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.

Bee Sting Allergy

Typical. I started to write an article on leukotrienes, the inflammatory derivatives of the omega-6 fatty acid, arachidonic acid, but ran across another powerful example to test my hypothesis to explain the cause of allergies. The leukotriene article will have to wait till another day.

Wikipedia is my source of choice for up-to-date summaries of biomedical information. I queried “leukotrienes” and immediately ran across the original name for these inflammatory compounds, “slow reacting substance of anaphylaxis”. I was initially distracted by the classic experimental use of snake venom and histamine to induce leukotriene production. Snake venom has the same enzyme, PLA2, as brown recluse spider venom (subject of a previous article) and honey bee venom, that releases arachidonic acid (ADA). ADA is an omega-6 fatty acid that is the starting material for inflammatory prostaglandins and leukotrienes.

The mention of honey bee venom in the Wikipedia article on anaphylaxis sent me on a quick check of the structure and sequence of the honey bee allergen. I initially found that the major allergen is a hyaluronidase. I quickly searched for a three amino acid sequence that I predicted would make it an allergen. It was just where I expected to find it. About two thirds of the way along the amino acid sequence I found, -TTSRKKVLP-. Three basic amino acids together, in this case -RKK-, argininine-lysine-lysine, form a strong heparin-binding domain, that I believe takes proteins into cells and during inflammation primes the immune system for allergic responses.

I have found the same strong heparin-binding domain associated with allergens of ragweed, dust mites and peanuts. The principal autoantigens of autoimmune diseases, such as lupus, celiac, etc. also display the same unusual sequences. In lupus, or example, nuclear proteins with the internalization signal provided by nucleic acid-binding domains (and nuclear localization signas) are autoantigens. This pattern is found with all allergens that I have examined. There are a few apparent exceptions, but in all of these cases, there is a closely related allergen from a related source that has the expected strong heparin-binding domain. It appears that in these cases, the less common allergen provides the initial exposure during the presentation phase of high inflammation, and the allergy is maintained by subsequent exposure to the more common allergen. After the establishment of the allergy, the strong heparin-binding domain is no longer needed, because antibodies bind to other parts of homologous allergenic proteins for internalization.

Just for fun, I have illustrated the honey bee allergen, hyaluronidase, to show both its strong heparin-binding domain (blue) along with its substrate hyaluronan (grey and red). Note that the substrate sugars are in the slot of the active site, which is lined with orange and yellow aromatic amino acids that provide flat, hydrophobic binding platforms for each sugar.

After this little distraction to provide further support for my explanation of the cause of allergies, I have to get back to looking at the role of leukotrienes in anaphylaxis, COPD, asthma and other inflammatory diseases.

Statins and Atherosclerosis

A recent study (JUPITER) on the statin Crestor was ended prematurely when the drug was shown to dramatically reduce vascular events. The statin was tested on patients with chronic inflammation as judged by elevated C-reactive protein, but with low LDL. These patients would not normally be treated with statins and therefore represent an immense new market for statins.

Statins are supposed to act by interfering with the synthesis of cholesterol and thereby lowering the serum concentration of the lipid carrier LDL. Lowered LDL is supposed to decrease vascular disease that is aggravated by accumulation of cholesterol at sites of inflammation on the surface of blood vessels.

Unfortunately the data linking cholesterol production, LDL levels and vascular disease is weak. Thus, it is possible to lower LDL and have no impact on cardiovacular disease statistics. The recent study on Crestor was interpreted as being support for the link between LDL levels and vascular disease, but I think it shows something very different.

There is increasing evidence that vascular disease is based on diet-based chronic inflammation and that statins have a mild impact on reducing inflammation. It follows then that statins will reduce inflammation enough to have an impact on vascular disease, independent of effects on LDL levels. The Crestor study actually showed that patients with low levels of LDL but chronic inflammation benefited from lowering of inflammation. The LDL levels were unimportant. Reducing inflammation was the point and using statins to reduce inflammation is unnecessarily expensive and ineffective. Adjusting diet makes a lot more sense.

Drug companies are already pushing for increased use of statins on larger segments of the US population to provide prevention from atherosclerosis, stroke and heart disease. This would be immensely expensive with marginal returns. It is also just treating the symptoms without addressing the cause.

The solution to cardiovascular disease is dietary. Omega-6 oils and low availability of omega-3 fish oils is the major cause of the chronic inflammation that is the major risk factor for cardiovascular disease. The major US vegetable oils, corn, soybean, cottonseed, safflower, need to be drastically restricted and olive oil needs to be encouraged. We need to recognize that saturated fats are safer than the omega-6 polyunsaturated fats that have replaced them. Elimination of omega-6 vegetable oils and use of fish oil supplements are cheap and effective ways of lowering chronic inflammation.

Cardiovascular disease is also based on decreasing muscle mass, sarcopenia, which is also the basis for increasing chronic inflammation inappropriately attributed to aging. People get less physical exercise as couch potatoes or with decreasing activity as they age. The result is replacement of muscle by fat, and fat is inflammatory. Obesity is an extreme of this trend that leads to high chronic inflammation identified as metabolic syndrome, the prelude to a suite of nasty degenerative diseases: diabetes, atherosclerosis, allergies, cancer, Alzheimer’s, etc.

The obvious bottom line is to avoid all of these problems with an anti-inflammatory diet and lifestyle.

Osteopetrosis

Bones like stones, that is osteo-petrosis. It is the opposite of osteoporosis, porous bones. Osteopetrosis is a disruption of bone metabolism so that mineralization by osteoblasts predominates over demineralization by osteoclasts.

Mice lacking almost all genes, i.e. knockout mice, are now available. To determine which body tissues require the inflammatory transcription factor, NFkB, NFkB knockout mice were engineered and their characteristics were carefully analyzed. Their bones kept mineralizing and mineralizing and mineralizing. NFkB function was required for the development and function of osteoclasts, the macrophage-derived cells that remove bone.

Inactivation of osteoclasts or inhibition of osteoclast development by bisphenols, such as Boniva, leads to a minor version of osteopetrosis. Killing osteoclasts leads to a reversal of osteoperosis. An omega-3 fatty acid deficient diet leads to osteoporosis in mice.

Omega-3 fish oil has been used to reduce inflammation induced bone loss associated with many diseases, e.g. arthritis, periodontitis and osteoporosis. In a recent study (below) the fish oils, DHA and EPA were added to mouse macrophages in culture and the impact on differentiation into osteoclasts was analyzed. DHA was more effective than EPA in reducing NFkB activation and TFA response leading to macrophage differentiation. The omega-6 fatty acid, ALA, enhanced osteoclast differentiation, consistent with many animal and human studies that show that the high omega-6 fatty acid content of such common vegetable oils as corn, soybean and safflower, are inflammatory and presumably encourage osteoporosis.

Osteoporosis increase in our population has paralleled the increase in use of omega-6 vegetable oils, and the increase in chronic inflammation. The first step in treating osteoporosis should be a shift to an anti-inflammatory diet lacking these vegetable oils. Olive oil is much safer.


Rahman MM, Bhattacharya A, Fernandes G. 2008. Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid. J Cell Physiol. 214:201-9.

Oxygen Starved

Hypoxia is the low oxygen environment of tissue lacking blood vessels. Oxygen is needed as the final, low energy electron acceptor of the aerobic metabolism of mitochondria, but it is also needed to make hydroxyproline, the special amino acid of collagen. Expression of genes in response to the oxygen available is controlled by a transcription factor, HIF.

The oxygen that we breathe is used as a place to dump the low energy electrons produced by mitochondria as ATP is using energy from high energy electrons of carbon and hydrogen present initially in glucose. In the absence of oxygen, fermentation can use glycolysis and dispose of the low energy electrons in the form of products such as lactic acid or ethanol. Aerobic metabolism, in the presence of oxygen, is much more efficient (more ATP per glucose), so fermentation is only used when oxygen is depleted, as in the case of exhausted muscles.

Low oxygen, hypoxia, is also encountered in tissues isolated from the oxygen-distributing vascular system. Cells that constantly secrete new cartilage, chondrocytes, are prevented from ready access to oxygen, because the tensile strength of cartilage would be sacrificed by mechanically weak blood vessels. This is paradoxical, because cartilage also contains large amounts of reinforcing collagen fibers, that require oxygen. Consumption of oxygen by aerobic metabolism must be restricted in chondrocytes to reserve diffusing oxygen for collagen synthesis. This also predicts that energy metabolism in chondrocytes exposed to ample oxygen, as in developing bone or wounds, must be drastically different from metabolism in mature chondrocytes that may be centimeters away from the nearest blood vessel.

Oxygen is used in collagen production to produce the hydroxyproline of the repeated triplets of amino acids that form the spiral threads of collagen. Every third amino acid is glycine and prolines that precede a glycine are converted to hydroxyproline by an enzyme that uses vitamin C. Thus, scurvy, a vitamin C deficiency, is characterized by weakened cartilage, e.g. loose teeth. The high hydroxyproline content of collagen also explains the low nutrition of gelatin, which is denatured collagen.

The trigger for shifting a cell to a low oxygen metabolism, is production of hydroxyproline in the transcription factor called, hypoxia-inducible factor, HIF. If oxygen is abundant, a particular proline in HIF is converted to hydroxyproline, and this modified HIF is quickly degraded in proteosomes. Lack of oxygen prevents modification of hydroxyproline and HIF successfully migrates to the nucleus, binds to the promoter sites of specific genes and changes the pattern of proteins in the cell.

HIF is important in all diseases that result in damage to blood supply. Tumors, for example stop growing when they reach a size that limits the oxygen that can diffuse from surrounding blood vessels to the center of the tumor. Hypoxia in a tumor causes release of angiogenic, blood vessel proliferating, cytokines from the tumor. HIF induces the production of angiogenic molecules. In the opposite direction, damage to cartilage that results in increased oxygen, will turn off HIF and prevent the secretion of mature cartilage by chondrocytes. Chondrocytes also secrete proteins that actively block the formation of blood vessels, e.g. endostatin, the end of one of the collagen.

Several drugs are now being developed to block HIF activity and starve cancers. These drugs will also modify the metabolism of many other cells that use HIF. It is worth noting that mice with the HIF gene knocked out do not survive long after birth.

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.

Bacterial Sex

A new study finds that women have more species of bacteria on their hands than do men. I don’t know what this means, but this brings up a question about the use of the term species in reference to bacteria.

Bacteria are bags of DNA and the proteins (and RNAs) that are coded by the genes that make up the DNA. Thus, excluding mutations, all of the progeny of a bacterium will have the same DNA sequence and proteins. Hereditary and evolutionary relationships between bacteria can be seen in their DNA sequences.

Problems arise in the definition of species, because of the promiscuous exchange of DNA between bacteria (and other organisms). Bacteria exchange genetic information on a prodigious scale. Viruses, for example, can infect one bacterium and transfer some of that bacterium’s DNA to a totally unrelated bacterium. Bacteria can also take up the DNA remains of ruptured bacteria. There is also a form of sexual, conjugal transfer of DNA between bacteria via proteinaceous bridges called sex pili.

These DNA exchanges are very wide spread in nature and some bacteria actively transmit their genes to plants, e.g. crown gall producing bacteria. Rotifers actively reconstruct their chromosomes after dehydration and will incorporate any plant or animals DNA that is present into their chromosomes as well. Humans also incorporate DNA from other species at a relatively modest pace. For example, the HIV or herpes viruses insert their genes into the chromosomes of infected human cells. As a consequence of similar historical events, human chromosomes are littered with DNA remnants from other species.

The upshot of all of this genetic exchange is the blurring of species boundaries, particularly in bacteria. Higher densities of bacteria mean more transfer, so the gut, for example, consists of bacteria defined by the cohort of genes necessary to occupy a particular biochemical niche. If bacteria identified by their biochemistry as E. coli in Boise, Idaho, are compared with E. coli identified the same way in Paris, their DNA sequences will be less similar than cats and dogs.

When I hear that women have more bacterial species on their hands than do men, I am perplexed. What do the numbers mean? How are the researchers defining the 4,700 different bacterial species that they found? I would have to say that, if the differences are real, then women have more biochemical niches on their skin than do men. The report also indicates that the one to two hundred different species on each hand of an individual also differ. This could reflect the unique colonization of each square centimeter of skin and the microstructure of bacterial populations. It does bring into question the use of the term species as applied to bacteria.

It would be interesting to see detailed DNA sequencing of bacterial populations applied to simulations of gut bacteria. I would expect that the biochemistry displayed by defined regions of the synthetic gut would become stable. This would indicate that particular species became established. But I would also expect that the DNA sequences of those “species” would continue to change, reflecting the exchange of DNA between the species. Introduction of new DNA into the system should result in a general drift of DNA sequences through the species even though the biochemical characteristics remain constant.

This discussion also applies to the inflammatory state of an individual as determined by diet and the corresponding gut flora that develops. Since there would be no advantage to bacteria to produce gut altering compounds that did not improve the reproduction of the bacteria, then the gut must initiate the exchange of molecules/nutrients and benefit from the communication. This biochemical communication is not understood, but is vitally important for health.

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.

Thalidomide Waste

Thalidomide suppresses TNF production and alleviates cachexia and anorexia of terminal cancer. Suppression of TNF is also effective in the control of numerous inflammatory diseases.

I have often wondered how cancer actually kills. By infiltrating and displacing cells of essential organs, a metastasizing cancer can kill by starvation, suffocation, etc. Brain cancers can build up pressure in the skull and cut off neural function needed to sustain life. But what about the loss of appetite and general wasting, anorexia and cachexia, associated with the terminal stages of cancer? As more people live longer with cancer, it seems to me that avoiding the wasting of the last stage is becoming more important. So what is wasting?

It seems to me that wasting is high level chronic inflammation. Inflammatory cytokines, particularly TNF (tumor necrosis factor) reach high levels and are characteristic of acute inflammation. TNF was initially called “cachexin” based on its association with wasting. Cytokine signaling is usually balanced and local, so chronic high level TNF marks a system out of control.

Inflammation suffers from stereotyping. We spend so much time trying to block inflammation that we sometimes lose sight of the essential requirement for inflammatory processes in normal immune function, wound repair and development. We notice this need for example in the disruption of the gut by aspirin, since inflammatory prostaglandins are needed for ongoing maintenance of the gastric and intestinal epithelium. Aspirin blocks COX2, the enzyme that produces inflammatory prostaglandins from omega-6 fatty acids, and that is how it leads to problems with causing bleeding.

A potent inhibitor of TNF, thalidomide, was initially banned, because it caused horrible birth defects when taken by pregnant women. We must be vigilant when using potent drugs to selectively eliminate problematic protein functions, because proteins always have multiple functions and multiple proteins have similar structures. Thus testing for the effectiveness of a drug, does not protect us from numerous underlying unintended consequences. All drugs interact and alter numerous, and in most cases unknown, functions within a cell.

Thalidomide was found to reduce TNF and was effective in the treatment of nausea and sleeping problems of pregnancy. Its teratogenicity gave it a terrible reputation for many years, so it was a long time before its potential was appreciated. Suppression of high chronic inflammation is very useful in extreme cases of arthritis, leprosy, multiple myeloma and many other diseases currently being examined. So, thalidomide is now being vindicated.

Upon seeing these observations of the effectiveness of thalidomide, I immediately thought about the possibility of alleviating cancer cachexia and perhaps even the physiological reinforcement of anorexia nervosa. A quick check of the biomedical literature confirmed that thalidomide is a very useful new tool in the treatment of terminal cancer. Thalidomide that can be tragic to embryos can provide comfort and improve the quality of life in its final stage.

Spider Inflammation

Happy Halloween! It's time for creepy, crawly things that bite... and cause inflammation.

The brown recluse has the bite that keeps on giving. Its venom contains a phospholipase that cleaves membrane phospholipids to release arachidonic acid (omega-6 fatty acid precursor of inflammatory eicosanoids) and lysophosphotidylcholine that binds to multiple receptors to signal NFkB-based inflammation.

It’s Halloween and a time to talk about things that bite and sting. After all, the symbol of Halloween is the Jack-O-Lantern, the flame within, i.e. inflammation. Venom is Nature’s answer to the need for instant inflammation.

The brown recluse is adapted for hunting, killing and eating. The killing bite is the subject tonight. The recluse injects a venom designed to get the biggest bark for the bite. It uses the least amount of protein to immobilize its prey/meal. Of course, since spiders are suckers, it helps if the venom also liquifies its meal.

The venom also has a secondary use as a defense. In this case, if something too big to eat encounters a brown recluse, the goal is to encourage the big beast, you or me, to leave. No pain, no gain.

Venom provides a convergence of enhanced meal making and avoidance of being made into a meal. The answer is, of course, enzymatic inflammation. Venom contains enzymes that convert common cellular components into potent pyrogens, inflammation inducers. The wicked witch of the world of inflammation is phospholipase A2 (PLA2).

Brown recluse venom PLA2 clips off the arachidonic acid from membrane phospholipids and leaves an amphipathic detergent, lysophospholipid (LPL). The LPL can destabilize membranes and rupture cells. It is also a potent activator of several cell receptors that signal inflammation via NFkB. The COX-2 induced by inflammation has a readymade substrate present, arachadonic acid, and produces inflammatory prostaglandins, that continue the signaling frenzy. The result is inflammation, pain and necrosis.

Recluse bites are hard to treat. I think that some people recommend topical application of gunpowder. I guess that would make sense, because one of the problems of recluse bites is actually constriction of blood flow by other venom components, and nitroglycerin patches apparently provide some relief. Both the patches and nitrates of the gunpowder cause vasodilation, and would remedy some of the circulation problems of the venom. Glucocorticoids are also partially effective, since they are anti-inflammatory. Then there is heparin. I would always try heparin, at least as a last resort, because heparan sulfate proteoglycans are an essential component of most extracellular signaling systems, e.g. cytokines. Heparin should disrupt or activate all of these systems. It is unpredictable. It turns out that heparin is helpful against the brown recluse. I would also like to try a topical potion made of Vick’s Vaporub (menthol, eucalyptol, camphor, turpentine), castor oil and heparin. That should fix about anything. It even smells good, sooths painful joints and makes your skin soft.

Happy Halloween. Enjoy trick or treating, but watch where you put your hands.

Cartilage as Rejuvenation

I have studied cartilage secreting (chondrocytes) for the last few years. Chondrocytes are normally derived by differentiation of mesenchymal stem cells (MSCs) that grow in the bone marrow. MSCs can differentiate to produce bone secreting cells (osteoblasts), muscle cells (myoctes), fat cells (adipocytes) and insulin-secreting cells (beta-pancreatic islet cells.) I chose to study a rat chondrosarcoma (RCS) cell line, because this is a type of cell that shares the properties of many other important cells and it will continue to grow in cell culture. Thus, I can dilute some RCS cells in a solution with all of the nutrients required for growth and the cells will stick to the surface of the plastic dishes that I use, grow and differentiate. If you stain the cultures for cartilage, you get the following micrograph.

At first the cells stretch out and move about the surface of the dish. Then they become progressively less adhesive to the surface and more spherical as they start to produce and secrete the polysaccharides (glycosaminoglycans, such as chondroitin sulfate and heparan sulfate) and proteins (collagen) of cartilage. Finally they produce thick layers of cells that are separated and embedded in cartilage. After a little more than a week in culture, the cells are moving through the cartilage matrix by enzymatically degrading the cartilage ahead of them and secreting new cartilage in their wake. The cells that eat their way to the dish surface separate the cartilage layer from the dish and the colonies of cells begin to slough off from the dish surface. Normal chondrocytes would eventually stop dividing under culture conditions, but the cancer line that I use continues to grow quite happily and can be diluted and plated continuously. Chondrocytes in cartilage live in cavities within the cartilage and are surrounded by heparan sulfate attached to proteins of their cell membranes, i.e. heparan sulfate proteoglycans.

Cut cartilage will grow back together as the chondrocytes mine, secrete and gradually knit the two surfaces together with strands of new cartilage. Movement through and renewal of cartilage, e.g. in the connective tissue of skin, is restricted if the collagen fibers that are assembled outside of the secreting cells are cross-linked. This is why sunbathing ages skin. High fructose corn syrup also accelerates cross-linking. This cross-linking is also what makes meat less tender. The cross-linking and toughness can be measured by inserting fluorescence-measuring probes into meat, because the protein cross-links fluoresce in UV light.

A consequence of the development of chondrocytes on the ends of bones, is that the nutrients for the cells change. Initially the chondrocytes enjoy the abundant glucose and oxygen of the blood stream and gradually they are remove further from blood vessels. (Note that cartilage actively inhibits vascularization, so there are no blood vessels in cartilage. This lack of blood vessels and associated enhanced risk of disfiguring infection, is a reason to discourage piercings that involve cartilage.) Chondrocytes snug in their little cartilage cavities no longer eat sugar or breath oxygen, they dine on cartilage and ferment.

What happens if you expose mature chondrocytes to a new source of rich nutrients? I think that the answer is rejuvenation. Quite literally, the chondrocytes regress and return to the lifestyle of their youth. Instead of producing mature, weight-bearing, dense cartilage, these rejuvenated cells start to produce the weaker matrix of their youth. This weak cartilage is readily damaged by abrasion and is not suitable for joint surfaces. This is one of the consequences of arthritis. Inflammation of cartilage brings rejuvenating, damaging nutrients to chondrocytes. The mechanical damage leads to destructive cycles of further inflammation.

Chondrocytes in cartilage also respond to mechanical stress and this stress maintains their maturity. Persistent weight bearing at the same orientation leads to bone production. Thus, after joint injury it is important to use the correct regimen of rehabilitation to maintain mobility of the joint and mineralization of the bone.

Palm Oil

One of my favorite chocolates is Truffettes de France. I tell myself that just one serving (1.5 oz) or five pieces will provide an excellent supplement to may daily plant anti-oxidants. I am almost tempted to consider it one of the five recommended servings of fruits and vegetables. Alas, examination of the ingredients lists palm oil first, sugar second and low fat cocoa third.

Palm oil is a story in itself. This oil is about half palmitic acid (C16), one third oleic (C18) and one tenth linoleic (C18). The soap made from palm oil was developed in the US by the Palmolive company. Notice the name, palm-olive, from the two prominent fatty acids, which are also named after their original sources, palm and olives.

Another rather shocking use of palm oil is with naphthalene to produce a sticky flammable mixture called napalm.

Oil palms were brought to Malaysia from West Africa in the beginning of the 20th century. When I stayed in Singapore in 1997 for a research sabbatical, the monsoon season was delayed so that the rainforests of Malaysia and Indonesia were drier than usual. Entrepreneurial plantation owners took advantage of the opportunity to send out thousands of workers to start fires. Fire is the tool used to clear forests and subsistence farmers from lands that cannot be used for profit, and to turn those cleared lands into plantations that use the landless as employees. The smoke was so thick throughout the whole region that street traffic in some areas of Malaysia and Indonesia had to be restricted for safety. I could see smoke even in the lecture halls at the National University of Singapore and my daughters had to check the smoke level outdoors before traveling to the university swimming pool. There were reports that birds fleeing the burning forests were lost at sea and took refuge on ships. Orangutans in Borneo Sumatran Tigers and the Asian rhinoceros are endangered by encroaching plantations.

My big worry about eating the delicious truffles is the linoleic acid. This shouldn’t be too much of a worry, even though it is an omega-6 fatty acid, because it is too short. Most vegetable fatty acids are shorter than the C20 needed to make inflammatory prostaglandins. The trouble is that short fatty acids can be elongated from 18 to 20 to 22 by cellular enzymes and omega-6 fatty acids block the elongation of omega-3 fatty acids. Omega-6 fatty acids that are heavily marketed by the US agribusiness industry, are essential in small amounts and a health risk when present in the diet in a higher ratio to omega-3 oils of greater than approximately three or four to one. The US diet is typically more than 10:1, highly inflammatory.

The single serving of five truffles contains about a gram, 1000 milligrams, of linoleic acid, so it will take about a single fish oil supplement capsule to balance that much omega-6 fatty acid. Maybe I will try to cut back to just a couple of truffles and eat more kale.

Osteoporosis Treatment

Osteoporosis is an imbalance between bone production and loss. Most loss is due to dietary use of omega-6 vegetable oils. Treatment should focus on elimination of demineralizing oils and minimizing inflammation.

It is hard for me to discuss osteoporosis without the image of Sally Field advocating the use of Boniva (the bisphosphonate Ibandronate, see figure) popping into my head. Advertising is very powerful. Bisphosphonates stop bone loss by killing osteoclasts, the cells that demineralize bone during bone remodeling.

Normally demineralization of bone by osteoclasts is followed by secretion of osteoid containing osteocalcin by osteoblasts. Osteocalcin initiates mineralization. Thus, a large fraction of bone is being rejuvenated by balanced osteoclast/osteoblast action at any given time. Cessation of this remodeling process in bone as in cartilage and other connective tissue, e.g. skin, gives the symptoms of aging.

Osteoporosis, loss of bone density, means that there is a net conversion of bone hydroxyapatite (calcium phosphate crystals) into blood calcium, followed by calcium loss in urine. This means that the continuing development and activity of osteoblasts and osteoclasts is out of control. Osteoblasts develop from stem cells that are also the origin of fat adipocytes. The transcription factor that controls the alternative destiny of these stem cells is PPARgamma. Omega-6 fatty acids are converted into molecules that stimulate PPARgamma and result in adipocyte production in bone marrow instead of osteoblasts. In general terms, vegetable oil (except olive oil) makes fat cells instead of bone cells.  This is particularly true in postmenopausal women.  It is no wonder that the emphasis on the use of vegetable oils to avoid saturated fats has resulted in a pandemic of osteoporosis.

Omega-3 fatty acids (fish oil) are anti-inflammatory and they do not stimulate the production of PPARgamma. As a consequence omega-3 fatty acids, DHA and EPA, enhance bone production and density, and are very important to maintain normal osteoblast production.

Osteoporosis treatments that block osteoclast development or activity can be expected to have long term side effects, because normal renewal of bone is being disrupted. Inhibiting bone demineralization can lead to a slowing of osteoporosis, but it does not get to the cause of the osteoporosis.

Because of the prevalence of diet-based chronic inflammation, one might expect that diet and lifestyle are also the foundation of most osteoporosis.  With both inflammation and osteoporosis vegetable oils appear to be the major culprit.  Aging is also associated with osteoporosis.  Most of the symptoms of aging can be attributed to mismanagement of chronic inflammation.  Now it turns out that osteoporosis is a dietary problem (vegetable oil) compounded by physical problems of inflammation that limit activity.  Loss of muscle mass, i.e. sarcopenia, and replacement with fat around organs and in bone marrow can be explained by diet-based chronic inflammation and inadequate weight-bearing exercise.

A major risk factor for osteoporosis is omega-6-rich vegetable oils, e.g. corn, soy, etc. In simple terms, the first step that I would recommend for anyone concerned about osteoporosis is to shift to an anti-inflammatory diet. Eliminate all vegetable oils, except olive oil, from the diet and supplement with omega-3 fish oils (short-chain omega-3 oils in most vegetable sources are much less effective.)

The biomedical literature is very clear. Osteoporosis is not normal, is not a part of aging and can be avoided. There are some genetic predispositions to osteoporosis, but most can be overcome by meaningful diet and lifestyle changes. An anti-inflammatory diet and lifestyle (sunlight for vitamin D and exercise) is the cheapest, safest and most effective way to prevent and treat osteoporosis.

Osteoporosis

Bone density is based on the balance between bone production and demineralization. Inflammatory cell signaling is required for release of calcium. Muscle building exercise favors increased bone density.

Newborns do not have fully formed bones in their limbs. The reason that milk has so much calcium, is that babies mineralize their cartilage bone scaffolds after they are born. Cartilage is made by chondrocytes (sisters of blood vessel endothelial cells and fat adipocytes, with the same stem cell parents) and the chondrocytes will continue to burrow through existing cartilage and make new cartilage, if mineralization does not take place. The cells that synthesize bone are called osteoblasts. They adhere to a framework of cartilage and begin to secrete collagen I, the major protein of bone and osteocalcin, the calcium binding protein that initiates the deposition of hydroxyapatite [Ca5(PO4)3(OH)]. As the bone forms, the osteoblasts become trapped in lacunae within the bone and stop secreting osteocalcin and begin to secrete hormones in response to the mechanical stress on the bone.

Bone is degraded by osteoclasts that colonize the completed bone after migrating from bone marrow. The total bone mass and density is determined by the dynamic balance between the deposition of bone by osteoblasts and disassembly of bone by osteoclasts. Approximately 10% of bone is being remodeled at any time and the porus trabecular bone in the pelvis, hips, wrist and spine is most actively remodeled. If there is an imbalance that leads to a bone deficit, it usually shows in weak trabecular bone.

Problems with low bone density, i.e. osteoporosis, can result from decreased estrogen (menopause), inadequate vitamin D/sunlight/dietary calcium, or medication, e.g. heparin or warfarin.

The ability of heparin to cause osteoporosis with prolonged use caught my attention. Heparin is anti-inflammatory and inflammation reduces heparin production. Thus, the inflammation caused by high blood glucose levels in diabetics results in loss of heparin production in kidneys and loss of protein from the urine. If heparin causes loss of bone mass, then it might be decreasing inflammation that is needed for bone accumulation.

Osteoclasts are activated by the RANK (receptor activator of nuclear factor κB) system. As the name states, RANK is a receptor that activates the inflammatory transcription factor NFkB. The cytokine that binds to RANK is the corresponding ligand, RANK-L, which is related in structure (and function) to TNF. RANK-L is secreted by osteoblasts, binds to RANK on osteoclasts, activates NFkB and stimulates bone demineralization. A protein called osteoprotegerin, is a soluble receptor of RANK-L that binds the bone and immobilizes the RANK-L and keeps it from activating osteoclasts.

Heparin could interact with many of these components. For example, the binding of RANK and RANK-L is mediated by heparan sulfate proteoglycans. The heparin deficiency that usually accompanies inflammation, and in this case excitation of osteoclasts, could be decreased by administration of heparin. Thus, demineralization would result in osteoporosis.

Warfarin-based osteoporosis could be based on upsetting vitamin K metabolism in osteoblasts. Vitamin K recycling is inhibited by warfarin and vitamin K is needed for a special modification of glutamic acids in particular proteins, such as osteocalcin. The action of osteocalcin in binding calcium is based on three glutamic acids that have been carboxylated using vitamin K. This is shown in the figure as three green calcium atoms bound to red dicarboxylic glutamic acids. You can also notice that the osteocalcin also has a substantial heparin binding domain (blue) at the top. Thus warfarin could cause osteoporosis by disrupting mineralization.

When I was trying to figure out the warfarin/osteoporosis relationship, I tried to find protein structures in the NCBI data base, which had warfarin bound. All I found was warfarin bound to human serum albumin, the protein that carries warfarin and many alkaloids through the blood. I was always suspicious of the use of heparin and warfarin somewhat interchangeably in many different settings in which the mode of action was assumed to be anticoaggulation of blood. I was not surprised when I found that the aromatic rings of warfarin (oxygens in red) were bound to arginines (blue) in a ligand-binding pit on the serum albumin.

A practical note on osteoporosis is that this disease is an exception to many of the degenerative and autoimmune diseases that are based on an inflammatory diet. Osteoporosis is more similar to the problem of gut injury by aspirin. Aspirin blocks COX-2 the enzyme that produces inflammatory and anti-inflammatory prostaglandins from omega-6 and omega-3 fatty acids, resp. Taking aspirin can block inflammation, but the integrity of the lining of the stomach and intestines requires inflammatory prostaglandins, so aspirin can also lead to a bleeding gut. Osteoclasts require NFkB signaling and other aspects of bone production may also require an inflammatory environment. This may explain why corticosteroids also lead to osteoporosis.

Deposition of bone is stimulated by weight bearing exercise that is consistent with the anti-inflammatory lifestyle.

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.

Asthma Nitric Oxide

Arginine is the source of nitric oxide using the enzyme NOS. Inducible NOS is expressed in response to triggers of inflammation mediated by NFkB. NO in turn activates guanylate cyclase to increase cyclic GMP and cGMP relaxes muscle actin and myosin.

Studies are being made to assess the status of asthma by measuring nitric oxide (NO) in respired air. Since NO is produced as a product of the action of induced nitric oxide synthase (iNOS), and iNOS is synthesized when the inflammatory transcription factor NFkB is activated, then the inflammation that is a characteristic of asthma should be signaled by the production of NO.

If NO is involved in asthma, then one would expect relaxation of muscle to occur. That is the opposite of what is observed; contraction of the muscle cells surrounding airways is responsible for airway constriction is asthmatic episodes. Thus, during asthmatic episodes NO must be reduced.

Since NO is made from the amino acid arginine, then the ability to produce NO can be decreased by reducing available arginine. Arginine can be reduced by the enzyme arginase. The product is ornitine that can in turn be converted into purtrescine, the simplest of the polyamines that are used as counterions in the secretion of heparan and chondroitin sulfates.

Arginase is produced in high levels in asthmatics, but not in normal lungs. Also elevated putrescine is found in asthmatic lungs, indicating that arginine is being converted by arginase into ornithine and on to purtrescine. Ornithine is the precursor for proline that is a key amino acid in collagen production and the increased extracellular matrix that thickens the tissue of asthmatic lungs is rich in collagen.

A major point of this discussion is that NO can only be effectively lowered if there is a limited supply of arginine. Thus arginine supplementation should be helpful to asthmatics. Nitroglycerine patches may also be a means of enhancing sources of NO. Another possible approach would be the use of NO donors conjugated to NSAIDS. These conjugates delivered in an atomized form could provide the lungs with both NO and anti-inflammatory agents. Asthmatics also respond well to rigorous application of the anti-inflamatory diet and lifestyle.

Palmitoleate: omega-7 lipokine

Palmitoleic acid is responsible for keeping people healthy (lipokine) and for the smell of old people (nonenal). Overproduction of this lipid by blocking uptake of fats results in resistance to type II diabetes and atherosclerosis.

If you listen to the commercials on television, you know that there are two sources of fats/cholesterol; you either make it in your cells or take from your diet. Recent research shows that mice that have been genetically modified to lack cytoplasmic lipid carriers, can’t store dietary fat, so they make more of their own lipids. Specifically, they make more palmitoleic acid (C16:1n7). By U.S. standards, those defective mice are very healthy. It turns out that palmitoleic acid acts as a lipid hormone that communicates between fat tissue and other organs, and maintains a healthy metabolic balance.

Lipids are hydrophobic and require protein carriers to be moved from their source, such as the intestines, through the blood and to be offloaded into tissues. Most of the lipids enter the diet as triglycerides, i.e. a three carbon glycerol with three fatty acids attached. Those fats are extracted from food with bile, which is a mixture of modified cholesterol salts that acts as a detergent to dissolve fats. The dissolved fats, in the form of chylomicrons (big fat droplets coated with a lipid layer and proteins) are produced by intestinal cells and released into the blood stream. During transit, a lipase removes the fatty acids from the triglycerides. As in all of the lipid transport systems, the protein carriers determine how the lipid contents are distributed.

If a fatty acid or triglyceride is added to a cell membrane, the lipid would get stuck in the membrane's double layer of phospholipids. Fat droplets in cells are nothing more than fats that are loaded into the membrane of a cellular vesicle until a droplet covered by a half membrane forms. The alternatives for lipid transport are the HDL and LDL (protein coated lipids of the blood), and the intracellular fatty acid-binding proteins. The proteins bound to the surface of LDLs and HDLs bind to receptors on cell surfaces and control transfer of lipids to and from cells. One example is apolipoprotein E4. This protein is intimately involved in determining risk for athersclerosis and Alzheimer’s. ApoE4 binds to its cell surface receptor via heparin. Note the blue basic amino acids that form a massive heparin-binding domain down one side of the protein.

Fatty acid-binding proteins (FABPs) are just globular proteins, with hydrophobic amino acids arranged in the center and hydrophilic, water-bonding, amino acids on the surface. I have drawn the structure of a FABP using a graphics program called Chimera to visualize X-ray crystallographic data in the National Center for Biomedical Information (NCBI) database. The continuous chain of amino acids is shown as a white ribbon and the surface of the protein is shown as a transparent overlay. The protein chain makes a cage with the hydrophobic parts of the protein pointing toward the center to make a hydrophobic-lined container for the trapped fatty acid (pink). The two ends of the protein vessel are held closed by interdigitation of tryptophans (yellow) and basic amino acids (arginine and lysine, dark and light blue). The FABP also has a nuclear translocation signal, a group of four basic amino acids and other concentrations of basic amino acids displayed linearly across the surface of the cage (not shown), that probably are involved in transport of the trapped fatty acid from the cell surface to the surface of the nuclear envelope, which is involved in phospholipid assembly.

Mice engineered to have the human ApoE4 gene develop atherosclerosis and type II diabetes. If the FABPs of the fat cells of these mutant mice have also been removed, then the mice are essentially normal. Removal of the FABPs blocks the uptake of dietary fatty acids and stimulates the production of endogenous lipids, including the omega-7 fatty acid palmitoleic acid. This fatty acid is a lipid hormone, lipokine that stimulates normal metabolism and provides protection against several inflammation-based diseases. Interestingly, palmitoleic acid accumulates abundantly in the skin of old people and is converted to nonenal that has the smell of old books.

Cao H, Gerhold K, Mayers JR, Wiest MM, Watkins SM, Hotamisligil GS. 2008. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 134(6):933-44.

Heparin Binding

This little video clip that I put together shows how a growth factor binds to its receptor. Heparin mediates the process. The growth factors bind to receptors in pairs with the heparin threaded through all four proteins and holding the complex together. The receptors would be embedded in the membrane at the bottom. This clip starts with a single receptor to which the heparin binds first, followed by the first growth factor.  The basic amino acids, arginine and lysine are shown in blue.  In the first image, the amino acid backbone is shown as a ribbon with blue bars.  In the second, the atoms of the amino acid side chains are filled in and the blue bars become blue atomic balls that reveal the surface of the protein.  The blue areas are positively charged and groups on the surface are heparin-binding domains.  The heparin is shown as a polysaccharide with atoms colored by element, O-red, S-orange, N-blue.  The proteins bind to the heparin like beads on a string.  Pairing of the receptors in a particular orientation determined by the heparin and growth factors, is responsible for triggering the cytoplasmic signal that the growth factor is present.

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.

Asthma

Asthma is chronic constriction and inflammation of the airways of the lungs. Aggrevation of these symptoms can result from allergens, exercise, stress or infection. Once lungs become asthmatic the episodes of symptoms can be controlled by drugs that usually include broncodilators. But what is the cause of asthma?

Risk factors suggest the source of the disease. Exposure to smoke or inhaled pollutants, antibiotic use, formula use, obesity (metabolic syndrome), use of chlorinated swimming pools all increase the likelihood of developing asthma. The common factor in all of the risks is chronic systemic inflammation combined with some irritation of the lungs. Systemic inflammation of asthmatics is frequently the result of altered gut flora. Use of formula or antibiotics, for example, lead to a disruption of normal newborn gut bacteria, replacement with hospital strains, e.g. Clostridia spp., and inflammation. Obesity can also lead to inflammatory gut flora. Systemic inflammation combined with lung irritants, e.g. allergens, chlorinated organic compounds, infections, can lead to allergic responses and in the lung this can mean asthma.

An asthmatic attack starts with spasmodic contraction of the airway muscles, followed by inflammation. Initially, so called cholinergic receptors located in the membranes of the smooth muscles that surround the airways of the lungs, are stimulated by the binding of neurotransmitters. The result is that these receptors trigger the cleavage of phosphoinositol diglycerides to release inositol with three phosphates (IP3) and a glycerol with two lipid chains (diacylglycerol, DAG). The IP3 (which mimics heparin, by the way) binds to receptors controlling the internal stores of calcium (in the endoplasmic reticulum), causing a surge of intracellular calcium. The rise in intracelllular calcium is what causes the muscle cell actin and myosin to contract and the rings of muscle on the airways to constrict. This is ridiculously simplified to show the basic components of stimulating receptors, IP3 increase, calcium increase and muscle contraction.

Early attempts to treat asthma used plant extracts that blocked the action of the cholinergic (as in acetylcholine) receptors. These are familiar plants such as tobacco, coffee, tea and cacao. The cholinergic blockers are nicotine (pictured and in computational model below), caffeine, theophylline and theobromine. Datura stramonium, Jimson weed, also produces atropine, that is another cholinergic inhibitor. There are modern forms of the cholinergic antagonists, which are used to treat episodic constriction.

I have illustrated in the model above, how nicotine binds to the cholinergic receptor, between a tryptophan (bottom, yellow) and two tyrosines (above, orange).

The IP3-calcium constriction system can also be stimulated by allergen-based mast cell release of histamine. The histamine binds to another set of receptors and triggers IP3 release. In this context, anti-histamines are moderately effective, but the initial response is so rapid, that the histamine phase is quickly passed.

The rapid constriction phase is followed six to eight hours later by inflammatory release of leukotrienes that also cause constriction via IP3. Leukotrienes share the same precursor, omega-6 arachidonic acid, as the inflammatory prostaglandins made by the cyclooxygenase, COX 1&2, but in this case an alternative enzyme, lipoxygenase, is used. The leukotriene receptors on muscle cells act similarly to the catechol receptors and stimulate constriction.

Another approach to preventing constriction or actively relaxing airway smooth muscles is by stimulation of adrenergic receptors that have the opposite effect of cholinergic receptors. The adrenergic receptors trigger cyclic AMP production, that compromises the IP3-calcium signaling. Cholinergic receptors are activated to minimize the damage to inhaled toxins or irritants. Adrenergic receptors open the airways to increase airway capacity for flight or fight in response to adrenalin. Some of the drugs to treat asthma stimulate adrenergic receptors and open airways.

Treatment of asthma is centered on constriction episodes and not on reversal or prevention of the disease. Thus, treatment or prevention of an asthmatic episode of airway constriction can utilize drugs that block steps along the cholinergic cascade (receptor, IP3 increase, calcium increase) or by stimulation of adrenergic receptors.

These treatments do not address the cause or reversal of asthma. The inflammatory basis of most asthma risk factors suggests that lowering chronic inflammation, particularly in young children, should be a high priority in preventing asthma. The association of asthma with the use of formula and antibiotics, implicates inflammation, as well as inflammatory gut flora, as the foundations of asthma and emphasizes the essential role of diet and lifestyle in the development of asthma.