to belly, their blood sugar rocketed, insulin surged, glucose gushed into cells, cellular metabolism peaked, cellular ATP pegged over, and superoxide spilled high energy electrons out of the saturated mitochondrial ETC. Cookies would be killers for humans, if superoxide production didn’t block insulin-based transport of glucose into most cells and channel the high blood glucose into fat deposition.

Marauding Naked Mole Rats

Cookie-fed humans become fat, lethargic and start to look like potatoes with legs, i.e. NMRs. Unfortunately, unlike NMRs, humans don’t have off switches for carb glutting. Humans evolved to run on fats, and can exploit occasional carb caches, because of an adaptive sweet tooth, but lack of evolutionary experience with gigantic carb caches, e.g. agriculture and supermarket cereal aisles, left humans maladapted for high carb diets. We can’t pull out the HFCS intravenous line and instead become couch potatoes waiting as potential victims for giant marauding NMRs (the healthcare industry). Fortunately, NMRs can keep the potatoes fat and feed on them indefinitely.

BPA in Thermal Printer Ink

2009-12-07T23:43:00.000-07:00

Bisphenol A Free Bottles, BPA-Free Pacifiers, BPA-Free Receipts

I previously poo-pooed the threat of the estrogen mimetic bisphenol A (BPA) from polycarbonate bottles, cans and pacifiers, because my quick calculations indicated that there was just too little BPA and too many other natural sources of estrogens that haven’t been problems. But it’s not the water that’s the problem, it’s the other plastic, your credit card.

Some Receipts Are Covered with BPA

In a recent article on the use of BPA for thermal printing it was claimed that some receipts have as much as 100 milligrams of BPA. I simply didn’t believe this, because 100mg is 0.1 gram, which is what I approximate as the weight of a cash register receipt. So, I emailed the investigator and he clarified. He encountered some coupons that were printed on 100 sqare inches of thermal printer paper. That is one whopping receipt, but even at that size, the coating with BPA was impressive and scary.

Thermal Printing Heats BPA with Second Reagent to Make Pigments

Thermal printing ink, e.g. BPA plus an acid-sensitive dye, smeared over the whole surface of the special thermal paper. Heating the paper in the printer head causes the BPA, which is a weak acid, to release its protons and react with the dye to produce a colored pigment. In order to make the printing visible, a lot of initially colorless ink has to be coated on the paper. That means that perhaps 5% of the weight of the thermal printer paper is BPA and that BPA is all on the surface and able to rub off onto your hands!

Don’t Touch the Receipts

A recent study of BPA exposure during gestation and subsequent stereotypical sex-specific behavior showed that women with higher BPA in their urine during their first trimester of pregnancy gave birth to babies that developed with less than their expected sex-specific behaviors. In other words, higher BPA in utero meant that boys behaved more like girls and vice versa. Most of the women tested had about 1 ppm BPA contaminating their urine. Some had a thousand fold more. Even if they ate polycarbonate bottles, they could not have had more than 1,000 ppm (1 ppm = one part per million = 1 microgram per gram = 1 milligram per liter, so 1,000 ppm = 1 gram per liter). This suggests that the women with funny, really average kids, were getting their BPA from some other source than bottles and cans contaminated with BPA.

Wash Your Hands or Wear Gloves When Shopping

I think that the culprit is the cashier. Why are some of these people so cheerful when they have to deal with so many louts in line? Maybe it is the BPA soaking into their finger tips from the BPA-soaked receipts that they are handing to you. You may have wondered why some people become fanatical about coupons. Maybe they are also taking in too much BPA. What about the kids playing with credit card receipts? BPA has been linked with precocious sexual development. Maybe it would be safer to let the kids play with cigarette butts.

Not All Receipts Have BPA

I have asked a few cashiers if their receipts are printed on thermal paper laced with BPA, but most don’t know or care. Many receipts are printed with ink, so they aren’t a problem. Either way, the cashier should know to avoid self-contamination or risks to customers. May you should ask the next time you hand over the plastic.

Superoxide Causes Insulin Resistance, Type 2 Diabetes

2009-11-24T00:29:00.003-07:00

Intracellular Nutrient Excess Produces Mitochondrial Electron Accumulation
(Article referenced below was brought to may attention by Cristian Stremiz - thanks)

Insulin resistance blocks insulin-based transport of glucose into cells that are already overloaded with nutrients. The spilling-over of excess high energy electrons in the mitrochondrial electron transport chain onto oxygen produces superoxide. Superoxide is the trigger to block the import of still more glucose. Thus, insulin resistance is a cellular defense against sudden death by superoxide and other reactive oxygen species (ROS).

High Energy Electrons of Glucose Are Used to Make ATP

Cells are biochemical machines that turn on genes to produce enzymes to convert the high energy electrons on the carbon and hydrogen atoms of glucose into ATP energy and molecular components of the cell. The high energy electrons are systematically depleted of energy, protons are pumped to produce a proton gradient across the inner mitochondrial membrane, ATP is made using the proton gradient and the low energy electron are passed off to oxygen molecules to make water. That is a quick summary of cytoplasmic glycolysis, the tricarboxylic acid cycle (mitochondrial matrix) and the mitochondrial electron transport chain. The final step of transferring the depleted electrons to oxygen to make water is how oxygen is consumed in respiration. Note that if everything works well, the high energy electrons of glucose, which could suddenly release all of their energy directly interacting with oxygen and start a fire, just produce water. Another bad alternative would be for the high energy electrons to bind to molecular oxygen making superoxide.

Cells Adjust their Glucose Individually to Match ATP Use

If the supply of ATP from the mitochondrial electron transport chain of a cell gets low, this triggers the migration of vesicles with glucose transport proteins to the cytoplasmic membrane. Since the number of transport proteins determines the rate of import of glucose, then more transporters means an increase in glucose and more ATP. Type 2 diabetes and insulin resistance represents the others extreme, i.e. what happens when cells get too much glucose, max out their capacity to make ATP and high energy electrons build up in the electron transport chain.

High Blood Sugar Triggers Insulin Production to Import the Glucose into Cells

Cells can also participate in body-wide metabolism coordinated by hormones, such as insulin. A sudden increase in blood glucose concentration triggers the pancreas islet cells to release insulin into the blood. The insulin binds to insulin receptor proteins on the surface of cells and that signal brings more glucose transport proteins to the cytoplasmic membrane. The cells import additional glucose and their metabolism increases and more ATP is produces. This lowers the blood glucose level. Some cells can continue to accumulate glucose in the form of glycogen or fat droplets, but other cells do not have this storage capacity. If glucose is supplied beyond the capacity of the cell to use it, then the mitochondrial electron transport chain begins to produce superoxide.

Superoxide Is a Reactive Oxygen Species (ROS)

Oxidation stress is the reason that plant antioxidants, vitamin C and N-acetyl-cysteine are recommended to avoid inflammation. One of the major sources of oxidation stress is the production of superoxide. Cells produce an enzymes, superoxide dismutase, to convert superoxide into hydrogen peroxide, and catalase to convert hydrogen peroxide into oxygen and water. Superoxide can also interact with nitric oxide to produce the nitric oxide radical. Unfortunately, superoxide can also produce hydroxyl radicals that can react with unsaturated lipids to produce lipid peroxides. Thus, superoxides can contribute to the production of many ROS, cause oxidation damage and trigger inflammation.

Many Different Processes that Produce Insulin Resistance all Produce Superoxide

The trigger for insulin resistance appears to be mitochondrial superoxide accumulation. A recent article used numerous mouse models of insulin resistance that mimic the typical human risk factors for insulin resistance and type 2 diabetes, e.g. excess nutrition, physical inactivity, pregnancy, polycystic ovarian syndrome, metabolic syndrome, inflammation, oxidative stress, anti-inflammatory corticosteroids, etc. and demonstrated that in each case mitochondrial superoxide accumulated. Moreover, mutant mice with lowered superoxide dismutase were more susceptible to insulin resistance and mutants producing an overabundance of superoxide dismutase were resistant to insulin resistance.

Insulin Resistance Is a Natural Defense Against Energy Excess

Superoxide sensing and insulin resistance protect cells against too much energy input and oxidative stress, but without the ability to reduce blood sugar, hyperglycemia leads to the suite of degenerative reactions that provide the symptoms of type 2 diabetes.

reference
Hoehn KL, Salmon AB, Hohnen-Behrens C, Turner N, Hoy AJ, Maghzal GJ, Stocker R, Van Remmen H, Kraegen EW, Cooney GJ, Richardson AR, James DE.Insulin resistance is a cellular antioxidant defense mechanism.Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17787-92. Epub 2009 Sep 30.

Bacterial Amyloid (Curli Fibers) Forms Biofilms

2009-11-17T18:42:00.005-07:00

E. coli Curli Stacks in Congo Red Staining Fibers

We can’t cure diseases, because we don’t understand basic chemistry (what is hydrophobic) and biology (which came first the biofilm or the bacterial cell wall?) Let’s look at a fundamental biological process, how bacteria form biofilms.

Biofilm Formation from Secreted Proteins and Polysaccharides

Investigators passed some E. coli through a special slide chamber so they could watch at high magnification as a single bacterium attached to the surface, divided to produce a colony of a few bacteria and then began to secrete proteins (curli fibers) and polysaccharides (colanic acid and cellulose) to make the biofilm matrix. The matrix stained red with Congo Red.

Congo Red Stains Amyloids, Cellulose and Rare LPS

Staining with Congo Red shows that the spacing of hydrophobic patches on the surface of the biofilm matrix matches the flat hydrophobic, aromatic rings of the dye, Congo Red. This particular dye is important, because Congo Red also specifically stains amyloid, e.g. beta amyloid of Alzheimer’s disease. But Congo Red also binds to cellulose, a linear beta 1,4-glucan polysaccharide. This seems paradoxical, because we are taught that the sugars of which a polysaccharide are made are hydrophilic. That turns out to be a half-truth.

Faces of Sugars Are Hydrophobic

The hydrogen bonding hydroxyl groups that make sugars water soluble and hydrophilic, radiate from a ring of carbons, and the faces of that ring cannot make hydrogen bonds. The faces of sugars are hydrophobic and in most cases will bind to hydrophobic surfaces, such as aromatic amino acids, e.g. tryptophan, tyrosine and phenylalanine. Thus, carbohydrate binding enzymes, such as shown in the figure bind cellulose (in grey and red) in a groove lined with aromatic amino acids (yellow and orange) so that each sugar orients over and sometimes sandwiched between aromatic amino acid residues. This also explains why Congo Red binds to cellulose, since the aromatic rings of the dye bind to neighboring glucose residues along the relatively flat cellulose strand. Most other polysaccharides and smaller sugars lack this spacing of sugars and they don’t stain red with Congo Red.

Basic Amino Acids Bind Hydrophobically

Another misperception is that basic amino acids, positively charged lysine and arginine, are hydrophilic. The nitrogen atoms that make these amino acids positively charged, can form hydrogen bonds, but the hydrocarbon tails that have these nitrogenous tips, are hydrophobic. Thus, basic amino acids and aromatic amino acids can stack to form tryptophan/arginine ladders in which they alternate. A prominent example of these interdigitations are the way that nuclear localization signals, a quartet of basic amino acids, bind to importin via its projecting, spaced tryptophans and drag proteins through pores into the nucleus. Similarly, the basic amino acids of heparin-binding domains extend across the hydrophobic faces of the sugars of heparin and hydrogen bond with their tips to the sulfates of the heparin. In each of these binding examples the binding is primarily hydrophobic.

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

Amyloids are proteins that stack together like stacking chairs, so that each protein is oriented in the same way all along the stack. In the case of the beta amyloid that makes up the toxic plaque in Alzheimer’s disease, each amyloid peptide is stacked like a hair pin on top of the next to make a fiber. At the bend in beta amyloid, is a basic amino acid and the amyloid fiber has a band of basic amino acids along its length. The spacing between the basic amino acids in an amyloid stack is just spanned by Congo Red, so amyloids are diagnostically stained red. This same spacing of basic amino acids fits the sugars in heparin. Thus, heparin can catalyze amyloid formation and is abundant in amyloid plaques in Alzheimer’s

Bacterial Biofilms Form from Amyloids and Polysaccharides

The E. coli cells that formed the biofilms that started this article secrete a protein, curli, that stacks as an amyloid into fibers. These fibers stained by Congo Red and bind to the cellulose that is also produced by the E. coli. It should not be surprising that biofilm formation is catalyzed by heparin and biofilm formation is a major problem in catheter infection, since heparin is used to coat catheters to keep them from forming blood clots. Amyloids are also formed from stacked seminal acid phosphatase proteins that form fibers in the presence of heparin and facilitate HIV infection.

Do Biofilms Foment Amyloid Production?

Basic amino acids, sugars, aromatic amino acids and plant phytochemicals all bind each other via their hydrophobic surfaces. It would not be surprising that bacteria that produce proteins and acidic polysaccharides that interact hydrophobically would also interact with host molecules with a similar spacing of hydrophobic surfaces, which are common in heparin-binding interactions and nucleic acid interactions. The bacteria in biofilms produce both proteins and polysaccharides that may catalyze amyloid production. The acidic biofilm polysaccharide, colanic acid, may replace heparan sulfate and curli should bind to heparin.

Berberine Binds Heparin and Blocks Amyloids and Biofilms

Just as bacterial products may compete for host heparin and heparin-binding domains, phytochemicals that interact with heparin, such as the phytochemical berberine, should disrupt heparin mediated molecular interactions, and by extension also biofilms. There is experimental evidence for berberine both disrupts amyloid formation and biofilm assembly.

Psoriasis, IL-17, Cathelicidin, TLRs, NFkB, Inflammation and Heparin Therapy

2009-11-12T11:32:00.002-07:00

Host DNA Released by Keratinocyte Apoptosis Binds LL-37 and Activates Dendrocytes

Psoriasis is an inflammation of the skin that leads to overproduction of keratinocytes resulting in a thick crust. Skin inflammation, in this case, is considered a result of autoimmunity, but an autoantigen has not been identified. It is more likely that psoriasis results from an autoinflammatory condition, in which inflammation produces a complex of self molecules that mimic bacterial DNA and trigger TLR/NFkB inflammation signaling. And of course, if this is going to be interesting, it has to involve heparin.

Vitamin D Binds to a Transcription Factor Receptor that Controls Antimicrobial Peptides
A significant component of the innate immune system is a group of antimicrobial peptide (defensins, cathelicidins, e.g. LL-37). These short polypeptides owe their natural antibiotic activity to numerous basic (positively charged, arginine and lysine) amino acids. The transcription factor that controls the expression of these peptides is the vitamin D receptor. Thus, various forms of vitamin D influence the amount of antimicrobial peptides produced in the mouth, skin and crypts of the intestinal villi. Oral vitamin D3 would be expected to directly improve defensin production in the gut and LL-37 production in the skin.

IL-17 Stimulates Skin Inflammation and LL-37 Production
A specific group of lymphocytes, called T helper 17 cells, produce IL-17. These Th17 cells accumulate in some sites of inflammation, such as psoriasis and their secretion of IL-17 is associated with ongoing inflammation and may contribute to LL-37 production, as well as apoptosis of keratinocytes in the thickening skin of psoriasis plaques.
http://www.ncbi.nlm.nih.gov/pubmed/19623255?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_PMC&linkpos=2&log$=citedinpmcarticles&logdbfrom=pubmed

Th17 Cells Are Produced in the Gut in Response to Segmented Bacteria
One of my readers brought to my attention an article that shows that one of the hundreds of species of gut bacteria, segmented filamentous baceria, stimulates the gut to develop T helper 17 cells that subsequently migrate to sites of inflammation.
http://www.medpagetoday.com/Gastroenterology/InflammatoryBowelDisease/16472
This emphasizes the link between the gut and inflammatory diseases and parallels other examples of gut influence on disease, such as the ability of Helicobacter pylori to affect asthma or parasitic worms to tame Crohn’s disease, allergies and asthma.

Inflammation Lowers Heparan Sulfate Production and Spreads LL-37
One of my students induced inflammation in cells in vitro and showed by quantitative PCR that genes involved in heparan sulfate proteoglycan production are selectively silenced. This observation explains in part the loss of heparan sulfate in kidneys and intestines that contributes to the leakiness of these organs in response to inflammation and the partial repair of these organs by heparin treatment. Decrease of heparan sulfate that normally coats cells and binds antimicrobial peptides, such as LL-37, would explain the enhanced movement of LL-37 in psoriatic skin.

LL-37 Binds to Host DNA and Triggers Toll-Like Receptors
DNA is released from keratinocytes in psoriatic skin and this host DNA binds the antimicrobial peptide cathelicidin LL-37. The LL-37/DNA complex mimics bacterial DNA and triggers the Toll-like receptors (TLR) on the surface of immune cells, dendrocytes, to activate NFkB, the transcription factor controlling inflammation.
http://www.ncbi.nlm.nih.gov/pubmed/19050268?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed

Heparin Treats Psoriasis
It seemed obvious to me that the heparin binding domains (Look at all the basic amino acids in blue in the illustration of LL-37.) of LL-37 were involved in DNA binding and the reason the LL-37 was binding to host DNA, was that heparan sulfate had been depleted as a result of local inflammation. It also seemed obvious that topical heparin should eliminate psoriasis plaques. So I did a Google search of psoriasis + topical heparin and got a hit on a 1991 patent application that claims a broad applicability for heparin use in curing symptoms of a wide variety of diseases, including psoriasis.
http://www.patentstorm.us/patents/5037810/description.html
The only topical form of heparin that I know of is Lipactin (available in Canada and Europe?), a treatment for coldsores, which makes sense because herpes viruses use heparan sulfate to infect cells.

Biofilms as Human Gut Mycorrhizals

2009-11-05T18:12:00.000-07:00

Are Biofilms Healthy Extensions of Intestinal Villi?

If soil is the stomach of the earth, then plant roots and mycorrizal fungi must be the intestines.

Mycorrhizal fungal hyphae extend from root hairs of plants into surrounding soil and enhance the uptake of phosphate and other nutritents. Many plants cannot colonize new soil without taking their fungal partners with them. It would seem obvious that the highly adapted human gut flora would include bacteria and fungi that actively communicate with intestinal epithelial cells. Perhaps that communication includes both nutrients, e.g. hydrogen, ammonia, etc., vitamins and bacterial wall components, e.g. LPS.

Plants Sit and Mine Soil, Humans Mine Nutrients Passed through Their Gut

I want to try to give a plant’s view of human digestion. Plants elaborate roots that branch repeatedly and the final extensions sprout hairs from individual epithelial cells. Mycorrhizal fungal hyphae further extend the reach of the plant into the soil for nutrients.

I think that a plant would look at us and see us stuffing soil/food into our mouths and watch it come out the other end. It would then try to figure out where are roots are, i.e. how we absorb the water and minerals from our moving internal stream of soil. The villi of the small intestines would look like root hairs, but where are the mycorrhizal fungi? Another problem is that the soil keeps moving past the root hairs and would break off fungal hyphae extending into the soil. Still another problem is the constant shedding of epithelial cells from the tips of the villi. The plant would be perplexed, but closer inspection would reveal that biofilms could solve the problems.

Biofilms Coat the Intestinal Villi

Biofilms coating and perhaps spanning the villi of the small intestines may enhance the transport of nutrients into the villi. This may be controversial and the biofilms may be more commonly limited to the smoother surface of the colon. The point here is that biofilms may enhance the intestinal uptake of nutrients from food. Biofilms may, therefore, be essential for health and extend the reach of the intestinal epithelial cells.

Bacterial Community Composition May Be Determined by Diet

A biofilm is composed of some type of linear polymer, such as DNA, heparan sulfate or bacterial acidic polysaccharides, with bacteria that bind to the polymer and to the intestinal epithelium. Diet determines the bacterial composition of the biofilm. Thus, the newborn starts without biofilms, gut development is finished by growth hormones in milk and a single species of Bifidobacteria excludes biofilm production, until solid food or formula initiates adult biofilms. The bacteria in the biofilm depend on diet, so the biofilms can be either beneficial or pathogenic.

Communication within Biofilms and with the Intestines

The bacteria respond to the presence of other bacteria by quorum sensing, which involves release of small molecules that alter the gene expression of other bacteria in the community. As a consequence, genes, e.g. antibiotic resistance, are exchanged and metabolism is altered. This is how Klebsiella nitrogenase and hydrogen production is controlled. The biofilm bacteria also produce compounds, e.g. vitamin D (?), that alter the behavior of the intestinal epithelial cells. The intestines can respond with inflammation to recognized pathogen components or by triggering development of cells of the immune system. The intestines are the home of most of the body’s immune cells.

Stimulation of Tregs

Helicobacter pylori adhering to the stomach lining increases the stomach’s quota of regulatory T cells that are involved in immunological tolerance. Presumably, the supply of Tregs in the intestines is also regulated by biofilms. Disruption of this system by chronic inflammation can deplete Tregs and lead to unrestrained immune attack that is observed as inflammatory bowel disease. Thus, Crohn’s disease and ulcerative colitis may be triggered by damaged biofilms.

Helicobacter Pylori, Gastric Ulcers and Cancer

2009-10-30T13:16:00.003-06:00

Stomach Pathogen or Immune Regulator?

Helicobacter pylori (Hp) has co-evolved with the human stomach. Hp has always been passed from mother to child as the child started premasticated solid foods. The advent of processed baby foods and antibiotics has eliminated Hp in 90% of the US population and coincides with a dramatic rise of allergies, asthma and autoimmune diseases (commonly explained by the hygiene hypothesis.)

Hp Is Stomach-Adapted

Hp is adapted for growth in an acidic environment. It produces ammonia to neutralize stomach acid. It also provided me with great perplexity in searching for heparin-binding domains in Hp proteins suspected of binding to stomach epithelial cells. I generalized that pathogens must have proteins on their surfaces that bind to the heparan sulfate proteoglycans of epithelial cells. I checked candidate Hp proteins and found histidines where I expected to find basic amino acids, either lysine or arginine. The “duh” moment came when I realized that the pH of the Hp milieu was acidic and hence histidine would have a positive charge and function like the other two basic amino acids. Hp was adapted to its stomach world.

Is Hp Good or Bad?

I have been trying to incorporate Hp as a pathogen into my view of gut function. After all, Hp causes stomach ulcers and gastric cancer. Several studies over the last few years have shown an association between Hp and asthma, but it is a negative association. Hp seems to provide protection from asthma and I think that it is likely that the protection extends to allergies and autoimmune diseases. It is also noteworthy that analysis of genetic predisposition to gastric cancer only reveals polymorphism in genes associated with inflammation, e.g. IL-1 or TNF.

Hp Lives on Hydrogen from Gut Biofilms

Further evidence of the integral nature of Hp as part of the natural gut flora is its use of molecular hydrogen (H2) as an energy source, i.e. high energy electrons for its electron transport chain to produce ATP or to power membrane transport. The source of the hydrogen is Klebsiella in biofilms in the intestines. The hydrogen diffuses into the intestinal blood supply and is circulated to the stomach lining where it provides energy for Hp. Attacking gut biofilms may starve Hp and feeding starch (indigestible branch oligosaccharides are unique food source only accessed by Hp pullulanase) enhances Hp hydrogen nutrients. [Since regulation of the Hp genes is not thoroughly understood, it is also possible that ample starch could shut down nitrogenase and starve the Hp.]

Hp Increases Tregs

Allergies and autoimmune diseases point to problems in self/non-self recognition, i.e. immunological tolerance. And tolerance is dependent on regulatory T cells. In this context, it is interesting that Hp stimulates the accumulation of regulatory T cells. The gut is the major repository of cells of the immune system. It seems to follow that by elimination of the stomach Treg population by curing Hp infections, the body may be deprived of it major resource to suppress immunological responses to innocuous antigens in foods, pollens, etc. and to self antigens. Coupling a shortage of Tregs with chronic inflammation may lead to allergies and autoimmune diseases. Another source of Treg depletion that may further compromise the immune system is circulating LPS, endotoxemia, that is associated with obesity (and leaky gut?)

Erectile Dysfunction Diet

2009-10-22T11:34:00.002-06:00

Inflammation Leads to Hypertension, Nitric Oxide Inadequacy and Impotence

Drugs for erectile dysfunction (ED), e.g. sidenafil (Viagra), compensate for inadequate nitric oxide (NO) production from arginine by inhibiting the enzyme, phosphodiesterase (PDE5), that hydrolyzes the cyclic GMP that mediates the NO-triggered process of vascular dilation.

Inflammation Is the Core of ED

Drug treatment to compensate for inadequate NO production is a multibillion dollar industry that avoids curing the underlying cause of the ED. All of the physiological predispositions to ED result in or derive from chronic inflammation. The major cause of ED, hypertension, frequently as a result of kidney disease, diabetes or metabolic syndrome, can be treated with diet and exercise. Of course the typically recommended diet is essentially the Anti-Inflammatory Diet, compromised by the unenlightened persistence in the counterproductive use of grain starches, high fructose corn syrup, omega-6 polyunsaturated fatty acids and low saturated fat.

Decreasing Testosterone Results from Declining Health -- not Age

Recent studies also indicate that testosterone levels do not normally decline with age, but rather with declining health. Healthy men have higher testosterone levels. I would suggest that reduction in serum testosterone could be used as a measure of chronic inflammation in men. This also suggests that many of the symptoms associated with aging in men actually reflect increasing chronic inflammation and reduced testosterone.

ED Diets Are Just the Anti-Inflammatory Diet Plus Veggies

A chronic high starch/sugar/HFCS diet with omega-6 oils in place of saturated fats, leads to chronic inflammation, high triglycerides, risk of metabolic syndrome and obesity. Of course, diabetics have an even lower tolerance for this type of diet. This diet, which is rather typical in many modern cultures, also provides a high risk of damage to endothelial cells lining the circulatory system and to ED. The opposite of the inflammatory diet is the low carb, high omega-3 fish oil, no vegetable oil, meat/fish/dairy, Anti-Inflammatory Diet. This is supplemented with exercise and high vitamin D. Foods labeled as beneficial to ED also include specific herbs, spices and leafy vegetables, because these contain organic chemicals that inhibit components of the inflammation system or are anti-oxidants.

ED and Biofilms

I would suspect that men with ED suffer from chronic dietary inflammation and one of the consequences of this type of diet is the accumulation of pathogenic biofilms. Hypertension, which is a contributor to ED and a consequence of chronic inflammation, is also associated with periodontal biofilms and kidney disease (aggravated by renal biofilms.) I suspect that endothelial cells of capillaries are compromised by biofilm-derived endotoxins that ultimately contribute to apoptosis, decrease in capillary beds and elevation of blood pressure. All of these assaults on endothelial cells undermine penile vasculature and contribute to ED.

Viagra Can Lead to Rosacea

Men taking Viagra or other PDE5 inhibitors typically have compromised vascular systems that are the basis for ED. Increasing the response to NO in men with ED produces an increased risk of rosacea. Withdrawal from PDE5 inhibitors stops the rosacea, which returns if the PDE5 inhibitor use is reinitiated. Thus, the flush that is the goal of Viagra therapy, leaves some redfaced.

ref:
Ioannides, D. et al. (2009) Phosphodiesterase-5 inhibitors and rosacea: report of 10 cases. Br. J. Dermatol. 160: 719-20.

Migraine Headache Diet

2009-10-15T13:04:00.007-06:00

Simple Guidelines to Lower Chronic Inflammation and Avoid Pain

If I stick to this Anti-Inflammatory Diet and Lifestyle, I don’t get migraine headaches any more. I can still get a migraine, if I let myself get very dehydrated or drift into carbohydrate excess, but I am shocked when it happens. I can still enjoy chocolate and coffee. Avoiding the headaches is under my control and the diet is healthy and easy to follow.

Chronic Inflammation Is the Foundation for Migraine Headaches

The details and rationale for the Basic Anti-inflammatory Diet and Lifestyle are discussed in many articles on this blog. The guiding logic is that migraine headaches are based on chronic inflammation, although in each individual case there may be specific health problems that contribute and trigger migraines. If the chronic inflammation is removed, then migraines can’t happen or are reduced in frequency and/or severity.

Common Migraine Guidelines Point to Inflammation as the Problem

Feverfew is present on all of the lists of traditional treatments to avoid migraines. Extracts of feverfew contain parthenolide, a sesquiterpene lactone, that has been shown in mouse studies to inhibit activation of NFkB, the inflammation transcription factor. Stress reduction, acupuncture, etc. all point to vagal stimulation to reduce chronic inflammation. I would also recommend that migraine sufferers investigate vagal stimulation exercises to augment the basic diet and exercise to eliminate chronic inflammation.

Anti-inflammatory Diet in a Nutshell

Vitamin D -- deficiency is common... even with adequate sun exposure
Low carbs -- starch is hyperglycemic, grain gluten intolerance is very common
Vegetable oils -- only olive oil is safe (trans fats are dangerous), butter is better
Fish oil -- omega-3 oils can reduce chronic inflammation
High fructose corn syrup -- eliminate all sources
Saturated Fats -- safer than polyunsaturated fats, major source of calories

Typical Meals for a Healthy Head

Breakfast -- eggs, bacon, sausage, stewed tomatoes, cottage cheese, coffee, yogurt (low sugar, no HFCS) (avoid cereal, pancakes, waffles, toast, etc.)

example: scrambled eggs with sausage, yogurt (unsweetened, blended with fresh raspberries, strawberries or blueberries, sweetened with honey) coffee mocha

Lunch -- soup, salad, chicken, ham, tuna, vegetables, modest amounts of fruit, etc. (avoid bread, buns, potatoes, pasta, rice), keep the carbs to less than 50 grams

example: homemade chili with extra ham; thin sliver of toast loaded with feta cheese, broiled and drizzled with extra virgin olive oil; salad with peppers, tomatoes and cubes of jalapeno cheese, olive oil/vinegar, herbs/spices

Dinner -- fish, meat, vegetables, 50 grams of carbs (avoid grains)

example: broiled salmon with crushed pinenuts, garlic, butter and lemon; sauteed sliced zucchini/miniature squashes; wedges of small potatoes, microwaved ‘till soft and fried in light olive oil and butter; strawberries painted with melted dark chocolate

Why Conventional Diet Wisdom Gives You a Headache

The government food pyramid was designed by the food industry and was never supported by evidence from the biomedical literature. Research shows that saturated fats actually lower heart disease. Polyunsaturated fats in common vegetable oils are a major source of chronic diet-based inflammation. Starch/sugar raises triglycerides, not dietary fats. Grains are a major source of inflammation, because of the high incidence of gluten intolerance, the high content of hyperglycemic starch (even in whole grain breads, etc.) and in the support of gut biofilms based on Klebsiella, a contributor to Crohn’s and other autoimmune diseases. Blood lipid levels were not associated with heart disease and lowering these levels with statins does not improve health. Lowering inflammation uniformly improves health, as well as eliminating migraines.

Biofilm Transformation, Helicobacter, Klebsiella

2009-10-12T23:54:00.006-06:00

Helicobacter pylori causes stomach cancer, but it feeds on hydrogen gas produced by Klebsiella pneumoniae in gut biofilms. DNA released by biofilm bacteria not only transfers antibiotic resistance, but it also provides protection against host antibacterial peptides, such a cathelicidins and defensins.

Exploding Labs

When I was working on host/pathogen interactions and plant disease resistance, I also became familiar with research on the formation of the plant equivalent of cancer, crown galls, and symbiotic bacterial nitrogen fixation. I mention this, because this also exposed me to the free-living bacterial nitrogen fixing system in Klebsiella and to the memorable urban legion of exploding labs. As the story goes, as bacteria convert atmospheric nitrogen gas into ammonia, nitrogen fixation, they use high energy electrons, e.g. from ferrodoxin, and lots of ATP, but they also produce hydrogen gas. In labs where they are researching nitrogen fixation, the excess hydrogen gas would accumulate on the ceiling until... boom! Now those labs are properly vented.

Helicobacter Uses Hydrogen as an Energy Source

Helicobacter pylori is considered the most common bacterial pathogen of humans and is the primary cause of ulcers and stomach cancer. H. pylori lives in the stomach by neutralizing stomach acid with ammonia. Another interesting ability of this bacterium is its ability to use hydrogen dissolved in circulating blood as an energy source. The high energy electrons from molecular hydrogen are transported to its electron transport chain, and the energy is used in membrane transport and ATP production. The circulating hydrogen is produced by gut bacteria.

Klebsiella Is not just a Soil Bacterium, Gut Gases

Klebsiella pneumonia is a lung pathogen and it also forms gut biofilms. Presence in the gut and the ability to produce hydrogen gas has some implications for hydrogen utilizing bacteria like H. pylori. Clearly, the stomach of someone with an abundant source of hydrogen fuel in their blood is a better target for H. pylori colonization. This explains why even at age 50, individuals who were exclusively breastfed have a lower incidence of H. pylori and stomach cancer, since even a single bottle of formula can shift an infant to adult, i.e. Klebsiella gut flora.

Klebsiella Needs Carbs to Produce Hydrogen

K. pneumoniae has been associated with Crohn’s Disease and Ankylosing Spondylitis. It grows in gut biofilms and produces pullulanase, an enzyme that can utilize the branched glucosides left over from the action of amylase on plant starch. So K.p. has an untapped food source and it needs lots of ATP to produce hydrogen gas. The nitrogenase needed for nitrogen fixation and hydrogen production is very sensitive to oxygen, so this means that K.p. needs a partially anaerobic environment and must get its energy from fermentation. Fermentation yields much less ATP than respiration using oxygen, which means that K.p. can only produce hydrogen with lots of glucose from starch.

Low Carb Diet Cures Crohn’s Disease

It turns out that the antigen causing Crohn’s disease is the pullulanse (with collagen mimetics.) As you should expect, it has a basic triplet. Eating a low carb diet reduces the flareups of Crohn’s disease, presumably by starving out the K.p.. It is interesting that nitrogenase is the antigen involved in Ankylosing Spondylitis.

Biofilms Promote Transformation and Antibiotic Resistance

Just as a footnote to the benefit of K.p. as a citizen of a biofilm community, H.p. should also live in those biofilms, since that is the source of the hydrogen it uses. Biofilms also stimulate the exchange of DNA, because the quorum sensing chemical signals trigger the release of DNA. The DNA is a component in the matrix that binds bacteria in the biofilm and can work in conjunction with bacterial acidic polysaccharides and host heparan sulfate. These acidic polymers tend to bind the basic antimicrobial peptides, e.g. defensins and cathecidins produced as a major non-adaptive defense against bacteria. Thus, the release of DNA triggered by quorum sensing, builds matrix, facilitates DNA transformation that is the foundation for the spread of antibiotic resistance in gut biofilms and provides resistance against antimicrobial peptides.

Cytstic Fibrosis Overproduces Tissue Transglutaminase and Contributes to Celiac

2009-10-06T11:00:00.004-06:00

Tissue transglutaminase (tTG or TG2) is produced in excess in some diseases, such as cystic fibrosis, and contributes to inflammation and disease symptoms. tTG also readily moves in and out of cells by virtue of its basic triplet and when in the cytoplasm, tTG is ubiquinated and degraded by proteosomes. I have previously pointed out that internalization and proteosome degradation are also the initial steps in processing of proteins for presentation by the immune system and antibody production, i.e. turning a cellular protein into an autoantigen involved in autoimmune disease.

Here is an image of a computational protein model of tTG I drew with Chimera. I have highlighted the basic triplet to show its exposure to facilitate transport.

Oxidative Stress Alters tTG and Triggers Inflammation

A recent article also links tTG intracellular chemical modifications (SUMOylation), which are linked to oxidative stress, to activation of NFkB and inflammation. Thus, tTG is a major player in controlling cell surface interactions with potentially toxic materials such as polyglutamine-rich gliadin, as well as triggering inflammation in response to oxidation stress.

Cystic Fibrosis Causes Overproduction of tTG

When I read that cystic fibrosis results in an increase in the production of tTG in lungs, I immediately thought of the role of tTG as an autoantigen in celiac disease and the progression of celiac into Hashimoto’s thyroiditis, which has the same autoantigen, tTG. I suspected that the overproduction of tTG and inflammation in cystic fibrosis should increase tTG autoantibody production and tTG-mediated autoimmune diseases of celiac and Hashimoto’s thyroiditis.

Extra tTG Leads to Autoimmune Celiac

A quick PubMed search of CF and celiac, revealed a study of comorbidity between CF and celiac in Norway. Just as expected, the two diseases occur together with a frequency three times higher than predicted by coincidence. CF stimulated tTG overproduction was driving the development of celiac.

references:
Luciani A, Villella VR, Vasaturo A, Giardino I, Raia V, Pettoello-Mantovani M, D'Apolito M, Guido S, Leal T, Quaratino S, Maiuri L. SUMOylation of tissue transglutaminase as link between oxidative stress and inflammation. J Immunol. 2009 Aug 15;183(4):2775-84.

Fluge G, Olesen HV, Gilljam M, Meyer P, Pressler T, Storrösten OT, Karpati F, Hjelte L. Co-morbidity of cystic fibrosis and celiac disease in Scandinavian cystic fibrosis patients. J Cyst Fibros. 2009 May;8(3):198-202.

Paradoxical Inflammation

2009-09-28T00:53:00.006-06:00

Anti-inflammatory Treatments Cause Inflammation in Some Diseases, e.g. Rosacea

I thought that the anti-inflammatory diet and lifestyle I outlined on this blog would be a general purpose starting point for the treatment of all diseases. Inflammation is the foundation for allergies, autoimmune diseases and cancer. Inflammation is a basic defense against infectious diseases and many tissues require signaling components integral to inflammation for their normal function, so it is possible to overdo anti-inflammatory treatment and produce immuno-suppression. But that is unusual. What I am talking about here is inflammation caused by vitamin D, omega-3 oils, potentially low carbs and inhibitors of NFkB, such as tumeric. This is Paradoxical Inflammation.

Rosacean Inflammation Is Paradoxical

The obvious example of a paradoxical inflammatory disease is rosacea. Rosacea seems to be a large group of diseases that manifest in facial inflammation. Excessive flushing of the face can become persistent and form pustules and swelling. The triggers for rosacean inflammation are legion and idiosyncratic. They include mundane social interactions, numerous foods, temperature extremes and, paradoxically, just about everything that I recommend to decrease chronic inflammation.

Bacteria in Tissue and Gut Biofilms Are Candidates

Why do otherwise anti-inflammatory foods and exercise make rosaceans red in the face? Even vagal stimulation that is uniformly calming to inflammation, can make a rosacean flush. This is very inconvenient. I can only invoke the typical players: cryptic bacteria, biofilms, vagus nerve stimulation and response, lymphocytes/macrophages, cytokines and neurotransmitters.

All rosaceans have demonstrated facial inflammation and have had long term exposure to antibiotics and NSAIDs. That combination suggests that bacteria have been transported from a leaky gut (NSAIDs) to the site of inflammation (the face). It is likely that cryptic bacteria inhabit the dermis near the blood vessels and resident lymphocytes/mast cells. This is also the location for axons from vagus nerves. Thus, vagus stimulation may result in the release of neurotransmitter acetylcholine to stimulate lymphocytes/mast cells with subsequent release of cytokines. In this case the cytokines are inflammatory.

Other sources of inflammatory cytokines are lymphocytes/mast cells activated by endotoxin release from cryptic bacteria triggered by immunological attack. In this case, the immunological attack can be initiated by disruption of the stasis invoked by the cryptic bacteria.

Activated Cryptic Bacteria Are Source of Inflammation

It is hypothesized that the cryptic bacteria remain in tissue, because they are able to induce a hibernation-like physiology in the tissue. Disruption of the hibernation would initiate an immunological assault. Disrupting agents typically include vagal stimulators, such as activators of the hot or cold sensors, e.g. capsaicin, castor oil or menthol. Interestingly, the cryptic bacteria require a residual level of inflammation to acquire nutrients from the host. Anti-inflammatories that inhibit NFkB may destabilize the bacterial/host interaction and result in an immunological attack on the bacteria. All of the attacks on the cryptic bacteria release inflammatory endotoxin.

Gut Biofilms Store Bacteria Recruited to Become Cryptic in Inflamed Tissue

During the course of the disease and following numerous antibacterial treatments, bacteria can be continually recruited from safe havens, such as gut biofilms. Antibiotic treatment of biofilms converts the biofilm community to antibiotic resistance through activated horizontal gene transfer. Moreover, harsh treatment of biofilm communities initiates shedding of bacteria that could migrate across the leaky gut adjacent to the gut biofilms and provide new emigrants into the inflamed face tissue. A likely resident would be Chlamydia pneumonia, which has been demonstrated to be carried by macrophages and offloaded at distant sites of inflammation.

How the Vagus Becomes Inflammatory

This brings up the question of why vagal stimulation shifts from anti-inflammatory to inflammatory in rosaceans. I don’t think that the vagus nerves change in either their activation or in the neurotransmitters that are released as a result of stimulation. This means that the cells that respond to the vagal acetylcholine must be changed. I think that the change is a depletion of Treg cells and the result is that acetylcholine receptors on the remaining T cells cause a release of inflammatory cytokines. These cytokines cause the release of NO by endothelial cells and vasodilation. Leaking of endotoxin from the resident cryptic bacteria causes persistent dilation and restructuring of the vasculature.

Helminth and Il-2 Therapy Reestablish Tolerance and Reverse Vagal Inflammation

Since I have been forced to explain paradoxical inflammatory diseases, I might as well speculate on exotic approaches that already suggest potential treatments. Ingesting parasitic worm eggs (helminth therapy) has proven successful in the treatment of inflammatory diseases such as asthma, allergies and IBDs. Interleukin 2 (Il-2), usually used as a complex with an anti-Il2 antibody, is also a productive treatment. In both of these cases, the treatment stimulates the proliferation of Treg cells, which appear to be deficient in many of the inflammatory diseases. These treatments should also lead to a lowering of inflammation in the gut and suppression of inflammation as a result of vagal stimulation. Inhibitors of acetylcholine receptors, e.g. scopolamine patches, might also be interesting to test to see if they inhibit rosacean flushes in response to typical vagal stimulants such as castor oil or menthol.

Addendum: Another possibility associated with the heavy use of antibiotics by rosaceans is intestinal (biofilm?) candidiasis. Yeast infections are common after prolonged antibiotic treatment. Interestingly, Candida produces resolvins from omega-3 fatty acids and the resolvins suppress neutrophil activity that would attack the yeast. Thus, many of the anti-inflammatory treatments would actually aggravate yeast infections and contribute to rosacea. Treatment for candidiasis (keeping in mind that yeast may be protected by biofilms) helps many rosaceans. Stripping biofilms may be useful if pro- and pre-biotics are used to displace Candida.

Vagus Nerve Controls Gut Inflammation II

2009-09-17T17:49:00.009-06:00

Inflammatory Mast Cells Silenced

In a previous article, I outlined the role of the vagus nerve in responding to infection/damage signals by producing signals that inhibit inflammation. In a recent article (ref. below), the role of the vagus nerve in gut inflammation was examined using real-time biophotonic labeling. Basically that means that a video camera sensitive to infrared can be used to detect infrared dyes produced when NFkB is activated -- the camera is able to visualize regions of inflammation in living mice. Using this technique, researchers were able to demonstrate that cutting the vagus nerve produced heightened inflammation in gut treated with an irritant. The vagus nerve appears to stimulate regulatory T cells that lower the activity of inflammatory cells.

Inflammation/NFkB Activation Visualized in Live Mice

The studies were performed in a mouse line constructed to express an infrared fluorescent protein in cells in which the inflammation transcription factor, NFkB, is activated. Mice of this strain were prepared with and without the vagus nerve intact leading to the intestines. The mice were then exposed to sodium dextran sulfate (DSS) to simulate inflammatory bowel disease symptoms.

Cutting the Vagus Nerve Permits Inflammation

Mice with intact vagus nerves exhibited much less inflammation in their gut than those without vagus innervation. The cut vagus experiments demonstrated that the vagus nerve was responsible for suppressing inflammation. Further experiments were performed to determine if the inflammatory and anti-inflammatory reactions could be transferred to other mice by transferring cells from the treated mice.

Regulatory T Cells (CD4+, CD25+) Block Inflammation

Transfer experiments showed that inflammatory T cells (CD4+, CD25-) from cut vagus, DSS mice would cause bowel inflammation in other mice, but that did not happen with the same type of cells from mice with intact vagus nerves. Further tests showed that either cutting the vagus or adding inflammatory T cells from a mouse with a cut vagus, reduced the population of regulatory T cells (CD4+, CD25+) in control mice treated with DSS. So, without the vagus stimulation, the regulatory T cell population declined in the presence of inflammatory signals.

Absence of Regulatory T Cells Can Explain Many Inflammatory Diseases

In many inflammatory diseases, e.g. celiac, Crohn’s disease, rosacea, there appears to be a deficiency of regulatory T cells. In the absence regulatory T cells, signals from vagus nerves will no longer produce anti-inflammatory suppression. In fact the same nerve signals may become inflammatory. This would explain why rosaceans will become inflamed by hot or cold stimulation that would normally lead to anti-inflammatory stimulation of regulatory T cells. Similarly, capsaicin, castor oil and menthol, which normally produce an anti-inflammatory response, produce inflammation in rosaceans.

[Vagal stimulation exercise links: here and here.]

reference:
O'Mahony C, van der Kleij HP, Bienenstock J, Shanahan F, O'Mahony L. 2009. Loss of vagal anti-inflammatory effect - in vivo visualization and adoptive transfer. Am J Physiol Regul Integr Comp Physiol. Aug 12. [Epub ahead of print]

Watson Makes Us Sick

2009-09-10T18:23:00.008-06:00

Common Textbook: Molecular Biology of the Cell, Lacks Coverage of Critical Molecular Interactions

One of the major reasons why healthcare practitioners are unable to cure diseases, is that their molecular view of disease is outdated. Their models of key signaling interactions lack critical molecules and fundamental types of chemical bonds are ignored.

The Major Textbook Used to Train Medical Students Lacks Essential Cellular Interactions

The most pervasive and perhaps the best text book on cell biology, The Molecular Biology of the Cell, first authored by James Watson, lacks a discussion of the bonding of aromatic amino acids (tryptophan, tyrosine, phenylalanine) with basic amino acids (arginine, lysine), carbohydrates, and aromatic phytochemicals, e.g. plant antioxidant or alkaloids. As a result, medical school graduates lack familiarity with the prominent interactions that dominate disease and drug treatments.

Hydrophobic Bonding to Aromatic Amino Acids Dominates Cell Molecular Biology

The dominating significance of aromatic hydrophobic bonds is the strength of these bonds, ca. 20 kcal/mol versus, the commonly considered weak bonds (hydrogen, ionic) at 1-2 kcal/mol, the same as the kinetic energy of water at body temperature. Thus, structures, such as alpha helices and beta sheets of proteins, require multiple weak bonds to be stable, but the hydrophobic bonding of tryptophan to a single arginine draped across its surface is stable.

Examples:

Tryptophan is the most highly conserved amino acid in protein structures (more than cysteine forming disulfide bonds!). This means that tryptophan is the most important amino acid in protein structure, and probably determines how proteins fold.

Carbohydrates have hydrophobic faces to their ring structures and typically bind to lectins, glycosidases and glycanases, via the hydrophobic surfaces of tryptophans or tyrosines in active sites.

Transport of proteins into nuclei is by binding of arginine or lysine residues of nuclear localization signals (basic quartets or neighboring basic pairs) to tryptophan hydrphobic residues projecting from the surface of LRR (leucine-rich repeat) importin molecules.

Heparin binds to basic amino acids in proteins via hydrophobic interactions. Aromatic dyes, such as berberine, bind to heparin through similar hydrophobic interactions.

Heparin binds to the basic amino acids arrayed in stacks of amyloid molecules and berberine blocks these interactions. Congo Red, a diagnostic dye for amyloids, is an aromatic molecule. Similar interactions occur with prions and the plaques of atherosclerosis.

Acidic polysaccharides form the matrix of biofilms. Heparin and nucleic acids can also serve this function. PEG, which disrupts hydrophobic interactions, can be used to disrupt binding of proteins to heparin, nucleic acids and biofilm polysaccharides.

Heparin binding mediates the interaction between most growth factors or cytokines and their cell surface receptors.

Many viruses and bacteria bind to cell surfaces via heparan sulfate.

LDL binds to LDL receptors via heparan sulfate. ApoE in diagram (arg and lys in blue, hydrophobic in pink.)

Antimicrobial peptides, e.g. defensins, have groups of basic amino acids. Heparin binding domains excised from proteins as peptides are antimicrobial.

Stomach proteases cleave around heparin-binding domains to produce antimicrobial peptides. Intestinal proteases cleave within heparin-binding domains and inactivate bacterial and viral agglutinins.

Life starts with heparin, i.e. heparin is leaked into fertilized eggs to remove the small, highly basic proteins used to package the sperm chromosomes.

Heparin is injected experimentally into nerves to silence IP3 signaling based on the binding of the hydrophobic face of inositol to basic amino acids, similar to heparin binding domains, of the IP3 receptors located on the surface of the ER.

The cytoplasmic domains of some receptor proteins have basic regions that interact with the IPs of the membrane surface, but subsequently serve to transport membrane-derived vesicles to the nucleus via importin carriers.

Heparin/heparan sulfate proteoglycans are secreted bound to basic molecules such as polyamines or histamine.

Heparan sulfate proteoglycans are continually secreted and taken up with a half life of six hours. This circulation is a major transport system of most cells. Amyloid/heparan aggregates on the surface of nerves and gliadin/tTG/antibody/heparan complexes on endocytes (celiac) may poison this system.

All allergens and autoantigens have a triplet of basic amino acids that may be involved in the initial aberrant presentation of these antigens as a result of the internalization by the carbohydrate-binding domain of mannose receptors on the surface of inflammation-stimulated immune cells.

Many neurotransmitters bind to their receptors via hydrophobic, aromatic interactions. These same receptors interact with hydrophobic, aromatic phytochemicals, e.g. “anti-oxidants.” Many spices, herbs, alkaloids and other phytochemicals have their abundantly complex interactions via these mechanisms.

Crystals of the tryptophan repressor involved in binding tryptophan and altering the expression of genes involved in tryptophan synthesis, shatter in the presence of tryptophan -- the tryptophan (yellow) strongly binds to basic amino acids (blue) in the tryptophan-binding domain of each repressor protein in the crystal and alters its shape.

Cure for Inflammatory Diseases

2009-09-02T16:13:00.013-06:00

Destabilizing Gut Biofilms by Simple Remedies

The intercommunication between the gut flora biofilms, the cells of the immune system juxtaposed with the intestinal endothelium and cryptic bacteria/tissue biofilms produces stable chronic inflammatory disease. Disrupting the gut biofilms may permit a resumption of effective immunity and remission.

Disrupting Biofilms to Treat ASDs

Cristian Stremiz brought to my attention the work of Dr. Anju Usman on the treatment of autism spectrum diseases by attacking inflammatory gut biofilms.

A Panacea

This approach, based on the use of common food components, to attack the gut biofilm matrix of acid polysaccharides, cations and proteins, should be generalizable to most inflammatory diseases. The interventions also provide facile explanations for the utility of numerous traditional cures such as vinegar, fiber, glucosamine, pectin, whey, proteases and probiotics.

Cures Act via Gut Flora Biofilms

There are numerous anecdotal reports of traditional, simple remedies working for essentially all diseases. Tantalizingly, many of these diseases are also occasionally successfully treated with antibiotics. The common thread seems to be the involvement of inflammatory gut flora and perhaps cryptic bacteria residing in the tissues displaying symptoms. Glucosamine works sometimes for arthritis, but little of the glucosamine that is eaten reaches the blood stream and the aching joints that seem to become less inflamed. Vinegar, pectin, and fiber have also been attributed with curative powers, yet none is likely to impact inflamed joints directly. Impacting gut biofilms is much easier to explain.

Biofilms of Bacteria Attached to Acidic Polysaccharides and Divalent Cations

Acidic polysaccharides are produced by bacteria and divalent cations cross-link the polysaccharides into a matrix. The bacteria have agglutinins to attach to the matrix. Gut pathogens produce agglutinins that they use to attach to the heparan sulfate (HS), the predominant acid polysaccharide of the intestinal epithelium. Mast cells of the intestines normally release heparin, which is a mixture of HS fragments, to stick to the agglutinins and block attachment to the HS of the epithelium. Numerous bacterial species form complex communities on the polysaccharide matrix and prevent access by antibiotics. Biofilms require 100X the antibiotic concentrations and a cocktail of different antibiotics to eradicate the bacteria.

Biofilms Disrupted by Competing Acid Polysaccharide Fragments and Cation Chelators

The Achille’s heal of biofilms is the ionic interaction between the acidic polysaccharide and divalent cations. This interaction can be attacked by both small fragments of similar acid oligosaccharides, by organic acids that can solubilize the cations, e.g. acidic acid in vinegar, or by chelators, such as EDTA. All of these treatments can remove the calcium, magnesium and iron that is essential to the matrix. Small molecules, such as glucosamine, chondroitin sulfate fragments, heparin, and pectin, can disrupt biofilms. Molecules that bind to heparin or nucleic acids, e.g. berberine, quinine (tonic), methylene blue, should also be effective in disrupting biofilms. [Note that the similarity between amyloid production and biofilms, means that treatments should overlap.] Lactoferrin is effective, since it both binds iron and binds to acidic polysaccharides via its heparin-binding domains.

Proteases Cleave Agglutinins

Stomach proteases, e.g. pepsin, specifically cleave proteins to release heparin-binding, acidic polysaccharide-binding domains that inhibit biofilm production in the stomach. Subsequently, the basic, antimicrobial peptides and agglutinins are cleaved by proteases, e.g. trypsin, that hydrolyze the binding domains. Eating proteases, such as nattokinase present in fermented soybeans, dissolves intestinal biofilms by attacking the agglutinins. The pathogenic E. coli and avian H5N1 also have these agglutinins. It is, therefore, wise to avoid establishing gut biofilms that can immobilize pathogens.

Probiotics Protect Against Biofilms

Resident gut bacteria that produce organic acids, e.g. lactic acid or acetic acid, provide protection against biofilm formation. Examples are the bacteria present in common forms of fermentation and food preservation, e.g. Lactobacillus sp., and the bacterium present in exclusively breastfed babies, Bifidobacter sp. Formula fed babies rapidly develop inflammatory biofilms, which explains their high rates of intestinal and respiratory diseases, as well as increased rates of inflammatory diseases.

Biofilm Inflammation Results in Inflammatory Bowel Disease, etc.

Gut biofilms support system-wide chronic inflammation that leads to allergies, autoimmune diseases, degenerative diseases and probably cancers. This attach on the gut also produces a leaky gut that supplies the bacteria that a moved by macrophages of the gut to all parts of the body. This may be how Chlamydia pneumoniae colonizes sites of inflammation throughout the body.

Attacking Gut Biofilms Is the First Step in the Treatment of All Inflammatory Diseases

Many inflammtory diseases, e.g. chronic lyme disease, rosacea, may be refractory to treatment with antibiotics, because of the reservoir of bacteria in gut biofilms. Attacks on gut biofilms with relatively non-intrusive treatments, such as vinegar, EDTA, lactoferrin and proteases, may lower the total resident pathogen load and make subsequent antibiotic treatment more effective.

Anti-inflammatory, Gluten-Free Diet for Celiac

2009-08-18T16:58:00.005-06:00

Low Grain Is Good for Everyone

I don’t think that I have an intolerance for grain, i.e. a gluten sensitivity, but it is so common and the biochemistry is so obvious, that it is only prudent to avoid wheat and related grain products. A low or gluten-free diet is also similar to the other common healthy diets, e.g. low carb and anti-inflammatory.

Gluten-free diets came to my attention recently in two ways. First, I saw Food, Inc., a documentary movie about abuses by multinational food processors. After that movie, I felt like I was a goose being readied for foie gras. Second, was a newspaper article on the expense of a gluten-free diet and the challenges of avoiding gluten.

I haven’t had to worry about wheat contaminating my diet, but I am sympathetic to the celiacs that I know who have to labor with a sloppy and exploitative food industry that uses the cheapest ingredients to compose the processed foods that are consumed in modern diets -- processed foods are complex blends of many different potential allergens from innumerable sources throughout the world.

A Celiac Diet Is Good for All
Fortunately, the answer to pervasive gluten is just a modest modification of the basic anti-inflammatory diet that I recommend on this blog. Unfortunately, people who have already developed gluten intolerance, have probably had the problem for years before diagnosis and that means that their intestines have already suffered major physiological alterations and they have problems absorbing nutrients and vitamins. Celiacs also, because of their chronic inflammation and autoimmunity, tend to readily develop food allergies and other autoimmune diseases. The recommended anti-inflammatory diet will help to avoid celiac, put celiacs into remission and avoid development of subsequent allergies and autoimmune diseases.

Vitamin D Is Usually Deficient (and a source of inflammation)
The basic anti-inflammatory diet starts with a return to optimal vitamin D with the use of an initial blood test, followed by high level supplements to reach a suitable level and then maintenance with D3 supplements of usually 2,000-5,000 IU per day. Depending on the D3 supplement, vitamin A will also need to be supplemented, because it interacts with vitamin D. Remember that sunshine is only effective in producing adequate vitamin D if you do not suffer from chronic inflammation. I would assume that all celiacs tend to be vitamin D deficient.

A Low Carb Diet Is Easier for Celiacs
The next component of the basic diet is low carbohydrates, that means a minimum of high glycemic foods, which means to avoid sugar and starch, do not cook vegetables more than necessary and don’t over-chew your veggies. This is good for celiacs, because it reduces the need for common grain foods that no one should eat: bread, cereal, pasta, etc. Everyone should lower their consumption of these wheat products in solidarity for celiacs and for general good health. Cereal is a very bad idea for children!

Most Vegetable Oils Are Unhealthy
Most vegetable oils contribute substantially to world-wide inflammation and celiacs don’t need the added burden of inflammatory omega-6 vegetable oils. Only olive oil and butter should be used. Saturated fats are safer than typical polyunsaturated vegetable oils.

Eat Wild Fish or Tons of Fresh Flax
Most people eat too little omega-3 long chain fatty acids, since these are most abundant in fatty fish, such as wild salmon (farmed fish are fed corn and have reduced omega-3 and increased omega-6 fats.) Few vegetable sources are available, since the omega-3 fatty acids are unstable and present in leaves rather than seeds. Flax seeds have short chain omega-3 fatty acids and must be freshly ground and consumed by the cupful, because the conversion to the long chains, in which they are useful, is very inefficient. Most celiacs will need to use fish oil (or krill oil, if fish is not tolerated) supplements (4-8 EPA/DHA capsule per day taken in a meal rich in fats for bile uptake) to balance the ubiquitous inflammatory omega-6 in their diets.

Grassfed Meat/Eggs Are Your Friends
Celiacs should seek out grass/pasture fed meats, eggs and wild caught fish. Corn-fed animals have higher levels of omega-6 fats and these contribute to dietary inflammation. Celiacs can usually eat meat and fish and these are very healthy foods. Red meat was not shown to contribute to degenerative diseases, it was the high carbs eaten with the meat that produced the inflammation that contributed to heart disease. (Remember that statins only decrease cardiovascular disease because they inadvertently lower inflammation, not because they lower serum lipids, LDL.)

No, No’s: HFCS and trans fats
High fructose corn syrup and trans fats are inflammatory and unhealthy for anyone, and should be avoided as much as wheat gluten. Fruits should be eaten as seasoning, since their fructose is not healthy and they also contain ample sucrose.

Most People Would Be Healthier on a Celiac Diet
The anti-inflammatory diet proposed here for celiacs should be uniformly healthy, since it provides optimal vitamins (D, C, B12, etc.), low starch/sugar/carbs, an optimal omega-3 to -6 fatty acid ratio, increased meat and saturated fats, and avoids HFCS and trans fats. The only major adjustment for celiacs would be avoidance of individual food allergens, more attention to vitamin supplements to compensate for poor absorption and replacement of wheat by rice, potatoes, etc. The low carbohydrate nature of the diet makes it more approachable, since typical carbs, such as bread and cereal are avoided and replaced with meat and vegetables.

I look forward to advice and suggestions from readers who have experience with gluten-free diets.

Recombining H1N1 and H5N1 Is Very Scary

2009-08-16T01:33:00.008-06:00

Avian Flu Acquired a Basic Internalization Domain in the 1990’s

Avian flu was simply for the birds until its hemagglutinin (the H or H5N1) acquired an extra four basic amino acids that provided another way into human cells.

Basic Amino Acids Accumulate in the Hemagglutinin

During the early 1990’s isolates of avian flu, H5N1 started to appear that eventually developed six basic amino acids in a stretch about 340 residues from the amino terminus. These basic amino acids are thought to be an adaptation to decrease inactivation by a host protease.

H5N1
~PQRE TRGLFG~ ABB88379 Mexico 1994
~PQRK TRGLFG~ ABQ84472 Italy 1993
~PQRK ETRGLFG~ ACH88842 USA 1993
~PQRKRKRKTRGLFG~AAC58990 Mexico 1995
~PQRE RKKRGLFG~ ABQ84473 Italy 1997
~PQRERRRKKRGLFG~ AAD37782 China 1996
~PQRK RKTRGLFG~ ACL79965 Mexico 1994
H1N1
~PSIQ SRGLFG~ AAF87275

The red area is the region that has accumulated the basic amino acids (R and K). Note that the novel H1N1, does not yet have this region.

The New Basic Region Looks Like an Internalization Signal

Those who have followed this blog know that I have an interest in heparin binding domains, groups of basic amino acids (K for lysine and R for arginine) of proteins that bind the common acidic extracellular polysaccharide heparin. Most recently I have been focusing on unusual triplets of basic amino acids that are found in the proteins of allergens and autoantigens. These basic triplets are similar to the basic quartets that are used as signals to move proteins from cytoplasm into the nucleus of cells, i.e. the nuclear localization signal (NLS).

Basic Sextet for Internalization and More

The newly evolved basic sextet, RRRKKR, should be readily transported into cells by the mannose receptor and then taken into the nucleus, because it would also act as a NLS. This should also mean that the new H5N1 viruses with this hemagglutinin should attach to numerous cells of the immune system and potentially transported to other areas of the body.

Is this Dangerous?

I don’t know what the likelihood of recombination between H5N1 and H1N1 is if a bird, pig or human is infected with both nor is the impact of acquisition of the basic sextet by H1N1 on virulence known, but the acquisition of the basic sextet occurred at the same time that H5N1 moved from birds to people and became lethal.

Basic Sextet May Explain New Entry for H5N1

H5N1 has recently been found to infect tissue that lack the sialic acid sugars that are the typical target for avian flu. The new targets are not known. I would start to suspect the mannose receptor that I have postulated to be involved in initiation of allergy and autoimmunity.

Cause of Autoimmunity and Allergies

2009-08-07T15:03:00.003-06:00

Inflammation-Induced Presentation by Mannose Receptor

Speculation on how innocuous proteins become the targets of our immune systems, and result in allergies, asthma, celiac, arthritis, lupus, and other inflammatory autoimmune diseases.

Inflammation

It all starts with chronic inflammation. In most cases diet is the predominant source of inflammation, but infections (bacterial, viral, fungal) may also contribute. Inflammation sets the stage for faulty processing of proteins at the focal location where lymphocytes and antigen-presenting cells are congregating.

Antigen Presentation

At this point a major mistake occurs. Cells that have been alerted to danger by inflammatory cytokines, start to internalize and process proteins in the vicinity, so that peptide fragments of the “antigens” can be displayed on their surfaces in the clutches of major histocompatibility complex proteins. The problem arises when self proteins are internalized, processed and displayed as candidate peptides.

Basic Triplets of All Allergens and Autoantigens

I have looked at dozens of allergens and autoantigens, and they all have one peptide sequence in common, a triplet of basic amino acids. The impetus for this article was finding this morning that one of the autoantigens for Hashimori’s thyroiditis is thyroglobulin, the serum carrier of thyroid hormones. I checked the sequence of thyroglobulin and it has two of the rare basic triplets.

Thyroiditis Autoantigen Is Thyroglobulin

I checked the Pubmed literature to see if thyroglobulin is naturally taken up by cells, since I have been trying to figure out the receptor for basic triplets. What I found was an article on the binding of thyroiditis autoantigens to mannose receptor. The idea of the paper was that the autoantigens in this case were heavily glycosylated and maybe the mannose receptor that is involved in antigen presentation of glycosylated antigens, would bind the selected autoantigens as well. I didn’t get much past the title of the paper.

Candidates for Allergen/Autoantigen Receptor

What I put together was the fact that antigen presenting cells use mannose receptor to internalize antigens. This places this receptor in the right place and the right time to be a receptor for basic triplets. But what would a basic triple binding protein domain look like and could mannose receptor be a candidate?

Structure of Thyroglobulin

I downloaded a structure for mannose receptor and began looking for a surface region that would bind carbohydrates, aromatics and basic amino acids. The critical amino acids in all of these cases are aromatic amino acids, phenylalanine, tyrosine and tryptophan. Tryptophan is at the heart of most carbohydrate binding proteins. Since the mannose receptor is a member of the carbohydrate-binding lectins, I expected to find tryptophans on the surface of the mannose receptor. I just looked for the tryptophan that binds the carbohydrates. It was exactly as I predicted. The mannose receptor should be able to form very stable tryptophan/arginine-like ladders with basic triplets.

I made a figure of the mannose receptor bound to a carbohydrate (red and grey). One of the exposed tryptophans (yellow) is bound to the carbohydrate. Several other tryptophans could be exposed and oriented toward a basic amino acid spread over the surface of the tryptophan in place of the carbohydrate. A similar kind of structure is used by importin to transport proteins with nuclear localization sites (NLS, basic quartets) into the nucleus. Many proteins with NLSs are also autoantigens, e.g. lupus.

I think that the peculiar circumstances that lead to allergy and autoimmunity result in the binding of self-proteins or allergens to mannose receptor and result in antibody production.

Why the Mannose Receptor?

Why does the mannose receptor make mistakes? The exposed tryptophan may also bind numerous plant products. In fact, the phytochemicals, e.g. alkaloids, flavonoids and terpenoids, are an abundant and varied group of chemicals that would bind to the exposed tryptophan of the mannose receptor and compete for binding with basic triplets. Protection offered by plant “anti-oxidants” may be due in part to this activity.

Inflammation and Vitamin D Deficiency

2009-08-05T00:21:00.006-06:00

Does Dietary Inflammation Reduce Skin Production of Vitamin D?

The media discovered the vitamin D deficiency pandemic last week. Amazingly researchers were recorded on camera saying that the D deficiencies are caused by insufficient exposure to ultraviolet in sunlight and inadequate consumption of vitamin D-laced milk. Have all of these people been avoiding the biomedical journals?

Have they noticed that my tan improved since I started eating anti-inflammatory?

Let’s shine some sunlight on these knowledge deficiencies:

Serum vitamin D levels have been dropping (as chronic inflammation has been increasing) over the last three decades -- has something changed in our diets?
Vitamin D deficiencies occur globally (not restricted to Northern latitudes or winter) -- related to diet?
Women are more vulnerable, because of cultural modesty in some countries, but males are still D-deficient.
A subset of people exposed to ample sunshine are still D-deficient.
Vitamin D deficient individuals also have elevated TNF.
Vitamin D deficiency and inflammation are risk factors in the same diseases.

It seems that the simplest conclusion is that chronic inflammation leads to vitamin D deficiency, even though vitamin D deficiency may also contribute to inflammation.

This also probably means that chronic inflammation makes it harder for skin to produce vitamin D during exposure to sunlight.

One would expect those who are inflamed to get sunburned more readily and people who eat plenty of omega-3 rich seafood probably produce more vitamin D, even if they are not in the sun as much.

Inflammatory starvation (or American fast food) diets high in starch and omega-6 vegetable oils, should produce vitamin D deficiency even on the Equator.

We should not be surprised that inflammatory degenerative diseases are associated with vitamin D deficiency. It would be interesting if vitamin D supplementation to eliminate deficiency, reduced inflammation and reversed degenerative disease.

Do statins reverse vitamin D deficiencies (and improve tanning) as they lower inflammation? [Statin lowering of LDL is unrelated to reduction in cardiovascular disease. Only the anti-inflammatory side-effect is important.]

Does NFkB activation (inflammatory signaling) inhibit vitamin D receptor activity and vice versa?

You can forget all of this confusion, if you just stick with an anti-inflammatory diet (that includes vitamin D supplements) and exercise frequently in the great outdoors.

Brilliant Blue Brains and Spinal Cords

2009-08-02T00:13:00.010-06:00

Hibernation-Suppression and Trauma-Induction of Inflammation

Inflammation/hibernation is a complex story at the foundation of chronic diseases. Inflammation is the common thread -- activation of the inflammation transcription factor NFkB.

Trauma Causes Life-Threatening Trauma

Trauma, everything from a bee sting to a horrific traffic accident that causes head and spine injuries, results in initial tissue damage and subsequent inflammation damage. The inflammatory response to punctures and abrasions is usually appropriate and self-limiting. The immune response to serious injuries is frequently more life-threatening than the initial damage.

Transplanted Organs Suffer from Inflammation

Organs removed for transplantation are subjected to a certain amount of necessary trauma and oxygen deprivation. If the organ was simply popped into a waiting recipient biochemically unaware of the process, the initial damage would be readily repaired in its new home. Unfortunately, some of the organs overreact and become damaged by their own immune/inflammatory reaction to the surgery.

Hibernation Reduces Trauma Inflammation

Organ transplants between animals that are hibernating, are much more successful, because the damaging inflammation is suppressed. Hibernation in animals or in human organs can be induced by the use of opioid peptides, e.g. DADLE, and subsequent surgical procedures are more successful. Hibernation also provides protection against experimental stroke. Apparently, the activation of the opioid receptor suppresses activation of NFkB and avoids inflammation.

Opioids and Steroid Hormones Block NFkB Activation and Inflammation

Steroid hormones can also provide protection against inflammatory damage resulting from head trauma. Thus, the ubiquitous steroid receptors may also block NFkB activation and inflammation.

Trauma Releases ATP that Triggers P2X7 and NFkB

Extracellular ATP can activate NFkB activity and inflammation, and ATP accumulation at trauma sites may be particularly dangerous for spinal injuries. Inhibitors of ATP binding to the purinic receptor P2X7, block inflammation and provide dramatic improvement in the return of function in animal models of spinal injuries. Most of the common inhibitors of P2X7 signaling must be injected directly into the traumatized tissue to block inflammation, because they can’t cross the blood-brain barrier. An exception is Brilliant Blue G.

Brilliant Blue G Blocks Trauma Inflammation

Brilliant Blue G, a.k.a. Coomasie Brilliant Blue, should be very well known to molecular biologists, because it is the commonly used stain for proteins separated on SDS-PAGE gels. I used that dye literally thousands of times to stain gels and I even tried it to stain the extracellular matrix surround cartilage-secreting cells, chondrocytes, grown in culture. I have included one of those pictures just for old times sake.

BBG can be injected IV into mice and the result is amazing. Not only do the mice become blue, but they recover much better from experimental spinal trauma. BBG in the blue mice blocks inflammation due to the surge in tissue ATP and the mice heal their trauma and regain function.

It would be amazing if BBG worked on people with spinal injuries. I expect the rapid development of a suitable drug to help spine and head trauma patients.

Can Manipulation of Hibernation Cure Chronic Diseases?

A big question is whether or not similar drugs might be used to block inflammation that supports cancer and other forms of chronic illness. Alternatively, in some instances the problem is that bacteria are suppressing local inflammation and inducing tissue hibernation to produce chronic illness. Under these circumstances, the induction of local inflammation or elimination of hibernation may make the bacteria vulnerable to attack.

references:
Borlongan CV, Hayashi T, Oeltgen PR, Su TP, Wang Y. Hibernation-like state induced by an opioid peptide protects against experimental stroke. BMC Biol. 2009 Jun 17;7:31.

W. Penga, M. L. Cotrinaa, X Hana, H Yua, L. Bekara, L. Bluma, T. Takanoa, G.-F. Tiana, S. A. Goldman and M. Nedergaard. 2009. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. PNAS 106:12489

National Healthcare and Diet

2009-07-25T10:39:00.006-06:00

Barack Obama's Anti-Inflammatory Breakfast Pulpit

Trying to improve the health of Americans by taking the advice of the healthcare industry is futile. Barack Obama must set the example of a healthy lifestyle.

The solution is to prevent the diseases that the healthcare industry is using as a source of profits and that means fundamentally changing diets and lifestyles. It has taken three decades to attack health by shifting from meat protein and saturated fats to starch, high fructose corn syrup and polyunsaturated (omega-6) vegetable oils. It will take a sustained, personal effort by President Obama to guide a relatively rapid return to a healthy, anti-inflammatory diet.

All of the degenerative and autoimmune diseases that form the core of current healthcare diagnosis and treatments are rooted in an inflammatory diet and lifestyle dictated by agribusiness and uninformed by science. The media nags about people eating too much and exercising too little. Our obese population is encouraged to lose weight by eating less. Food fat is demonized. Statins are prescribed with religious zeal to lower blood lipids to reduce cardiovascular disease. All of this “health” advice is wrong and unsupported by the biomedical literature.

It is about time for an authority figure, i.e. The President of The United States of America, to use some leadership skills and teach people how to eat and live. That would be much easier than trying to get doctors to order fewer tests from their own medical test companies or order fewer images through their own imaging companies. Are the pharmaceutical companies going to suggest that their pills should cost less and be pushed less frequently? Will the insurance companies step out of their lucrative middleman role between doctor and patient? It is more reasonable for The President to use his bully pulpit to change the U.S. diet and lead us back to health.

All that is needed is for President Obama’s image at the breakfast table to be judiciously used by a private, non-profit organization on a website:

Barack’s Breakfast

This would provide an opportunity for the President’s health agenda to be presented to the world through his prescription (and explanation of health benefits) for each morning meal:

Slow food
Local food
Low carbon footprint
Low carbs
High omega-3 to -6 fatty acid ratio
Praise eggs and saturated fats
Warn about grains

Vitamin D deficiency
No HFCS
No trans fats

Each meal would come with a source and description of each ingredient and its benefits. YouTube videos of the meal preparation could show the techniques needed. Occasionally The President could be seen enjoying the meal and animation could be used to show why hypoglycemic ingredients were used. Maybe The President would show solidarity to the diabetic victims of industry food fights by getting his finger pricked for a blood sugar test after a meal. It would be good to see him complain about the inaccuracy of several different typical meters. Imagine the close-up of all of the lancet marks needed to convince him that the readings are making sense!

This single approach would cost the American people nothing to implement and would save billions of dollars in healthcare expenses over a few years, as citizens of all socio-economic classes changed to diets that were less inflammatory, and degenerative and autoimmune diseases quickly declined.

Low Carbs Lower Triglycerides

2009-07-21T16:00:00.006-06:00

Blood Triglycerides Depend on Diet Carbs, not Fats

I don’t know why the medical community keeps pushing the low fat diets to modify blood lipids. The medical literature shows that a low fat, high carbohydrate diet (more than 50 grams of starch/sugar in a meal) produces high triglycerides, and a low carb diet ( less than 50 grams per meal), regardless of saturated fats and meat, produces lower triglycerides.

In seems reasonable that fats in the diet should mean fats, triglycerides, in the blood, but that ain’t so. It’s the rise in blood insulin in response to a rise in blood sugar due to high glycemic index foods in a meal, that yields high blood triglycerides.

The low carb, low triglycerides facts of life were brought to may attention by my wife’s blood chemistry. She knows better, but refuses to follow my preacherly suggestions about an anti-inflammatory diet. She follows most of the use of supplements and prohibitions about vegetable oils, but loves carbs. She eats two thick slices of bread in a sandwich and I cut a thinner slice and eat mine open-faced. I can’t eat her pancakes or French toast. Ok, I eat lots of dark chocolate, but I don’t have flavored syrup in my lattes.

She was stressed by a high triglycerides (292 mg/dl) in her blood work and her doctor wanted to start her on meds. I was sympathetic. Not really. I actually said, “carbs, carbs, carbs,” until she threatened me. I nagged heavily to just junk the junk and wait on the meds. She started counting grams of carbs with each meal. Actually she tried to average over the whole day, I nagged, she finally relented and stuck to the plan. No more than 50 grams of carbs in any meal. (I think 30 grams, would actually be better.)

One month later, her blood work showed triglycerides down 57% to 127 mg/dl. Individual results may vary, but this is pretty straightforward. Carbs are important -- avoid them. The food pyramid is for chumps. The highest glycemic food you will encounter is a French baguette (95), compared to pastas in the 30s or table sugar at 70.

The facts are:

Saturated fats in meat are no big deal, and much better than...
Vegetable oils (most are rich in omega-6 oils, except olive oil) are inflammatory.
Fish oil (omega-3 DHA/EPA) is anti-inflammatory (unless there is also too much vegetable oil.)
Starch and sugar increase blood triglycerides and are only needed to gain or keep body fat. Losing weight is much easier without starch/sugar.
Most people are deficient in vitamin D and C (even with plenty of solar exposure).
High fructose corn syrup is ten times more damaging than starch/sugar, and is especially bad for diabetics. It doesn’t raise blood sugar as much as starch, it just causes damage, e.g. glycation, at an extraordinarily high rate. It also ages skin by accelerating cross-linking of collagen. Very bad stuff even in fruit juices.
Eating plant anti-oxidants protects unsaturated fats as they pass through the oxidizing environment of the stomach, so nuts are better unroasted and eaten with veggies.

Autoimmunity, Allergies and Basic Triplets

2009-07-21T14:13:00.003-06:00

Basic Triplets Only in Primate Forms and Allergens

I have examined the proteins, autoantigens, that are the focus of a dozen autoimmune diseases and a similar number of allergens. All of these proteins have basic triplets that I previously associated with heparin-binding. I have had two recent revelations. First, these triplets appear to not be involved with heparan sulfate proteoglycans for internalization. In fact, HSPGs don’t appear to be involved, even though the process is inhibited by heparin. So this suggests a transport system, perhaps using the LDL receptor or the mannose-6-phosphate receptor, (or a protein with an acidic triplet or quartet.)

The second interesting observation is that the mouse, cat, dog, etc. versions of the human autoantigens lack the basic triplets. This suggests that these diseases cannot occur in non-primates by the same mechanisms. So what is the role of these basic triplets in humans? They didn’t evolve to cause problems under unusual conditions of chronic inflammation, so what is their adaptive advantage?

I think that the answers to these questions could yield the identification of a fundamental cellular transport process and associated cellular phenomena that could be worth a Nobel prize in medicine and it could be unravelled by a group of high school kids doing some straightforward bioinformatics.

Oh. I just remembered that IL-1 beta, the inflammatory cytokine, has a basic triplet. I just checked NCBI, and that basic triplet is found in all of the mammalian IL-1 betas. Oddly, the soluble receptor for IL-1 beta has an acidic quartet. I looked up the protein structure of the IL-1 beta bound to the IL-1 receptor and did a quick illustration using Chimera:

I put the surface on the IL-1 beta and left the IL-1R wrapping around it in as a ribbon. The basic triplet of IL-1 beta is in blue and the acidic quartet of the receptor is in red. One of the basic amino acids is stabilized by hydrophobic bonding over the face of a tyrosine, in yellow. Clearly, there is a simple ionic, plus-minus charge, bonding between the basic and acid amino acids. I don’t normally see this interaction between basic heparin-binding domains and other proteins. Other proteins that bind to heparin-binding domains use aromatic amino acids to make hydrophobic bonds with the hydrophobic arms of the basic amino acids, e.g. importin and nuclear localization signals or tryptophan/arginine ladders. The use of simple acid-base connections (with the projection of each stiffened by adjacent prolines) shows that this interaction is selected to be irreversible.

I don’t know all of the ramifications of basic triplets, but they are very important and are the basis for most modern allergic and autoimmune diseases.

Celiac Causes Allergies and Autoimmune Diseases

2009-07-17T23:58:00.005-06:00

Anti-Tissue Transglutaminase Can Lead to Hashimoto’s Thyroiditis

Celiac, gluten intolerance, causes intestinal inflammation and immunological presentation of the common intestinal protein, tissue transglutaminase (tTG). The result is anti-tTG autoantibodies that stimulate an immune attack on intestines and other tissues.

Heparan-Binding Proteins Involved in Autoimmunity and Allergy

Those familiar with my blog know that I am obsessed with heparin-binding protein domains. The reason that I am focused on these parts of proteins, is because most cells rapidly sweep heparan sulfate polysaccharides across their surfaces from sites of secretion to sites of internalization. During inflammation, proteins with strong heparin-binding domains, consisting of triplets of basic amino acids, e.g. KRK (lysine-arginine-lysine), are internalized along with the heparan sulfate. The result is an aberrant presentation of these internalized proteins to the immune system and production of inappropriate antibodies, e.g. autoantibodies.

Basic Triplets in Hasimoto’s Autoantigens

One of my hobbies is checking for the unusual occurrence of basic triplets in autoantigens and allergens. I have found dozens of examples. The most recent is associated with Hashimoto’s Thyroiditis. I knew that attack on the thyroid was common in celiacs, because the celiac autoantigen tTG (it has a basic triplet) is also present in the thyroid and the celiac autoantibodies to tTG also cause an attack on the thyroid. But the autoantigen for Hashimoto’s Thyroiditis is thyroid peroxidase (TPO).

I was momentarily perplexed, but then examined the TPO amino acid sequence and immediately found a couple of basic triplets (KKR and KRK).

MRALAVLSVTLVMACTEAFFPFISRGKELLWGKPEESRV
SSVLEESKRLVDTAMYATMQRNLKKRGILSPAQLLSFSK
LPEPTSGVIARAAEIMETSIQAMKRKVNLKTQQSQHPTD
ALSEDLLSIIANMSGCLPYMLPPK...

Hashimoto’ Thyroiditis Linked to Celiac

Then, I did a PubMed search for “celiac and Hashimoto’s”. As expected, there is a recent paper (see below) that shows that celiac commonly leads to Hashimoto’s Thyroiditis.

An obvious explanation is that the initial attack on the thyroid by anti-tTG autoantibodies of celiac leads to thyroid inflammation and presentation of TPO, with a second round of autoantibodies produced to TPO resulting in Hashimoto’s Thyroiditis. Celiac may be the initial autoimmune trigger for many other autoimmune diseases and allergies.

Autism has been associated with maternal autoimmunity and placental abnormalities. Guess where tTG is found in high abundance?

reference:
Bardella MT, Elli L, Matteis SD, Floriani I, Torri V, Piodi L. Autoimmune disorders in patients affected by celiac sprue and inflammatory bowel disease. Ann Med. 2009;41(2):139-43.

Chronic Disease, Cryptic Infections, Hibernation

2009-07-16T00:09:00.008-06:00

Suppression of Inflammation and Surviving Cytokine Storms

There are numerous unanswered questions in modern medicine. What is aging, for example? Why do people become more inflamed as they age? What’s with all of the chronic, degenerative diseases? Why is lipid metabolism (LDL, HDL, triglycerides) linked to degenerative diseases, along with immune system function and inflammation? I am only going to start the answers here.

I might as well continue to be cryptic and give you the string of words/concepts I am trying to connect to answer the other questions:
Hydrogen sulfide (H2S), endorphins, hibernation, nuclear receptors (PPARs), antibiotics, chronic inflammatory diseases (fibromyalgia, arthritis, chronic fatigue, Lyme, Morgellon’s, Alzheimer’s, prostatitis, pancreatitis, cancers, etc.), autoimmunity, leaky gut/kidney/brain barrier, autism and H1N1.

First a word of advice: Beware of assuming that molecules are specific, i.e. with unique interactions, and that a small molecule will bind to one and only one protein target. [There are lots of bizarre exceptions to the assumption: Aldolase acts as a structural protein for Toxoplasma motility. Fluorescein is added to make protein fluorescent, but the fluorescein is also transported into cells on its own, i.e. fluorescein and rhodamine labeling can give different results. Heparin binds to most extracellular proteins and it is mostly a hydrophobic interaction -- heparin is not just for clotting anymore.]

Observations from the literature:

Maternal autoimmunity is linked to autism.
Autism is linked to leaky gut and chronic inflammation.
Gut/kidney/brain barriers are based on integrity of extracellular matrix (heparan sulfate) that is compromised by inflammation.
Chronic diseases require inflammation and circulating inflammatory cytokines (TNF, IL-1, IL-6) are elevated..
NSAIDs induce leaky gut and release of bacteria toward liver.
Phagocytosis of bacteria leads to transport of some bacteria, e.g. Chlamydia pneumoniae to other sites of inflammation, e.g. gut to joints.
Opiods can induce hibernation in rodents.
Sulfides can induce hibernation in rodents.
H1N1 my cause lethal pneumonia by lung cytokine storm.
Inflammatory cytokines and inflammation result from activation of NFkB.
Hibernation involves PPARs (another nuclear receptor transcription factor).
Omega-3 fatty acids reduce inflammation via COX-2 prostaglandins, but also by binding to PPARs.
For most of the diseases under consideration, suppression of inflammation will eliminate symptoms.
Antibiotics can impact all of these diseases in unpredictable ways. In some cases complete remission can be achieved and in other cases antibiotics can produce lethal cytokine storms.
Bacterial cell wall components, e.g. lipopolysaccharide, lipid A, are intensely pyrogenic, i.e. inflammatory.

Cryptic Bacteria in our Tissues

The role of bacteria in numerous diseases, including cancers, has been proposed since the early isolation of bacteria from human tissues. Many of these bacteria are difficult to culture and have variable forms viewed by microscope. Because these bacteria are difficult for microbiologists to handle with conventional approaches, their existence and significance has always been questioned. Use of antibiotics to treat chronic, inflammatory conditions has seemed inconsistent with the unproven existence of a bacterial cause. Thus, there is surprise when the inappropriate use of antibiotics leads to a cure.

Cryptic Bacteria Suppress Local Inflammation and Promote Chronic Inflammation

I think that the fundamental problem is the assumption that human tissue is sterile, i.e. free from microorganisms, such as bacteria, unless there is overt infection. Part of the sterile assumption derives from the intense inflammatory response to bacteria. In order for bacteria to survive in tissue, the bacteria must suppress inflammation and the tissue must tolerate the slow leaching of inflammatory bacterial materials.

Chronic Disease Hypothesis

Based on the cryptic bacterial infection hypothesis, many, if not all chronic diseases are initiated by inflammatory events that release bacteria into the blood stream carried in phagocytic cells. The cells migrate and take up residence at a region of inflammation. The bacteria produce molecules that produce tissue hibernation and quell local inflammation in response to the bacteria. The bacteria are, however, a source of ongoing irritation to the tissue and a chronic inflammatory disease results.

Eradication of Cryptic Bacteria

Antibiotics would be a typical choice for killing infecting bacteria. In the case of cryptic, chronic infections, however, application of therapeutic antibiotics may be problematic. The established infections may have produced privileged locations isolated from the vascular system and protected by a bacterial community, e.g. a biofilm. Alternatively, the death of the bacteria and release of pyrogenic factors my produce life-threatening inflammation, that requires careful support.

Hibernation in Rodents Provides Treatment Clues

The compromise of tissue inflammation in response to cryptic bacteria is similar to the physiology of rodent hibernation. In both cases, systemic inflammation is suppressed. At the cellular level, this means that other signaling pathways silence the inflammatory NFkB expression pattern. One of the major nuclear receptors that is activated in hibernation is PPAR. PPAR is activated by opiods and H2S, which also induce hibernation in rodents. There are numerous analogs, inhibitors and H2S donors that could be used to disrupt hibernation (free local suppression of inflammation) or reduce symptoms by suppressing systemic inflammation.

Inflammation Compromises Tissue/Blood Barriers

Inflammation causes a disruption of the integrity of the endothelial extracellular matrix at sites of local inflammation. NFkB activation shuts down the expression of genes involved in heparan sulfate proteoglycan (HSPG) synthesis makes the tissue/blood barrier leaky. Locally this facilitates the recruitment of lymphocytes and neutrophils for defense, but systemically it leads to leaky gut/kidney/brain barriers that permit bacteria to cross.

Convergence of Therapies to Attack Cryptic Infections

The central approaches to attack cryptic infections are a combination of antibiotics and suppression of cytokine storms. These approaches are used in Marshall’s Protocol [http://bacteriality.com/ ], which also exploits a vitamin D receptor antagonist, Olmesartan, that also inhibits NFkB and inflammation.

A similar protocol has been developed by Dr. Michael Powell to inhibit hibernation and attack cryptic infections:
http://www.faqs.org/patents/app/20090163448

These approaches are similar to the lengthy use of antibiotics for the treatment of chronic Lyme disease.

It is very interesting to note that some of the most effective treatments for a long list of degenerative chronic diseases, autoimmune diseases and cancers, use essentially the same protocol that should attack cryptic bacteria and provide support for ensuing inflammation.

Flu Susceptibility and Anti-Inflammatory Fish Oil

2009-07-07T11:05:00.003-06:00

Omega-3 Oils Reduce Inflammation, but May Increase H1N1 Infection Risk

The goal seems to be to reduce inflammation and reduce disease, but it isn’t that simple. Inflammation is not bad. Chronic inflammation is the problem for degenerative diseases. After all, inflammation is just what we call the mobilization of our immune system to fight infection. The problem is that inflammation needs to be properly controlled to be invoked only when needed, to be kept localized and to be brought to a proper conclusion.

A recent article extended studies of fish oil and various types of infections, to influenza. It used a mouse model that focused on the local, lung aspects of flu infection. Some mice were fed fish oil in a 4:1 ratio to corn oil (fish group) and the controls were just fed corn oil (corn group), as the lipid part of the diets.

Both fish oil and corn oil groups got sick when exposed to flu virus. The lungs of the fish treated group were less inflamed, but there was more virus and an increased death rate. The fish oil effectively reduced inflammation, but the inflammation in the corn oil, inflamed, mouse was useful in controlling the spread of the virus. Does this mean that chronic dietary inflammation is protective?

How close does this mouse system model human H1N1 infections? A lot can be learned from animal models, but not all aspects of the human disease are reflected in this model. There is no single H1N1 strain, for example. Flu viruses mutate thousands of times faster than even the most variable bacteria. Thus, people in various parts of Asia may be experiencing a different H1N1 than people in South America. Some H1N1 infections involve organs other than the lungs and cytokine storms can also be deadly.

If H1N1 is raging, is fish oil a good idea? It would be prudent to reduce other sources of inflammation, by eating an anti-inflammatory diet and getting plenty of exercise. The answer would seem to be to use only enough fish oil to reduce remaining symptoms of chronic inflammation, e.g. aching joints. The mouse model may have reduced the ability to produce an inflammatory response beyond elimination of chronic inflammation.

Most people who eat a high carb diet, with the typical inclusion of vegetable oils, starch and high fructose corn syrup would probably benefit from fish oil supplements, even in the context of influenza risk. It would take a lot of fish oil to compensate for the other inflammatory parts of their diet. Obesity is both a symptom of dietary inflammation and a source of chronic inflammation. Reluctance to engage in physical activity is another indicator of inflammation.

It would be helpful if epidemiologists studying the H1N1 swine flu pandemic would determine if chronic inflammation is a risk or benefit in surviving the disease. It would also be helpful to know what simple dietary or other interventions, e.g. nicotine, caffeine, would be helpful for various symptoms of the disease.

ref:
Schwerbrock NM, Karlsson EA, Shi Q, Sheridan PA, Beck MA. Fish Oil-Fed Mice Have Impaired Resistance to Influenza Infection. J Nutr. 2009 Jun 23. [Epub ahead of print]

An Autoantigen for Pancreatitis

2009-07-04T00:24:00.007-06:00

Pancreatic Secretory Trypsin Inhibitor (PSTI) Has Internalization Basic Triplet

Pancreatitis is an inflammation of the pancreas resulting from lack of adequate inhibition of proteases. Autoantibodies against PSTI would explain some forms of pancreatitis.

I was researching the maintenance of baby gut flora by mother’s milk, when the reference discussed here was brought to my attention by my wife, who happens to be a lactation consultant. The paper showed that PSTI is present in colostrom, the first milk that a baby gets, before the true milk comes in. PSTI protects the new gut from digestion by its own pancreatic proteases, since PSTI is a protease inhibitor that sticks to the gut.

I naturally assumed that PSTI stuck to the gut by heparin-binding domains that would stick to the heparan sulfate proteoglycans on the gut surface. [Recall that it is via these HSPGs that viruses and bacteria infect the gut and the HSPGs in turn are protected during infections by the release of heparin from mast cells. The heparin in the guts of cattle and pigs are used to make commercial heparin to block blood clotting.] So I looked up the structure (above, with basic amino acids in blue and basic triplet on right) sequence of human PSTI at NCBI:

>gi|190694|gb|AAA36522.1| PSTI
MKVTGIFLLSALALLSLSGNTGADSLGREAKCYNELNGCTKIYD
PVCGTDGNTYPNECVLCFENRKRQTSILIQKSGPC

The basic triplet (RKR,arg-lys-arg), from my perspective, should result in presentation to the immune system during high levels of inflammation, and as a consequence result in autoantibodies against PSTI. The result would be the neutralization of the protease inhibitor and damaging production of active protease to attack the pancreas, i.e. pancreatitis.

It would be fairly easy to test this hypothesis by looking for the anti-PSTI antibodies in some people with pancreatitis. Other autoantibodies, e.g. against tissue transglutaminase, might also be checked, because the inflammation that produced one autoantibody may produce others and both PSTI and tTG are produced in the intestines. In fact, celiac may be the cause of some cases of autoimmune pancreatitis.

Note added in proof:

I just checked the literature on PubMed and found that PSTI is in fact an autoantigen in pancreatitis and produces antibodies against PSTI:
Raina A, Greer JB, Whitcomb DC. Serology in autoimmune pancreatitis. Minerva Gastroenterol Dietol. 2008 Dec;54(4):375-87.

and
I found that pancreatitis is often found associated with celiac (gluten intolerance):
Patel RS, Johlin FC Jr, Murray JA. Celiac disease and recurrent pancreatitis. Gastrointest Endosc. 1999 Dec;50(6):823-7.

ref:
Marchbank T, Weaver G, Nilsen-Hamilton M, Playford RJ. Pancreatic secretory trypsin inhibitor is a major motogenic and protective factor in human breast milk. Am J Physiol Gastrointest Liver Physiol. 2009 Apr;296(4):G697-703.

Diet, Carbs, Fat and Weight Loss

2009-06-30T14:57:00.003-06:00

Glucose and Insulin Are Required to Gain or Retain Body Fat

The essential fact of weight gain or loss is that fat (triglycerides, TGs) stored in fat cells (adipocytes) is made from glycerol (from glucose) and fatty acids (from the blood or recycled from previously stored fat droplets.) Adipocytes must import glucose to make glycerol and TGs.

Glucose Is a Carbohydrate

Glucose is a sugar or carbohydrate. Once again the word tells the story, i.e. carbohydrates consist of carbons that each have a hydrogen, -H, and a hydroxyl -OH attached. Since each glucose has six carbons, then it can be roughly approximated as (H-C-OH)6. This is convenient notation, because this lets glycerol be considered as a three carbon sugar, i.e. (H-C-OH)3.

Glycolysis Can Convert Glucose into Two Glycerols

The central metabolism of all cells is called glycolysis, literally breaking glucose. The products of glycolysis are usually high energy electrons (carried by NADH), two pyruvates and ATP (chemical energy). Active cells with access to oxygen can use their mitochondria to accept the high energy electrons and pyruvate, and generate lots of ATP and CO2. Fat cells can bring in glucose by glucose transporters, make glycerol instead of some of the pyruvate and use the rest of the pyruvate by mitochondria to make fatty acids (instead of ATP.)

Fats Have Three Fatty Acids Attached to Glycerol: Triglycerides

Fats, triglycerides, are made in a series of biochemical reactions catalyzed by enzymes. The enzymes first make glycerol phosphate from glycerol and NADH, from glycolysis. Then the fatty acids are attached. The fatty acids can come from three sources: from glycolytic pyruvate, from fatty acids offloaded from the blood stream and from constantly recycled stored fat. Regardless of the source of fatty acid, new glycerol phosphate is needed for TG production in fat cells

Insulin Is Required for Glucose to Enter Adipocytes

Glucose is transported into adipocytes by specialized proteins embedded in their cytoplasmic membranes. A few glucose transporters are always present to supply enough materials and energy for basic maintenance of the adipocytes. Even with very high blood sugar (glucose), the glucose uptake of the adipocytes will not be increased without an increase in the number of glucose transporters in the cytoplasmic membrane. Insulin released into the blood by the pancreas in response to an increase in blood sugar, stimulates the adipocytes to introduce more glucose transporters into their membranes and glucose is actively transported into the adipocytes. The adipocytes then convert the glucose into glycerol, fatty acids and ultimately stored fat.

Without High Blood Sugar and Insulin, Adipocytes Lose Fat

Fatty acids in adipocytes are constantly be converted to fats and then released from storage as they are enzymatically removed from the fats to reappear as free fatty acids. As a consequence, the absence of extra glucose in the adipocytes will mean a shortage of glycerol and a net accumulation of free fatty acids from stored fats. Excess fatty acids will mean that fatty acids will be removed from rather than deposited in adipocytes.

Weight Gain Occurs with High Blood Sugar Regardless of Fats Eaten

Fat will be produced in adipocytes if there is high blood sugar and insulin production, because the fat already stored in the adipocytes will be recycled into fat, any fat in the diet will be converted into stored fat and glucose transported into adipocytes will be converted into fat. In addition, protein in the diet and potentially in muscle, will also be converted into stored fat.

Weight Loss Occurs with Low Carbohydrate Diets Regardless of Fats Eaten

Fats that are eaten without sufficient carbohydrates to cause a rise in blood insulin, are metabolized for energy in liver and muscle. Excess fat ends up being secreted into the gut by the gall bladder and lost as feces. Type I diabetics without insulin cannot get enough glucose into their cells to make fat and cannot gain weight without insulin. The amount of glucose, as simple carbohydrates (sugar or starch), needed to raise blood sugar to trigger a rise in insulin production is typically 30 to 50 grams per meal. Hunger, a response to a drop in blood sugar, prior to the next meal is a typical indication of insulin production and fat storage.

Lyme Spirochete Binds to Heparan in Blood Vessels

2009-06-26T01:49:00.002-06:00

Borrelia burgdorferi Sticks to Host Cells via Heparin-binding Proteins

A research group at the University of Calgary has watched the binding of fluorescent Borrelia burgdorferi spirochetes, the Lyme disease pathogen, to the surface of blood vessels in mice. (ref. below) A small heparin molecule was shown to block this interaction between the spirochete surface protein BBK32 and the heparan sulfate proteoglycans of the endothelial cells of the skin blood vessels.

The heparin-binding domains in the spirochete protein, BBK32 are easy to spot in the amino acid sequence of this protein that I downloaded from the NCBI protein database:

>gi|19072701|gb|AAL84596.1| BBK32 [Borrelia burgdorferi]
MKKVKSKYLALGLLFGFISCDLFIRYEMKEESPGLFDKGNSILET
SEESIKKPMNKKGKKIARKKGKSKVSRKEPYIHSLKRDSANKSN
FLQKNVILEEESLKTELLKEQSETRKEKIQKQQDEYKGMTQGSL
NSLSGESGELKETIESNEIDITIDSDLRPKSSLQDIAGSNSISYTDE
IEEEDYARYYLDEDDEDDEYYEDDYEEIRLSNRYQSYLEGVKYNV
DSAINTINKIYDTYTLFSTKLTQMYSTRLDNLAKAKAKEEAAKFTK
EDLEKNFKTLLNYIQVSVKTAANFVYINDTHAKRKLENIEAEIKTL
IAKIKEQSNLYEAYKAIVTSILLMRDSLKEVQGIIDKNGVWY
Basic amino acids are K=lysine, R=arginine

The minimal heparin binding pairs, e.g. KKVKSK are shown in red and the strong heparin-binding triplets, e.g. KRK, are shown in blue. Notice that one triplet is augmented with several pairs to further enhance heparin binding.

I would also expect that BBK32 would be internalized into host cells and transported into the nucleus, where it may alter transcription, a la HIV-TAT. The existence of multiple, strong heparin-binding domains may also serve to bind the BBK32 (or the spirochetes) to multiple different heparan sulfate proteoglycans and interfere with the HSPG circulation system. This may have a toxic effect.

reference:
Norman MU, Moriarty TJ, Dresser AR, Millen B, Kubes P, Chaconas G. Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host. PLoS Pathog. 2008 Oct 3;4(10):e1000169.

The Cause of Allegies and Autoimmune Diseases

2009-06-24T12:14:00.004-06:00

Keyhole Limpet Hemocyanin (KLH): Internalized Antigen

Scanning the literature for a common protein that can be used as an experimental antigen, it becomes quickly obvious that a favorite is KLH. This would seem to be an odd choice -- why a keyhole limpet protein? But that is the wrong question.

Why is KLH such a good antigen, i.e. why is it readily presented to the host immune system? If you have been reading my posts, you might be thinking about triplets of basic amino acids and that is the answer.

As soon as I remembered the prominent use of KLH as an antigen, I checked the NCBI protein database and immediately found an unusual KKK (triple lysine) near the amino terminus of hemocyanin II ( it comes in two pieces). This triplet explains why KLH is such a good experimental antigen, because it is internalized into antigen presenting cells by its strong heparin-binding domain. Other components, adjuvants, are typically added to the KLH for injection to make sure that a strong local inflammation occurs.

Autoantigens Have Strong Heparin-Binding Triplet

I also learned that Hashimoto’s thyroiditis is an autoimmune disease mediated by the autoantigen thyroid peroxidase. A quick search reveals that thyroid peroxidase is an autoantigen, because it also has a triplet of basic amino acids that can enhance presentation under inflammatory conditions. Grave’s disease of hyperthyroidism is an autoimmune disease in which the thyroid receptor (with a basic triplet) is an autoantigen. The same kind of triplet of basic amino acids was found when I searched today for fire ant antigens and mosquito antigens.

I have also looked for the triplets in protein databases. The triplets are rare in cytoplasmic and extracellular proteins. The proteins that have triplets are usually identified as autoantigens in some disease. The triplets are common in nuclear proteins, since heparin-binding and nucleic acid-binding share the same basic amino acid domains. The nuclear internalization signal also results in rapid cellular internalization, e.g. HIV-TAT, heparanase, IGF-binding proteins. Nuclear proteins are common autoantigens in lupus.

Inflammation Plus Heparin-Binding Internalization: Allergy, Autoimmunity

Chronic inflammation can produce antibodies against proteins (foreign or self) with strong heparin-binding domains (triplets or sometimes neighboring pairs of basic amino acids, lysine or arginine). The generalization explains why particular proteins in pollens, foods, insects, pets, mites, asthma, MS, lupus, celiac, etc. produce antibody responses.

Diet, Nutrition and Health

2009-06-23T14:12:00.005-06:00

These are my generalizations (some would say prejudices) from 40 years of experience in plant biochemistry and molecular biology:

Plant Secondary Compounds Are Defensive and Toxic

The development of plant secondary compounds (all of the compounds that are not part of metabolism or structures) is in response to pathogens, herbivores and pollinator/disseminator attractants -- development of these compounds has nothing to do with humans. Examples: Nicotine and caffeine are very toxic to herbivores and are present in plants for protection. Humans learn to play with toxic plant chemicals, just as they have learned to play with fire and explosives.

Plants Are Not There For Us

People have learned to exploit local plants for protection against local human pathogens, but there is no selective advantage to plants (except for domesticated plants) for useful plants to grow near humans. This logic would suggest that rats and mosquitoes, that flourish near human habitations, are there because of their human utility. Human live near places were useful plants grow.

Grains Are Unhealthy

One of the biggest problems with food processing is separating the inflammatory parts = starch and omega-6 oils, from the nutritive parts, the so-called anti-oxidants, vitamins, proteins, etc. Grains, even so-called whole grains, are simply too enriched for starch and inflammatory oils to be healthy. They are not safe to eat in large amounts. Leafy plant parts are healthy, but even those parts are not good in large amounts from a single plant species. Humans are browsers, because the plant secondary compounds are uniformly toxic, but can be tolerated better in a mixture of different toxicities.

Starch Is Inflammatory

Starchy foods should be treated like a fish. The starch should be pared away and discarded, like the fish gut and bones. (The guts and bones could actually be processed to make them nutritious. Not so with the starch. The starch should be fermented.) The potato skin should be eaten and the rest discarded, just as an aphid secretes as honey dew the extra sugar it sucks in from a plant leaf.

Cereals Are Inflammatory

Breakfast cereals are a dietary abomination. They contribute immensely to obesity, inflammation and chronic disease. Oatmeal for cardiovascular health is a total fraud. The fiber might be useful, but the high starch causes cardiovascular disease. Grains/cereal are the foundation of the chronic disease pyramid.

Fructose is Toxic

Fruit juices are another fraud. The juice (fructose) should be removed and discarded. The fructose is very unhealthy. Mice are given type II diabetes for research purposes by feeding them fructose (especially high fructose corn syrup.) Fructose is avoided in the beef industry, because it causes rapid cross-linking of collagen and leads to tough meat. The same thing happens in humans who eat fructose, it causes aging of the skin and other tissues. High fructose corn syrup is a commercial addiction -- it is hugely profitable as a sweetener -- and that is why it is still used, even though it is grossly unhealthy. It will eventually be removed from the market after the industry is protected from subsequent law suits. It is equivalent to the tobacco industry -- too lucrative to eliminate.

Phytic Acid

The active ingredient in fiber that provides its benefits is phytic acid, the same chemical that people are trying to eliminate. Phytic acid acts as a chelator. I don't think it is actually a problem. The problem comes from extracting cations from the phytic acid before it is eaten. Phytic acid should go in saturated, so that it doesn't contribute to deficiencies. The actual problem is that the diet is already low in minerals, because of eating processed foods that are mineral deficient.

Enzymatic Detoxification: P450, Glycosylation and Secretion

Humans are adapted to plant secondary metabolites by the abiltiy to enzymatically detoxify [using p450 and glycosylating (adding glucuronic acid)] and secrete the toxic compounds. These chemical modifications that occur in the intestines and liver are usually effect. They also work on drugs and that is how we eventually clear these compounds from our systems. Grapefruit and black pepper inactivate these enzymes and alter the way we metabolize plant toxins and drugs. The detox enzymes can also convert innocuous compounds into toxins and carcinogens. That conversion is the basis for using liver enzymes in the Ames Test for carcinogens. The activity of the enzymes is dependent on recent diet, so it would make sense to gradually change the amount and type of vegetables that are eaten in a meal to permit the detox system to adjust.

Glucose and Insulin Cause Fat Accumulation

Fat accumulation is dependent on dietary carbohydrates and insulin. Fat and serum lipids accumulates with a high carbohydrate diet and decrease on a low carbohydrate diet. This is more important than the number of calories consumed.

Inflammation Not Serum Lipids Cause CVD: Statin Are Unnecessary

Inflammation is the source of chronic degenerative diseases. Serum lipids are only secondary factors. Statins lower serum lipids, but do not impact cardiovascular health unless they also lower inflammation. Lowering inflammation lowers serum lipids and decreases cardiovascular disease. Statins appear to be a very expensive way of treating cardiovascular disease dependent on their side effect on inflammation. Modest dietary and lifestyle changes are much more effective, cheap and safe than statins.

Insulin-like Growth Factor, Diabetes Autoantigen

2009-06-21T00:56:00.003-06:00

IGF Binding to Heparin is Basis for Receptor Interaction, Internalization and Immunization

Examination of the protein sequence of insulin-like growth factors reveals strong heparin-binding domains (triplet of basic amino acids) that are also associated with internalization. Similar heparin internalization domains are also found on allergens and autoantigens. It was a small leap to expect that IGFs would also become autoantigens under inflammatory conditions that minimize heparan sulfate proteoglycan production.

Triplets of Basic Amino Acids Internalize Proteins

In several articles on this blog, I have discussed proteins that are internalized by their heparin binding domains. Heparin binding domains consistent only of a pair of basic amino acids, e.g. RK, flanked by one or more basic amino acids within a hydrophobic sequence of protein, are not sufficient to mediate internalization on heparan sulfate proteoglycans. A triplet of basic amino acids is usually required. Simple inspection of amino acid sequences is sufficient to identify these regions.

Internalization Triplet Identified in Insulin-like Growth Factor Binding Proteins

I noticed in a paper that insulin-like growth factors bind to epidermal growth factor receptors. I have previously written an article showing that EGF1 binds to its receptor via heparin, i.e. both the EGF and the receptor have heparin-binding domains. So I suspected that IGFs also had heparin binding domains. Inspection of the sequences readily identified simple heparin binding domains with pairs, but not triplets of basic amino acids. A search of the literature confirmed that heparin mediated IGF binding to receptors. A further search indicated that the heparin binding domains from proteins that bind and control the activity of IGFs could mediate internalization of proteins into cells and also into nuclei.

Internalization Triplets Are Associated with Allergens and Autoantigens

I have previously noted that all allergens and autoantigens have internalization triplets of basic amino acids. The presence of these triplets in IGF binding proteins suggested that IGF binding proteins might also be autoantigens. A quick check of the literature showed that antibodies against IGFs themselves frequently occur in type I diabetes. This suggests that the IGF-binding protein complexes are internalized and IGFs are immunologically presented during inflammation to produce anti-IGF antibodies. It is interesting that the other autoantigens for type I diabetes, e.g. transglutaminase, also have the expected internalization triplets.

references:
Maruyama T, Murayama H, Nagata A, Shimada A, Kasuga A, Saruta T.
Anti-insulin-like growth factor-1 autoantibodies in type 1 diabetes. Ann N Y Acad Sci. 2002 Apr;958:267-70.

Miao D, Yu L, Eisenbarth GS. Role of autoantibodies in type 1 diabetes. Front Biosci. 2007 Jan 1;12:1889-98.

Goda N, Tenno T, Inomata K, Shirakawa M, Tanaka T, Hiroaki H. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5. Exp Cell Res. 2008 Aug 1;314(13):2352-61. Epub 2008 May 29.