200th Post — Diet, Inflammation, Disease & Gut Flora

— all 200 Posts —

I started posting to Cooling Inflammation on 21 Aug, 2008 with How Your Diet Makes You Sick or Healthy.  My impetus for writing was my growing awareness that diet was the major reason why people were sick, and that health myths were preventing people from being healthy.  Inflammation originated by diet-inflicted injury and people attributed their sickness to genetics, environmental toxins and pervasive pathogens. 

My Path to the Obvious

My research background started with plant biochemistry, including carbohydrate structural analysis and polyphenol chemistry.  At that stage I was interested in understanding how plants protected (phytoalexins) themselves from pathogens, and I expected to use this perspective to explore human innate immunity.  From there, I went on to enzymology and protein characterization, biofilm structure, plant genetic engineering and breeding, monoclonal antibody production, mycotoxin detection, stem cell analysis, passive immunity in neonates, computational modeling of collagen and heparin binding, and heparan sulfate proteoglycan inhibition by inflammation.  These were temporary foci and the research imperatives, in retrospect, prevented me from seeing the bigger pictures, although they did leave me with a broad skill set.

Perspective: Water and Surface Tension

When I finally decided to slow down, smell the flowers and start having kids, I switched from research to teaching, from university to small liberal arts college.  For the first time, I actually thought about what I was teaching and my first revelation was that after teaching biochemistry for twenty years, I didn’t understand water and surface tension.  I could provide the platitudes from the Molecular Biology of the Cell, but I couldn’t do it mechanistically with colliding, sticky, energetic water molecules in my mind or at the blackboard.  I had to develop functional explanations of hydrogen bonds, entropy and thermal energy, that translated into the structuring of a layer of water molecules responsible for hydrophobic interactions and surface tension.  I extended that to include an explanation of the two layers of water holding together cytoplasmic membranes, the tube of structured water that holds together the cylinder of stacked bases in DNA or the shrink wrapping water layer surrounding proteins.

Perspective: Heparin Binding and Amphipathy of Sugars and Basic Amino Acids

As the kids got older, I started to dabble in research again and my expertise in carbohydrate chemistry led me into cartilage (mostly the glycosaminoglycan, GAG, chondroitin sulfate) synthesis and ultimately another GAG, heparan sulfate proteoglycans (HSPGs).  I was attracted to the dynamic HSPGs, that recycled with a half-life of six hours and formed layers around chondrocytes that secreted cartilage as they burrowed/ate through living cartilage.  I learned that the heparin filled granules of mast cells could be stained with berberine, which similarly stained the heparin in basement membranes of tissues and amyloids of Alzheimer’s, atherosclerosis and diabetes.  I was led by protein modeling of collagens to the binding of heparin to proteins and the revelation that basic amino acids (heparin binding domains) and sugars (heparin) are amphipathic, i.e. they have both hydrophobic and hydrophilic regions.  This is also true of plant polyphenolics.  Thus, polyphenolics, “basic” amino acids, “hydrophobic” amino acids, and sugars will all stack together.

Amphipathic Interactions

• DNA bases stack.
• Heparin binding sites of proteins are basic amino acids (Arg, Lys).
• Sugar binding sites in enzymes and lectins are hydrophobic amino acids (Trp, Tyr, Phe).
• Nuclear translocation signals, quartets of basic amino acids, bind to receptors with tryptophans.
• Tryptophans are the most highly conserved amino acids in the same proteins across great evolutionary distances.
• Hydrophobic bonding between tryptophan and a sugar or basic amino acid is ten times greater than hydrogen or ionic bonds.
• Tryptophan/Arginine ladders zip regions of proteins together.
• Polyphenols can disrupt cellular protein interactions by binding to receptors for carbohydrates/heparin, steroid hormones, amyloids, etc.
• Heparin holds dozens of hormones to receptors and changes the shapes of proteins, e.g. clotting and complement.
• Most nucleic acid binding proteins will also bind to the more negatively charged heparin.
• Bacteria use a pair of lysines to mark proteins for export.
• Peptides containing the basic amino acids of heparin binding domains (also produced by the specificity of gastric proteases) are antimicrobial, e.g. defensins, and so are plant polyphenols.
• Many drugs are active because they are domesticated plant polyphenols.

From Heparin Binding to Antigen Presentation

As soon as I realized that basic amino acids were involved in heparin binding, I started to look for the basic amino acids (R for arginine and K for lysine in amino acid sequences) in proteins known to bind heparin.  After study of hundreds of structures, it became obvious that heparin binding domains were simply a pair of basic amino acids (RR or KK or RK) with another within a distance of six amino acids.  No particular structure was necessary, as I later deduced, since binding to the heparin provided the structure.  In fact, in many X-ray crystallographic structures, the heparin binding regions on the surface of the protein are missing, because they are not in a defined shape.  I suspected that protein antigens involved in autoimmunity and allergy might be brought into cells for presentation to the immune system by interacting with HSPGs on the surface and so started to check them out for heparin binding domains.  I was very skillful at picking out pairs of Ks or Rs within sequences of hundreds of amino acids by that time, so I was shocked to see that the first dozen antigens that I checked, all had a triplet of basic amino acids.  I had discovered that autoantigens and allergens utilize a basic triplet analogous to the basic quartet used in nuclear translocation!  This also explained why proteins that interact with nucleic acids and are transported into the nucleus with a basic quartet are also prominent autoantigens.

Gut Flora and Immunity

Twenty years ago I read a curious description of leprosy that said that the course of infection could be either innocuous or devastating depending on whether the aggressive or the suppressive part of the immune system dominated.  I remained perplexed until I realized that diet and gut flora were the major determinants.  I was aware of the importance of diet at the outset of this blog, because it was clear that diet trumped genetics.  I was also aware thirty years ago in my studies of passive immunity, that milk contained bifidus factor, now known to be milk oligosaccharides, that controlled the growth of Lactobacilli that in turn controlled the development of the neonate immune system.  It was also known that bacteria-free mice had impaired immune systems.  It still took me several years for the relationship between diet, gut flora and immunity to make sense.  I began searching the literature for connections between gut flora and development of the immune system and soon noted experiments that linked filamentous bacteria with aggressive components and Clostridium spp. with Tregs.  A further refinement was linking resistant starch, a soluble fiber, with Clostridium.

My Current Views are Summarized in Three Health Diagrams

Diet, Gut Flora, Inflammation, Antigen Presentation, Tregs and Autoimmunity

Protein from the body and from food don’t normally stimulate the immune system, because there in no inflammation, the proteins lack basic triplets that enhance presentation, and antibody production and aggressive T cells are suppressed by Tregs.  Diet can throw the balance toward autoimmunity and allergy, by producing inflammation, e.g. hyperglycemia/AGE or high omega-6 fatty acids/prostaglandins, and starving gut flora needed for Treg production by eating processed food lacking soluble fiber.  The combination of inflammation and Treg deficiency causes proteins, either self or potential allergens, which have basic triplets to be presented to the immune system and stimulates attack by the immune system.

The Cure is to Cool Inflammation and Stimulate Tregs with Diet and Bacteria

I have provided an outline with The Anti-Inflammatory Diet to avoid inflammation, to stimulate existing gut flora with soluble fiber and encourage Treg production.  Mark Sisson, on Mark’s Daily Apple has provided an excellent dietary guide that also provides starch guidelines.  If you already have symptoms of autoimmune disease or allergies, then Richard Nikoley provides gut flora repair advice on Free the Animal, and Dr. B G provides more details on Animal Pharm.

Autoimmunity and allergies are not genetic destiny and they can be cured with diet and bacteria.

More Inconvenient Truths

I am writing this shouting summary of bottom lines in response to recent good news and bad news. The good news is that Michael Pollan is speaking in Boise, near my home town. The bad news is the recent press coverage of the JUPITER study on statins.

Michael Pollan is one of my heros. He speaks simply and clearly about the role of national agriculture policy in promotion of hazardous foods that lead to profits in the healthcare industry, but death and disease for the US population. Pollan also provides wise advice to solve our problems.

A new statin, Crestor, was shown in the JUPITER study to significantly reduce the risk of cardiovascular events, e.g. heart attacks, stroke, death, in a study population with normal LDL and elevated C-reactive protein, an indicator of inflammation. The press supported the drug maker’s interpretation that the statin provided benefit by lowering LDL in a population with chronic inflammation. What is missing is the clarification that lowering LDL is unimportant in reducing cardiovascular risk. Lowering inflammation lowers cardiovascular risk and there are more appropriate ways of lowering inflammation than using very expensive drugs. It is much cheaper, healthier and effective to switch to an anti-inflammatory diet and lifestyle!

After reading thousands of articles in the biomedical research literature, here are a few of my obvious bottom lines. Diet affects your health and the most fragile stages of development and most fragile organs, are the most sensitive to abuse. Therefore, damaging diets are most harmful to fetuses, newborns, brains, the cardiovascular system and reproductive systems.

• Formula promotes inflammatory bacteria in newborn guts resulting in lower intelligence, disrupted immunity, infections, allergies, obesity, degenerative diseases and autoimmune diseases. Breastfeeding is the only anti-inflammatory answer for infants.
• The US diet (hyperglycemic starch/sugar, high omega-6 to omega-3 fatty acid ratio, HFCS, low vegetable anti-oxidants, low vitamin D/sun exposure, low vitamin C, grain-fed meat instead of fish) is inflammatory.
• The Mediterranean Diet (small portions of starch, low omega-6 oils, no HFCS, high vegetable anti-oxidants, routine sun exposure, adequate vitamin C, fish and grass-fed meat) is anti-inflammatory.
• Inflammatory diets lead to infertility (female and male), problems during pregnancy (e.g. preeclampsia is an omega-3 fatty acid deficiency) and prematurity/low birth weight.
• Mental illnesses of many different types benefit from anti-inflammatory diet and lifestyle. Diet-based brain inflammation may be a major predisposing factor.
• All of the prevailing drug therapies for cardiovascular disease benefit from anti-inflammatory diet and lifestyle. Most of the drugs that reduce cardiovascular events rely on anti-inflammatory activities. Inflammation is the primary cause of cardiovascular disease, not elevated blood lipids/cholesterol.
• Vegetable oils (corn, soy, cottonseed, safflower) are rich in omega-6 fatty acids and are dangerously inflammatory. These polyunsaturated oils are less healthy than saturated fats. Olive oil is the most healthy.
• Reasonable routine exposure to the sun could eliminate inflammatory vitamin D deficiencies.
• Obesity is inflammatory, but diet-based inflammation may also be a major contributor to obesity.
• Genetic predisposition to specific diseases is triggered by diet-based chronic inflammation.
• Diseases and disabilities associated with aging are symptoms of mismanaged chronic inflammation typically resulting from decreasing muscle mass and increasing fat.
• Sensible diet and lifestyles could dramatically improve quality of life and reduce healthcare expenditures in the US.

Prescription: eliminate vegetable oils, eliminate HFCS, eliminate trans fats, use olive oil, reduce starch, eat vegetables, eat more fish and less meat, get daily sun, use fish oil supplements, get frequent muscle-building exercise, and stay lean.

Arthritis Antibodies

Antibodies can be used to attack the signaling (TNF) molecule that mediates the autoimmune attack on arthritic joint tissues. These anti-TNF antibodies minimize inflammatory signaling, reduce joint inflammation and also reduce bone attrition.

Inflammation is an activated state of a tissue in which inflammatory cytokines, TNF, IL-1, IL-6 are secreted by T-cells and the tissue responds by expressing genes that cause characteristic vascular dilation and accumulation of migrating cells of the immune system. One particular type of blood cell, a macrophage, can also migrate to the site of inflammation and develop, in response to signals from the inflamed tissue and resident bone secreting cells, osteoblasts, into osteoclasts that degrade bone. Thus, inflammation of joints can result in bone destruction and increase in serum calcium.

TNF is particularly pivotal in the development of osteoclasts and bone destruction. Thus, drugs, such as thalidomide, that block TNF production, also block the symptoms of arthritis. Antibodies can also be developed that bind to TNF and some of these antibodies have been chemically and genetically modified to make them useful as drugs. Examples are Infliximab and Andalimumab. These are proteins that bind to and inactivate TNF. In a similar alternative strategy, a portion of the TNF receptor was engineer to serving as a neutralizing molecule to bind TNF in inflamed tissue. All of these TNF inactivators can reduce symptoms and provide effective therapy for arthritic joints.

The unanswered question in the use of TNF inactivators is, “What caused the inflammation of the joint in the first place?” Inactivation of TNF can provide a temporary return to approximately normal tissue function, but the symptoms are expected to return.

Thus, we come to the unifying question of what causes inflammatory disease mediated by the immune system and directed at normal tissue components. Two obvious candidates are diet and infectious agents.

Food ingredients can exacerbate or ameliorate the symptoms of inflammatory disease, and particular diets determine the risk of acquiring these diseases. Diet is a major factor in inflammation of any source. Bacterial or viral infections frequently precede inflammatory conditions.

The association of infection with inflammation remains controversial, but there is growing evidence that bacteria in particular reside in almost all inflamed tissues. Moreover, there is abundant anecdotal evidence of effective use of antibiotics in numerous inflammatory diseases, including arthritis, inflammatory bowel disease, atherosclerosis and cancers of various types.

I expect that elucidation of the link between chronic inflammation, diet and bacterial infection will provide increasingly effective and simple therapies for most diseases in the near future.

Inflammation Score

Most people need some feedback to monitor the impact of their diet and exercise on their health. I tried to point out some of the major contributors to chronic inflammation with a little check list. See how you score (choose one of the list for each category) and give me your feedback on the how you think each part contributes to inflammatory diseases.

Fat Content ____
lean 0
extra abdominal fat 4
obese 8

Carbs ____
small meals, no cereal for breakfast 0
fistful of starch with each meal 2
pasta/rice/potato as a meal 4

HFCS ____
high fructose corn syrup banned from your diet 0
don’t avoid HFCS, but avoid soft drinks 2
have replaced sucrose with HFCS, enjoy soft drinks 4

Unsaturated Fats ____
have removed vegetable oils (except olive oil) from your kitchen 0
use canola oil 2
have replaced saturated fats with corn oil 4

Trans fats ____
eat no trans fats 0
avoid trans fats on your chips 2
don’t know what trans fats are 4

Fish oil ____
supplement with two or more fish oil (DHA/EPA) capsules per day 0
eat at least two helpings of oily fish per week 2
avoid all fish products 4

Antioxidants ____
know that coffee, tea and chocolate are good sources of vegetable antioxidants 0
eat five servings of fruits and veggies 0
take vitamin C supplement, because you avoid veggies 2
avoid veggies; meat and potatoes type 4

Exercise ____
take a stroll after meals and maintain your muscle mass 0
run when you feel guilty 2
couch potato 4

If you smoke, add an extra 15 points

Add ‘em up. How much are you stoking the inflammation furnace?
0-5 Cool! You will never look your age.
6-10 You are getting warm. Hope that you don't have any genetic predispositions to disease.
11-15 You may postpone inflammatory illness until middle age. The flame is lit. Pick your disease.
16-25 If you aren’t showing a chronic disease, you will soon.
26+ You can reverse your disease symptoms with the inflammatory diet and exercise.

Farmer and Chief

Michael Pollan has articulated the relationship between U.S. agricultural policy, diet, health and lifestyle. In an open letter to the new President-Elect in the October 9, 2008, issue of The New York Times, he reiterates his logic in a plea for reasonable action. Farmer in Chief is at once an indictment of the greed of our food processors, a shrug at the myopia of our medical system, and a hopeful guidance for a future path that provides a sensible alternative.

Mast Cell Heparin

Mast cells are sentinels in tissues. They respond to invading pathogens by releasing their stored histamine, enzymes and heparin. The heparin modifies the activity of enzymes and cytokines.

What are mast cells and why are they loaded with heparin (left)? Mast cells start in the bone marrow, like many other components of the immune system. They then move into the blood stream and offload in most of the tissues that typically encounter pathogens and parasites. Thus, the typical commercial source of the mast cell-produced heparin is pig intestines or cow lungs, i.e. since heparin is made and stored in mast cells and mast cells are abundant in lungs and intestines, those are the sources of crude heparin. Proteins bound to the crude heparin are removed as the heparin is cleaned up to be used as an anti-clotting drug.

Mast cells are sentinels near the surface of mucus membranes that line the airways of the lungs and the digestive tract. Diseases of the lungs and intestines, e.g. asthma and inflammatory bowel disease, that have an inflammatory and/or autoimmune component yield high levels of mast cells in the affected tissues. Pathogens or parasites coming in contact with mast cells trigger the sudden release of vesicles full of histamine, enzymes and heparin.

Heparin stored in vesicles in mast cells can also be readily visualized by staining the mast cells in microscope sections using the fluorescent dye berberine (left). Berberine binds quite specifically to heparin and is also used in herbal medicine as a treatment for many inflammatory diseases, such as arthritis. It would be very interesting to know whether berberine has any effect on asthma.

Mast cells display a variety of receptor proteins on their surfaces. Protein receptors work by binding target molecules, ligands, changing their shapes and transmitting a signal through the cytoplasm. A key aspect of the signal transmission is the requirement for the ligand binding to bring together receptors in pairs. The pairing of receptors during ligand binding is facilitated by the binding of heparin to both ligands and receptors. Two ligands, e.g. cytokine peptides, such as TNF, can bind to adjacent sites on a heparin molecule and this pair can then bind to two receptors brought together on the surface of a cell. The receptors bind to the ligand and to the heparin. Some ligands will bind to their receptors without heparin, but the presence of heparin greatly accelerates and intensifies the reactions.

Heparin is synthesized in the vesicles of mast cells and binds to enzymes, e.g. tryptase, also present in the vesicles. The tryptase enzyme proteins form tetramers with heparin wrapped around the edge (left, edge view showing one pair of tryptase proteins with heparin bound diagonally to blue heparin-binding domains; other pair of tryptase proteins is hidden).

Interestingly the active site for each tryptase in the tetramer faces a hole where the four proteins come together. Thus the tetramer can degrade small peptides, but large proteins cannot get access to the blocked active sites. Monomers change shape and are no longer active.

Activated mast cells release their vesicle contents with some enzymes active and their bound heparin is replaced by the heparan sulfate attached to adjacent cells. Other enzymes are initially inactive bound to heparin and are activated by dissociation of the heparin once they are released from the vesicles. In both cases some of the heparin is released from the mast cells into the surrounding tissue. The free heparin can bind to cytokines released from other cells and the combined pairs of cytokines bound to heparin can in turn bind to appropriate receptors on other cells. The abundance of heparan sulfate bound to other cells will determine whether additional heparin is required for receptor responses from particular cytokines. Cells with abundant heparan sulfates will sweep heparin binding ligands toward receptors aggregated in lipid rafts, as the heparan sulfate proteoglycans are internalized for recycling.

Mast cells can be activated by allergens, because of IgE receptors. IgEs are antibodies that trigger allergic responses. The IgEs produced by antibody producing B lymphocytes circulate in the blood serum and bind to mast cell receptor proteins. Allergen molecules bind to the IgE-receptor complexes, trigger the activation of the mast cells and release histamine. The histamine binds to receptors on other cells and produces the symptoms of allergy or asthma.
Heparin can be sprayed into the lungs of asthma sufferers and reduce symptoms. This suggests that the ratio of heparin to cytokines is important and that cytokine signaling required for asthma episodes of airway constriction can bind individually to different heparin molecules and minimize mast cell triggering and histamine release.

Asthma also responds to a general decrease in chronic systemic inflammation. Thus, an anti-inflammatory diet and lifestyle, can reduce episodes and potentially reverse symptoms. Omega-3 oils and glucosamine, for example are both effective.

Tryptase model: Sommerhoff CP, Bode W, Pereira PJ, Stubbs MT, Stürzebecher J, Piechottka GP, Matschiner G, Bergner A. 1999. The structure of the human betaII-tryptase tetramer: fo(u)r better or worse. Proc Natl Acad Sci U S A 96(20):10984-91.


Berberine staining of mast cell heparin: Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, Takahashi HK, Morgan ES, Dvorak AM, Fehling HJ, Rodewald HR. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 25:6199-210.