Anti-Inflammatory Diet

All health care starts with diet. My recommendations for a healthy diet are here:
Anti-Inflammatory Diet and Lifestyle.
There are over 190 articles on diet, inflammation and disease on this blog
(find topics using search [upper left] or index [lower right]), and
more articles by Prof. Ayers on Suite101 .

Wednesday, December 30, 2009

2009: What I Learned Last Year

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.

Wednesday, December 9, 2009

Humans vs. Naked Mole Rats

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

  • 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.

Monday, December 7, 2009

BPA in Thermal Printer Ink

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.

Tuesday, November 24, 2009

Superoxide Causes Insulin Resistance, Type 2 Diabetes

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.

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.

Tuesday, November 17, 2009

Bacterial Amyloid (Curli Fibers) Forms Biofilms

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.

Thursday, November 12, 2009

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

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.$=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.
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.$=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.
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.

Thursday, November 5, 2009

Biofilms as Human Gut Mycorrhizals

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.

Friday, October 30, 2009

Helicobacter Pylori, Gastric Ulcers and Cancer

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?)

Thursday, October 22, 2009

Erectile Dysfunction Diet

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.

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

Thursday, October 15, 2009

Migraine Headache Diet

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

  1. Vitamin D -- deficiency is common... even with adequate sun exposure
  2. Low carbs -- starch is hyperglycemic, grain gluten intolerance is very common
  3. Vegetable oils -- only olive oil is safe (trans fats are dangerous), butter is better
  4. Fish oil -- omega-3 oils can reduce chronic inflammation
  5. High fructose corn syrup -- eliminate all sources
  6. 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.

Monday, October 12, 2009

Biofilm Transformation, Helicobacter, Klebsiella

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.

Tuesday, October 6, 2009

Cytstic Fibrosis Overproduces Tissue Transglutaminase and Contributes to Celiac

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.

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.

Monday, September 28, 2009

Paradoxical Inflammation

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.

Thursday, September 17, 2009

Vagus Nerve Controls Gut Inflammation II

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.]

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]

Thursday, September 10, 2009

Watson Makes Us Sick

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.


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.

Wednesday, September 2, 2009

Cure for Inflammatory Diseases

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.

Tuesday, August 18, 2009

Anti-inflammatory, Gluten-Free Diet for Celiac

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.

Sunday, August 16, 2009

Recombining H1N1 and H5N1 Is Very Scary

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.

~PQRE TRGLFG~ ABB88379 Mexico 1994
~PQRK TRGLFG~ ABQ84472 Italy 1993
~PQRKRKRKTRGLFG~AAC58990 Mexico 1995
~PQRE RKKRGLFG~ ABQ84473 Italy 1997
~PQRERRRKKRGLFG~ AAD37782 China 1996
~PQRK RKTRGLFG~ ACL79965 Mexico 1994

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.

Friday, August 7, 2009

Cause of Autoimmunity and Allergies

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.


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.