Rosacea: Alzheimer’s of the Face

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

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

Amyloids:

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

Anti-microbial Peptides:

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

Abeta Is Anti-microbial Like LL-37

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

Implications of Abeta as an AMP Like LL-37

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

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

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

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

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

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

Heparin, Growth Factors and Rosacea

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

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

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

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

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

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

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

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

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

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

Human Gut Flora Genes

Thousands of Species, Millions of Genes

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

Major Findings:

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

Feces from 124 Europeans

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

Illumina Multiplex Sequencing of PCR Amplified Fragments

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

Comparison to 89 Existing Human Gut Bacteria Genome Sequences

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

Polysaccharide/sugar Metabolism Common among Gut Flora

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

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

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

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

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

ABO Oligosaccharides, RBCs, VWF, Endothelial Cells, Megakaryocytes

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

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

ABO Antigens Are Oligosaccharides, Not Proteins

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

Glycosyl Transferases - Enzymes that Assemble ABO Oligosaccharides

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

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

Dominance Means Functional Enzyme, Recessive Means Dysfunctional

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

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

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

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

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

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

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

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

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

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

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

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

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

Last Week of the Eades Cure

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

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

The First Weeks of The Cure

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

The Middle Weeks of The Cure

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

The Last Weeks of The Cure

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

Gut Flora Matter

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

Hunger Comes with Carbs

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

BMs Are Bacterial Motivated

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

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

The Cure Works

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

Arthritis, Autoimmunity and Arginine Deimidation

Celiac and Antibody Production Against Tissue Transglutaminase as a Model

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

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

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

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

Arthritis Is Mediated by Autoantibodies to Peptidylarginine Deiminase and Citrullinated Proteins

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

PAD Also Has a Triplet of Basic Amino Acids for Internalization

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

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

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

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

The Eades Cure Week 4

Meals, Alcohol, Caffeine, but no Dairy

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

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

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

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

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

Constipation, Gut Flora and Health

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

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

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

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

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

Feces Is Primarily Bacteria

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

Pathogenic Gut Flora

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

Disruption of Gut Flora Leads to Disease

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

Gut Controls Immune System

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

Change Your Gut Flora and Change Your Health

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

Anti-Inflammatory Diet Should Support Healthy Gut Flora

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

Cure for Middle-Aged Middle

My First Two Weeks with the Drs. Eades Diet

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

The Cure Reduces Visceral Abdominal Fat

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

Basic Outline of The Cure

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

After the First Two Weeks of Protein Shakes

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

Weight Loss Was Effortless

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

Reduced Need for Fish Oil with Altered Gut Flora

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

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

Rosacea, Brain Cooling and Niacin Flush

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

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

Rosacea is Persistent Vasodilation of the Face with Accumulation of Neutrophils

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

Facial Blood Circulation to Cool the Brain

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

Nerves to the Face

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

Cathelicidin, Vitamin D Receptor, DNA Complexes, Autoinflammation

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

Niacin Flushing Implicates Arrestins

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

Gut Flora, Biofilms and Cryptic Bacteria

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

Treatment with Anti-Inflammatory Diet

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

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

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

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

2009: What I Learned Last Year

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

What We Eat Contributes More to Disease Risk than Genetics

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

Diet-Based Inflammation Is the Major Risk

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

Anti-Inflammatory Diet and Lifestyle Is the Cure

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

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

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

Living with Inflammation

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

Resolve to Eat Your Way to Health

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

Humans vs. Naked Mole Rats

Blood Sugar, Insulin, Superoxide, Couch Potatoes

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

Mole Rats:

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

Humans:

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

Naked Mole Rats Are as Unique as Humans

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

NMRs Know When to Stop

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

Humans Are Runners

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

Humans Kill for Fat

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

Sweet Tooth Is Deciduous

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

Marauding Naked Mole Rats

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

BPA in Thermal Printer Ink

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

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

Some Receipts Are Covered with BPA

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

Thermal Printing Heats BPA with Second Reagent to Make Pigments

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

Don’t Touch the Receipts

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

Wash Your Hands or Wear Gloves When Shopping

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

Not All Receipts Have BPA

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

Superoxide Causes Insulin Resistance, Type 2 Diabetes

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

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

High Energy Electrons of Glucose Are Used to Make ATP

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

Cells Adjust their Glucose Individually to Match ATP Use

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

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

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

Superoxide Is a Reactive Oxygen Species (ROS)

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

Many Different Processes that Produce Insulin Resistance all Produce Superoxide

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

Insulin Resistance Is a Natural Defense Against Energy Excess

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

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

Bacterial Amyloid (Curli Fibers) Forms Biofilms

E. coli Curli Stacks in Congo Red Staining Fibers

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

Biofilm Formation from Secreted Proteins and Polysaccharides

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

Congo Red Stains Amyloids, Cellulose and Rare LPS



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

Faces of Sugars Are Hydrophobic

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

Basic Amino Acids Bind Hydrophobically

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

Amyloid Binds Congo Red by Stacked Heparin-Binding Domains

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

Bacterial Biofilms Form from Amyloids and Polysaccharides

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

Do Biofilms Foment Amyloid Production?

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

Berberine Binds Heparin and Blocks Amyloids and Biofilms

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

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

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

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

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

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

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

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

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

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

Biofilms as Human Gut Mycorrhizals

Are Biofilms Healthy Extensions of Intestinal Villi?

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

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

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

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

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

Biofilms Coat the Intestinal Villi

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

Bacterial Community Composition May Be Determined by Diet

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

Communication within Biofilms and with the Intestines

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

Stimulation of Tregs

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

Helicobacter Pylori, Gastric Ulcers and Cancer

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?)