Loren Cordain, Ph.D.

1
Potential Therapeutic Characteristics of
Pre-agricultural Diets in the Prevention and
Treatment of Multiple Sclerosis
Loren Cordain, Ph.D. Colorado State
University Fort Collins, CO USA
2
The Hominin Fossil RecordPlio-Pleistocene Diets
Homo sapiens
H. heidelbergensis
0
H. erectus
Paranthropus boisei
H. neanderthalensis
1
H. antecessor
Australopithecus habilis
P. robustus
2
H. ergaster
Au. garhi
Au. rudolfensis
P. aethiopicus
3
Au. africanus
Kenyanthropus platyops
Au. bahrelghazali
Ardipithecus ramidus
4
Millions of Years
Au. afarensis
Au. anamensis
5
Orrorin tugenensis

• As many as 20 hominin species may have existed
  since the evolutionary split between hominins and
  pongids (5-7 MYA)
• No universal diet existed, but rather varied by
  ecologic niche, season, geographic locale,
  availability of edible foods

6
7
Sahelanthropus tchadensis
8
Wood B. Palaeoanthropology hominid revelations
for Chad. Nature 2002418133-35
3
Plio-Pleistocene Hominin Diet The Known Foods
That Couldnt Have Been Eaten
Highly Processed, Refined Foods
Minimally Processed, Wild Plants
Minimally Processed, Wild Animals
What are the Health Implications?
4
These foods comprise (gt70 energy) in typical
Western DietsBut were virtually unknown in
Ancestral Human Diets
Breads, Cereals, Rice and Pasta
Dairy Products
Added Salt
Refined Vegetable Oils
Refined Sugars (except honey)
Fatty Meats
Alcohol
Cordain et al. Am J Clin Nutr 200171682-92
5
Refined sugars, grains, vegetable oils and dairy
70.9 of energy in the U.S. food supply
Meats, Fish
Eggs
Nuts, Seeds Legumes
Refined Sugars
Fruits
Refined Vegetable Oils
Dairy
Grains
Vegetables
Miscellaneous

• Refined sugars, grains, vegetable oils and dairy
  represent Neolithic Industrial era foods that
  were not present in traditional ancestral human
  diets
• By default, their inclusion displaces minimally
  processed, wild plant and animal foods.

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
6
Evolution of the Western DietNeolithic (10,000
to 5,500 yrs ago) Food Introductions
SUCROSE
FIRST SALT MINES
FIRST DAIRYING EVIDENCE
WINE BEER
WHEAT BARLEY DOMESTICATED 10,000 YRS AGO
SHEEP, GOATS, COWS DOMESTICATED
Years ago
present
Human Generations
0
66
100
133
167
200
233
267
300
333
33
7
Evolution of the Western DietIndustrial
Revolution (200 yrs ago)
HFCS
HYDROGENATED OILS
REFINED VEGETABLE OILS
REFINED GRAINS
FEEDLOT PRODUCED MEATS
SUCROSE
Year
2007
Human Generations
0
2
3
4
5
6
7
1
8
Generations (30 yrs) in the Evolution of Humanity

• Generations Total
• Homo habilis (1st Homo species ) 76,667 100.0
• Homo erectus (modern body size) 60,000
  78.2
• Modern Homo sapiens (cranial size) 6,666
  8.7
• Agricultural Revolution (cereals) 333
  0.4
• Advent of Dairying (milk, cheese etc)
  200 0.26
• Industrial Revolution (refined sugars,
  7 0.009
• refined cereals, oils, canned food)
• Food Processing Industry (junk food)
  4 0.005

Conclusion 99.6 of all Homo generations had
no evolutionary experience with commonly consumed
modern foods introduced during the Neolithic!
9
Neolithic and Industrial Era Foods Nutritional
Implications

• As Neolithic Industrial Era foods displace
  minimally processed, wild plant and animal foods,
  they adversely affect the following nutritional
  factors
• 1. The Glycemic Load
• 2. The Fatty Acid Balance
• 3. The Macronutrient Balance
• 4. The Trace Nutrient Density
• 5. The Acid/Base Balance
• 6. The Sodium/potassium Balance
• 7. The Fiber Content

Disruption of these 7 nutritional components
fundamentally underlies much of the chronic
diseases in the Western World
10
Plio-Pleistocene Hominin Diet The Known Foods
That Couldnt Have Been Eaten(Cereals)
Contribution of Cereals To Total Energy in the
U.S. Diet

• Item total energy
• Whole grains 3.5
• Refined grains 20.4
• TOTAL 23.9
• 85 of all grains are consumed as refined grains

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
11
Ancestral Human Diet Foods That Couldnt Have
Been Eaten(Cereals)

• Cereal grains which are the seeds of grasses
  (Gramineae) in their wild state are
• 1. Small
• 2. Difficult to harvest
• 3. Minimally digestible without (a) grinding to
  break down cell walls (b) cooking to gelatinize
  starch granules

Cordain L. Cereal grains humanitys double
edged sword. World Review of Nutrition and
Dietetics 19998419-73
12
Ancestral Human Diet Foods That Couldnt Have
Been Eaten(Cereals)

• Thus, the appearance of crude grindstones and
  mortars in the Middle East (Natufians) and
  elsewhere (10-15,000 years ago) heralds the
  beginnings of humanitys use of cereal grains as
  a staple food

Bar-Yosef O. The Natufian culture in the Levant,
threshold to the origins of agriculture. Evol
Anthropol 1998 6159-177. Wright K. The origins
and development of ground stone assemblages in
Late Pleistocene Southwest Asia. Paleorient
19911719-45
13
High Glycemic Foods

• ALMOST ALL REFINED GRAINS HAVE HIGH GLYCEMIC
  INDICES
• Rice Chex Cereal 89
• Corn flakes 84
• Pretzels 83
• Rice Krispie Cereal 82
• Rice Cakes 82
• Rye bread 76
• Waffles 76
• Total Cereal 76
• Graham crackers 74
• Cheerios 74
• Bagels 72
• Short grain white rice 72
• Corn chips 72
• White bread 70
• Whole Wheat bread 69

HIGH G.I. FOODS gt 70 MEDIUM G.I. FOODS 55-70 LOW
G.I. FOODS lt 55
Foster-Powell K et al. Am J Clin Nutr 2002765-56
14
High Glycemic Load Carbohydrates Promote
Diseases of Insulin Resistance
The Metabolic Syndrome

• Type 2 Diabetes
• Hypertension
• Coronary Heart Disease (CHD)
• Dyslipidemia (Reduced serum HDL cholesterol,
  elevated triglycerides, elevated VLDL, elevated
  small dense LDL cholesterol)
• Obesity
• Gout

Liu S et al. Dietary glycemic load and
atherothrombotic risk. Curr Atherosclerosis Rep
20024454-61 Ludwig DS. The glycemic index.
Physiological mechanisms relating to obesity,
diabetes and cardiovascular disease. JAMA
20022872414-23.
15
Plio-Pleistocene Hominin Diet The Known Foods
That Couldnt Have Been Eaten(Dairy)
Contribution of Dairy Products To Total Energy in
the U.S. Diet

• Item total energy
• Whole milk 1.6
• Low fat milks 2.1
• Cheese 3.2
• Butter 1.1
• Other 2.6
• TOTAL 10.6

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
16
Ancestral Human Diet Foods That Couldnt Have
Been Eaten(Dairy)
Ever tried to approach a wild animal? How About
Milking It?
17
Ancestral Human Diet Foods That Couldnt Have
Been Eaten(Dairy)
Copley MS et . Proc Natl Acad Sci
20031001524-29
FIRST DAIRYING EVIDENCE
SHEEP, GOATS, COWS DOMESTICATED
Years ago
present
Human Generations
O
100
150
200
250
300
350
400
450
500
50
Hiendleder S et al. Proc R Soc Lond B
2002269893-904 (SHEEP) Luikart G et al.
Proc Natl Acad Sci 2001985927-32 (GOATS)
Loftus RT et al. Mol Ecol 1999 82015-22 (COWS)
18
Evolution of the Western DietNeolithic Food
Introductions (Dairy)
Despite a low glycemic load, dairy products
paradoxically have insulin indices similar
to white bread

• ALMOST ALL REFINED GRAINS HAVE HIGH GLYCEMIC
  INDICES
• Rice Chex Cereal 89
• Corn flakes 84
• Pretzels 83
• Rice Krispie Cereal 82
• Rice Cakes 82
• Rye bread 76
• Waffles 76
• Total Cereal 76
• Graham crackers 74
• Cheerios 74
• Bagels 72
• Short grain white rice 72
• Corn chips 72
• White bread 70
• Whole Milk 27
• Yogurt 24

Foster-Powell K et al. Am J Clin Nutr 2002765-56
Ostman EM et al. Am J Clin Nutr 20017496-100
19
The Displacement of Game Meats Fish by Dairy
Foods Increases Saturated Fats at the Expense of
Polyunsaturated Fats Monounsaturated Fats
Total Fats
Total Fat (74 fat)
(49 fat)) (46.0 fat)
(19.0 ) (by energy)
Increased Saturated Fat Increased risk for
Syndrome X, CHD, certain cancers
20
Plio-Pleistocene Hominin Diet The Known Foods
That Were Rarely Eaten(Fatty Meats)
Salami 74 Fat, 22 Protein
Bacon 77 Fat, 21 Protein
Ground Beef 64 Fat, 33 Protein
Pork Ribs 72 Fat, 26 Protein
Hot Dogs 82 Fat, 14 Protein
T-bone Steak 68 Fat, 30 Protein
21
Wild vs. Domestic Animals
Caribou

• Body fat in wild animals waxes and wanes
  seasonally
• With the advent of animal husbandry 10,000 years
  ago, it became possible to attenuate or prevent
  the seasonal decline in body fat by
  provisioning captive animals with plant food
• It also became feasible to consistently slaughter
  the animal at peak body fat

Increased Saturated Fat Increased risk CHD,
Syndrome X, certain cancers
22
Total ?-3 Fatty Acids in Wild, Grass and Grain
Fed Animals Muscle Meat (100 g sample)
Total n- 3 fatty acids (mg)
Diseases linked to reduced ?-3 fatty acids
Syndrome X,CHD, cancer, autoimmune diseases, all
inflammatory (itis) diseases
Cordain et al. Eur J Clin Nutr 200256181-91
23
Total Salt (NaCl) in the U.S. Diet (Grams per Day)
Plio-Pleistocene Hominin Diet The Known Foods
That Were Rarely Consumed (Added Salt)

• Source grams/day
• Added in processed foods 7.2
• Table salt and cooking use 1.4
• Naturally occurring in foods 1.0
• TOTAL 9.6

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
24
Plio-Pleistocene Hominin Diet The Known Foods
That Were Rarely Consumed (Added Salt)
The Mountain of Salt (Cardona, Catalonia, Spain)

• Salt was known to be gathered on a dry lake bed
  in China 8,000 years ago
• First inland salt mines appear in Europe 6,000
  years ago
• Hunter gatherers living near the ocean dipped
  food in seawater and used dried sea salt
• Inland hunter-gatherers rarely used salt on a
  regular basis

The first known salt mine in Europe (6,200 -
5,600 years ago)
Weller O. Antiquity 200276317-18.
Diseases linked to salt consumption
Hypertension, stroke, osteoporosis,
kidney stones, Menierres Syndrome, stomach
cancer, insomnia, motion sickness,
asthma, exercise induced asthma
25
Contribution of Refined Sugars to Total Energy
in the U.S. Diet
Plio-Pleistocene Hominin Diet The Known Foods
That Were Not Consumed (Refined Sugars)

• Item total energy
• Sucrose 8.0
• High fructose
• corn syrup 7.8
• Glucose 2.6
• Syrups 0.1
• Other 0.1
• TOTAL 18.6

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No.
55 U.S.D.A. Economic Research Service, 2002.
Food consumption (percapita) data system,
sugars Sweeteners, Washington D.C.
26
Evolution of the Western DietIndustrial Era
Food Introductions (Refined Sugars)
Per Capita Sugar (Sucrose) Consumption in the
Netherlands (1745-1937)
Per capita consumption (lbs.)
1937
Year
27
Plio-Pleistocene Hominin Diet The Known Foods
That Were Not Consumed (Refined Sugars)

• Diseases linked to refined sugars
• Metabolic Syndrome (Type 2 diabetes, CHD,
  dyslipidemia, obesity, gout, hypertension
• Dental caries
• Certain cancers

Cordain L et al. Hyperinsulinemic diseases of
civilization more than just syndrome X. Comp
Biochem Physiol Part A 200313695-112.
28
Contribution of Refined Vegetable Oils to Total
Energy in the U.S. Diet
Plio-Pleistocene Hominin Diet The Known Foods
That Were Not Consumed (Refined Vegetable Oils)

• Item total energy
• Salad, Cooking Oils 8.8
• Shortening 6.6
• Margarine 2.4
• TOTAL 17.8

Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
29
Per Capita Change in Refined Vegetable Oils in
the U.S. (1909-99)
kg
Total vegetable oil consumption has increased 459
since 1909
Salad, Cooking Oil consumption has increased 1340
since 1909
Margarine consumption has increased 488 since
1909
Shortening consumption has increased 237 since
1909
Gerrior S, Bente I. 2002. Nutrient Content of the
U.S. Food Supply, 1909-99 A Summary
Report. U.S.D.A, Center for Nutrition Policy and
Promotion. Home Economics Research Report No. 55
30
Plio-Pleistocene Hominin Diet The Known Foods
That Were Not Consumed (Refined Vegetable Oils)

• Vegetable oils are high in ?-6 fatty acids, but
  low in ?-3
• Diseases linked to high ?-6/ ?-3
• Metabolic Syndrome (Type 2 diabetes, CHD,
  dyslipidemia, obesity, gout, hypertension),
  cancers, autoimmune diseases, virtually all
  inflammatory (itis) diseases

31
Recommendations for a Contemporary Diet Based
Upon Paleolithic Food Groups
Fish Seafood
Lean Meats
Fresh Fruits
Fresh Veggies
Nuts/Seeds
32
Neolithic Food Introductions Potential Factors
underlying Multiple Sclerosis
Dietary Lectins ?
Milk and Dairy ?
Legumes Beans
Legumes Peanuts
Tomatoes
Whole Grains Wheat
33
What is Multiple Sclerosis ?
Damaged Myelin in Multiple Sclerosis

• Multiple Sclerosis is an autoimmune disease in
  which the immune system destroys the covering
  (myelin sheaths) of nerve cells in the brain
  spinal cord (CNS)
• Activated T cells attack proteins within the
  myelin which appear to be foreign
• While healing may occur (remission), plaques
  (scarring) frequently develop throughout the CNS
  which impair motor and sensory function
• M.S. afflicts 1 in 1,000 people or 300,000
  people in the U.S.

Nerve Cell
Brain Signal normal
Normal Myelin
Nerve Fiber
Muscle Fiber
T Cells
Damaged Myelin (plaques)
Brain signal is blocked because of
damaged myelin
34
What Causes Multiple Sclerosis (Genetics
Environment)

• It is well recognized that both genetic endowment
  and environment play important roles in MS
  pathogenesis
• The former being stronger the latter still
  unidentified 1.
• The MHC locust where the HLA DRB11501,
  DQA10102, and DQB10602 haplotype is well
  validated as the major genetic risk factor for
  MS2.
• The low concordance of MS in monozygotic twins
  (25.3) 3 clearly is indicative that
  environmental factors must be involved.

1. Poser CM. Clin Neurol Neurosurg 2006108
227-33 2. Hafler DA et al. Nat Rev Immunol
2005583-91 3. Willer C et al. Proc Natl Acad
Sci 200310012877-82
35
Multiple Sclerosis Etiology Suspected
Environmental Factors

• Infection Epstein Barr virus, Chlamydia
  pneumoniae, Mycoplasma pneumoniae, viruses
  causing influenza, measles, rubella
• Geography Higher latitude increases risk
  sunlight UV exposure vitamin D?
• Physical trauma, surgery, psychological stress,
  certain toxins, occupational exposures, cigarette
  smoking
• Vaccinations
• Dietary Factors Saturated/PUFA, antioxidants,
  milk consumption, wheat consumption

Epstein Barr virus
1. Poser CM. Clin Neurol Neurosurg 2006108
227-33 2. Hafler DA et al. Nat Rev Immunol
2005583-91 3. Willer C et al. Proc Natl Acad
Sci 200310012877-82
36
Multiple Sclerosis Etiology Key Questions?

• WHY do T cells not recognize myelin proteins as
  self and attack it like a foreign protein?
• I Because they become sensitized (outside the
  CNS) to foreign proteins which structurally
  resemble myelin MOLECULAR MIMICRY

Damaged Myelin in Multiple Sclerosis
Nerve Cell
Damaged Myelin (plaques)
Muscle Fiber
T Cells
Molecular Matches
f
s
w
g
a
e
g
q
r
Myelin Basic Protein
f
g
w
g
a
e
l
n
m
Escherichia coli bacteria
37
Multiple Sclerosis Etiology Key Questions?

• WHERE do T cells become sensitized to foreign
  proteins that structurally resemble myelin?
• II They become sensitized (outside the CNS)
• A. Gut, mouth, nose (mucosal
  linings)
• B. Direct entry into blood
• By Viral, bacterial and dietary proteins
  (antigens)

Gut Bacteria
T Cells
Molecular Matches
f
s
w
g
a
e
g
q
r
Myelin Basic Protein
f
g
w
g
a
e
l
n
m
Escherichia coli bacteria
38
Multiple Sclerosis Etiology Key Questions?

• HOW do T cells become sensitized (activated) to
  foreign proteins that structurally resemble
  myelin?
• III There is no single foreign antigen that
  activates T cells in Multiple Sclerosis, but
  rather multiple factors acting synergistically
• 1. The immune system must first be primed by a
  prior viral or bacterial infection whose
  molecular structure mimics a myelin protein (many
  viruses and bacteria mimic various myelin
  proteins)
• 2. In most, but not all cases, there must be
  genetic susceptibility (E.g. HLA DRB11501,
  DQA10102, and DQB10602 haplotypes)

Molecular Matches
f
s
w
g
a
e
g
q
r
Myelin Basic Protein
f
g
w
g
a
e
l
n
m
Escherichia coli bacteria
39
Multiple Sclerosis Key Questions?
HOW do T cells become sensitized (activated) to
foreign proteins that structurally resemble
myelin?
T Cells
Gut Bacteria
III cont.

• 3. There must be a secondary and continued
  stimulation of T cells outside the CNS by foreign
  proteins which mimic a myelin protein or proteins
  
• 4. In addition to the mimicking proteins
  (immunogens), an adjuvant (immune system booster)
  must also be simultaneously present to develop
  immunity (autoimmunity)

Immunogen Only
No Immunity
Immunogen Adjuvant
Development of Immunity
40
Multiple Sclerosis Key Questions?
HOW do T cells become sensitized (activated) to
foreign proteins that structurally resemble
myelin?
III cont.
Gut Cross Section

• 4. If the foreign protein is derived from
  the gut , it must cross the gut barrier and
  reach circulation to activate T cells in the
  lymph
• 5. Activated T cells must be able to then
  cross the blood brain barrier (BBB) to destroy
  myelin tissue
• 6. There must be continual and chronic
  activation of other immune components
  (inflammatory cytokines) for myelin destruction
  by T cells

Gut bacterial proteins cannot normally cross
gut Into circulation
circulation
Brain CNS
circulation
BBB
41
Multiple Sclerosis Etiology Dietary Lectins
Legumes

• Lectin Latin verb (legere) to select
• Originally defined by ability to agglutinate
  (clump) erythrocytes
• More recent definition ability to reversibly
  bind a specific mono or oligosaccharide
• Lectins are omnipresent in plant kingdom likely
  evolved as toxic defensive mechanisms to ward off
  predators
• Most dietary lectins are benign and non toxic to
  humans
• Primary exceptions Grain and legume lectins
  which bind to gut tissue

Whole Grains
42
Common Food Lectins which May Bind Gut Tissue and
Their Concentrations

• Wheat germ 300 350 mg/kg wheat germ
  agglutinin (WGA) (1)
• Whole wheat flour 30-50 mg/kg WGA (2)
• White flour 4.4 mg/kg WGA (2)
• Kidney beans (Phaseolus vulgaris) 1,000-10,000
  mg/kg phytohemagglutinin (PHA) (3)
• Soybeans 200 2,000 mg/kg soybean agglutinin
  (SBA) (3)
• Tomatoes lt10 mg/kg tomato lectin (TL) (3)
• Peanuts 110 mg/kg peanut agglutinin (PNA) (1)

1. Vincenzi S, et al. J Agric Food Chem. 2002
Oct 2350(22)6266-70. 2. Matucci A et al. Food
Control 200415 391-95 3. Peumans WJ, Van
Damme EJM. Trends Food Sci Technol 19967132-39
43
Necessary Qualities of Lectins to Influence
Multiple Sclerosis

• Must survive cooking and processing
• Must survive digestive enzymatic degradation
• Must bind gut tissue
• Must cross gut tissue barrier
• Must resist immunological and hepatic disposal
• Must arrive in peripheral circulation intact in
  physiological concentrations
• Must interact with one or more mechanisms known
  to influence multiple sclerosis

Peumans WJ, Van Damme EJM. Trends Food Sci
Technol 19967132-39
44
Ingested Peanut Lectin Rapidly Enters Peripheral
Blood in Humans
Appearance of PNA in serum following consumption
of either 200 g whole raw peanuts (n2) or 200 g
roasted salted peanuts (n5)
Immunoblot detectable PNA (µg/mL serum)
Time after peanut ingestion (h)
Wang Q, Yu LG, Campbell BJ, Milton JD, Rhodes,
JM. Identification of intact peanut lectin in
peripheral venous blood. Lancet 19983521831-32
45
Gut Binding Lectins Demonstrated to Enter
Peripheral Circulation

• The entry of dietary lectins into peripheral
  circulation has been sparsely examined in both
  humans and animals
• It is quite likely that all lectins capable of
  binding gut epithelial tissue enter circulation
• There may be multiple common entry pathways
• 1. M cells
• 2. Paracellular entry
• 3. Trans-membrane receptors

In Vitro
Animals
Humans
Yes 4 Yes 1 Yes 1
Yes 2
Yes 3
Yes 4 Yes 5
TL
PNA
PHA
WGA
1. Kilpatrick DC, et al. FEBS Lett.
1985185299-305. 2. Wang Q, et al. Lancet.
19983521831-2 3. Pusztai A, et al. Biochem Soc
Trans 198917481-2. 4. Lochner N, et al. Pharm
Res. 2003 May20(5)833-9. 5. Pusztai A, et al.
Br J Nutr. 1993 Jul70(1)313-21.
46
How Do Lectins Cross Gut Barrier

• Lectins arriving intact in gut must get past
• 1. Mucus lining
• 2. Glycocalyx Trans-membrane glycoconjugate
  layer extending from apical surface of epithelial
  cells
• Glycocalyx dimensions (400- 500 nm
  thick) pore diameter (7.4 28.8 nm)
• Function Size selective diffusion
  barrier that excludes bacteria, viruses
  foreign material from contacting enterocyte
  membrane
• 3. Cell membrane (Either absorptive enterocytes,
  M cells, goblet cells, Paneth cells, or
  enteroendocrine cells)

Mucus
Glycocalyx
Microvilli or Brush Border
47
How do Lectins Cross Gut Barrier
Villus
1. Endocytosis of Lectins by M Cells
Single Lymphoid Follicle Within a Peyers Patch

• Specialized epithelial cell found only in
  lymphoid follicle
• Function take up luminal material
  (microorganisms, dietary antigens) transport it
  to underlying lymphoid tissue
• Lack dense villi
• Glycocalyx thin or lacking
• Maintain apical surface glycoproteins which bind
  gut antigens
• Entry pathway exploited by certain pathogens

M
M
M
E
E
M
M
E
Crypt
M cell
Enterocytes
T B lymphocytes
48
2. Paracellular Transport of Lectins via
IncreasedEnterocyte Permeability Leaky Gut
How do Lectins Cross Gut Barrier
Dietary Lectins (WGA/PHA)
Brush Border (Villi)
GUT LUMEN
Enterocytes
Tight Junctions
1. Sjolander A et al. The effect of
concanavalin A and wheat germ agglutinin on the
ultrastructure and permeability of rat intestine.
Int Arch Allergy Appl Immunol 1984 75,
230236. 2. Greer F Pusztai A (1985)
Toxicity of kidney bean (Phaseolus vulgaris) in
rats changes inintestinal permeability.
Digestion32, 4246. 3. Pellegrina CD et al.
Plant lectins as carriers for oral drugs Is
wheat germ agglutinin a suitable candidate?
Toxicol Appl Pharmacol 2005207170-78
49
How do Lectins Cross Gut Barrier
3. Entry via Trans Membrane Receptor
PHA
or
WGA

• WGA and PHA gain access to circulation via
  enterocyte endocytosis at the EGF-R
• The gut EGF-R is expressed luminally in humans 1
  and binds galactose specific PHA 2 and neuraminic
  acid specific WGA 3
• Other legume, cereal and tomato lectins likely
  use this pathway because of common sugar binding
  affinities

Gut Lumen (Inside)
EGF Receptor
EGF Receptor
Into Lymph
WGA
Enterocyte Cytosol
WGA
EGF Receptor
Into Circulation
1. Hormi K et al. Cell Tissue Res
1994278439-50 2. Rebbaa A et al. J Neurochem
1996672265-2272 3. Lochner N, et al. Pharm Res.
2003 May20(5)833-9.
50
Necessary Qualities of Lectins to Influence
Multiple Sclerosis

• Must survive cooking and processing
• Must survive digestive enzymatic degradation
• Must bind gut tissue
• Must cross gut tissue barrier
• Must resist immunological and hepatic disposal
• Must arrive in peripheral circulation intact in
  physiological concentrations
• Must interact with one or more mechanisms known
  to influence multiple sclerosis

Peumans WJ, Van Damme EJM. Trends Food Sci
Technol 19967132-39
51
Lectins Must Interact With Mechanisms Influencing
MS Molecular Mimicry
B/F
I II T Cells must become sensitized
(outside the CNS) to foreign proteins which
structurally resemble myelin MOLECULAR MIMICRY

B/F
WGA
Bacterial/Food Peptides
B/F
Lectin
WGA
Gut Interior

• Lectins simultaneously bind bacterial and/or food
  antigens in the gut EGF Receptor
• Both the (Lectin bacteria or food antigen)
  enter the lymph/ circulation where
• They may activate T Cells
• Multiple gut bacteria display molecular mimicry
  with myelin proteins

EGF Receptor
EGF Receptor
Into Lymph
WGA
B/F
B/F
Enterocyte Cytosol
WGA
EGF Receptor
Westall FC. Molecular mimicry revisited gut
bacterial and multiple sclerosis. J
Clin Microbiol 2006442099-2104
Into Circulation
Activate T Cells
52
Lectins Must Interact with Mechanisms Known to
Influence MS Adjuvants
III

• 3. There must be a secondary and continued
  stimulation of T cells outside the CNS by foreign
  proteins which mimic a myelin protein or proteins
  
• Lectin transport mimicking gut proteins into
  circulation
• 4. In addition to the mimicking proteins
  (immunogens), an adjuvant (immune system booster)
  must also be simultaneously present to develop
  immunity (autoimmunity)
• WGA and Tomato Lectin 1 simultaneously act as
  adjuvants vehicles for immunogen transport
• Lectins likely transport gut bacterial cell wall
  proteins (adjuvants)2 into circulation

Immunogen Only
No Immunity
Immunogen Adjuvant
Development of Immunity
Lectin
WGA
1. LaVelle EC et al. The identification of
plant Lectins with mucosal adjuvant
activity. Immunology 200110277-86. 2. Visser
L. Proinflammatory bacterial peptido- glycan as
a cofactor for the development of central
nervous system autoimmune disease. J Immunol
2005174808-816.
53
Lectins Must Interact with Mechanisms Known to
Influence MS Inflammation
Blood

• Pathogenic mechanisms contributing to MS include
• 1. Leukocyte chemotaxis into the CNS (Crossing
  the BBB)
• 2. Production of inflammatory mediators resulting
  in
• Oligodendrocyte damage, demyelination and
  neuronal injury
• MS characteristically elevated cytokines IFN?,
  TNFa, TNFß IL-2, IL-1ß, IL-6, IL-8, IL-12, IL-18

MHC Class II
APC
Naïve Autoreactive T cell
TCR
Inflammatory Cytokines (TNFa, IFN?, IL-12, IL-2)
Myelin peptide
Monocyte
Adhesion Molecules ICAM-1 VCAM-1
Activated T cell
BBB (Endothelial cells)
Microglial cell
CNS
B cell
Complement
Inflammatory cytokines (TNFa, IFN?, IL-12, IL-2)
Autoantibody
Holmes S et al. Exp Rev Mol Med 20057 1-17
54
Lectins Stimulate Inflammatory Cytokines in
Vitro Mononuclear Cells
IL-12 (48 hrs)
INF-? (24 hrs)
Arbitrary U/ml
Arbitrary U/ml
IL-2 (24 hrs)
Arbitrary U/ml
Muraille E et al. Cell Immunol 19991911-9
55
Lectins Stimulate Inflammatory Cytokines in
Vitro Mononuclear Cells (cont.)

• PHA stimulates TNF a TNF ß, IL-1 ß, IL-2, IL-6,
  IL-8, IL-12 and INF ? 1-4
• WGA stimulates IL-12 and INF ? 3
• SBA stimulates IL-6 5

1. van den Borne BE et al. J Rheumatol
19972455-60 2. Isler P et al. Eur Cytokine
Netw 1993415-23 3. Muraille E et al. Cell
Immunol 19991911-9. 4. Baran J et al. Clin
Diag Lab Immunol 20018303-13. 5. Jenkins DJ et
al. Metabolism 200251919-924.
Specifically In MS Patients PHA Stimulates

• PHA stimulates TNF a IL-1 ß, IL-2, and INF ?
  6-10

6. Hollifield RD et al. Autoimmunity
200336133-41 7. Gusev EI et al. J Neurol
1994241500-510 8. Chofflon M et al. Eur
Cytokine Netw 19923523-31 9. Martino G et al. J
Neuroimmunol 199562169-76 10. Guillen C et al.
Immunopharmacol Immunotoxicol 1999 21527-49
56
Lectins Stimulate Inflammatory Cytokines in
Vitro Mononuclear Cells (cont.)

• INF ? is pivotal in MS pathogenesis and
  progression
• Relapses are preceded by increased INF ? in
  cerebrospinal fluid and by peripheral lymphocytes
  
• Administration of recombinant INF ? leads to
  disease worsening
• Conversely, IFN ß is a well established
  immunomodulatory drug for MS treatment
• Both PHA and WGA increase INF ? production in
  vitro

Cosentino M et al. J Neuroimmunol
2005162112-121.
57
Lectins Must Interact with Mechanisms Known to
Influence MS Leukocyte Chemotaxis

• Pro-inflammatory cytokines (TNF a, IL-1 ß) induce
  VCAM-1 and ICAM-1 expression in endothelial cells
  
• Hence increase influx of monocytes and T
  lymphocytes from blood into peripheral tissue and
  CNS 1,2
• IL-8 is elevated in leukocytes of untreated MS
  patients 3 and is responsible for BBB disruption
  and migration of leukocytes into CNS 4
• PHA elevates TNF a, IL-1 ß and IL-8 in vitro

Blood
MHC Class II
APC
Naïve Autoreactive T cell
TCR
Inflammatory Cytokines (TNFa, IFN?, IL-12, IL-2)
Myelin peptide
Monocyte
Adhesion Molecules ICAM-1 VCAM-1
Activated T cell
BBB (Endothelial cells)
Microglial cell
CNS
B cell
Complement
Inflammatory cytokines (TNFa, IFN?, IL-12, IL-2)
Autoantibody
1. van den Borne BE et al. J Rheumatol
19972455-60 2. Bullard DC et al. J Immunol
2007178851-7 3. Lund BT et al. J Neuroimmunol
2004155161-71 4. Mirowska-Guzel DM et al. J
Neuroimmunol 2006176134-140
58
Lectins Stimulate Leukocyte Chemotaxis

• PHA stimulates IL-18 production in T cells 1
• IL-18 up-regulates VCAM-1 and ICAM on endothelial
  cells 2
• Direct cell to cell contact of T cells with
  (monocytes/ macrophages) in peripheral tissue
• Causes up-regulation of TNF a and IL-1 ß 1
• Neutralizing antibody to IL-18 prevents monocyte
  activation in direct cell to cell contact 1 and
  prevents EAE 3

Image of Endothelial VCAM-1 In Vivo
1. Dai SM et al. Arthritis Rheum 2004 50
432-43 2. Morel JC et al. J Biol Chem
200127637069-75 3. Fukaura H et al. Nippon
Rinsho 2003611416-21
59
Lectins Stimulate Leukocyte Chemotaxis
Lectins May Promote Influx of Monocytes and T
Cells Into the Intima by Disrupting the Glycocalyx

• The endothelial glycocalyx, a highly hydrated
  mesh of membrane-bound negatively charged
  proteoglycans, glycosaminoglycans,
  glycoproteins, and glycolipids, has a thickness
  of around 500 nm
• The hairy structures form the endothelial
  glycocalyx.
• Shields the endothelial cells from the flowing
  blood and forms a mechanical barrier against
  adhesion of leukocytes to endothelial surface

(bar1?m).
Electron microscope overview of a alcian blue
8GX-stained rat left ventricular myocardial
capillary.
Van den Berg, Vink, Spaan, Circulation Research
2003, 92 592-594
60
Lectins Stimulate Leukocyte Chemotaxis

• Inflammatory agents (TNF a, thrombin) and growth
  factors (EGF) cause rapid shedding of glycocalyx
  1,2
• Facilitating Influx of leukocytes

Glycocalyx
Crystal Structure of TNFa
1. van den Berg BM et al. Am J Physiol Heart
Circ Physiol 2006290H915-H920 2. Subramanian
SV et al. J Biol Chem 199727214713-20.
61
Lectins Stimulate Leukocyte Chemotaxis
Lectins May Promote Influx of Monocytes and T
Cells Into the Intima by Disrupting the Glycocalyx

• It is likely that dietary lectins disrupt and
  reduce the thickness of the glycocalyx and
  thereby facilitate influx of leukocytes into the
  intima
• WGA, PNA (neuraminidase), bind myocardial,
  retinal and brain arteriole glycocalyx 1
• Tomato lectin binds variety of human arteriole
  glycocalyx 2
• WGA 3 and PHA 4 bind the EGF-R which causes rapid
  shedding of the glycocalyx 5

The Arterial Glycocalyx
1. Lawrenson JG et al. J Anat 200019655-60 2.
Debbage PL et al. Histochem Cell Biol
2001116349-59 3. Lochner N, et al. Pharm Res.
2003 May20(5)833-9. 4. Rebbaa A et al. J
Neurochem 1996672265-2272 5. Subramanian SV et
al. J Biol Chem 199727214713-20.
62
Potential Mechanisms of Lectin Involvement in
Multiple Sclerosis
Lectins Up-Regulate Matrix Metalloproteinases
(MMPs)

• MMPs are elevated in MS and are implicated in
  disease etiology 1,2 by
• 1. Disrupting BBB and facilitating influx of
  leukocytes
• 2. Degrading Myelin Basic Protein
• PHA up-regulates MMP-9 in human monocytes 3
• WGA up-regulates MMP2 in human monocytes, but not
  MMP-9 4

(plt0.05)
ns
1. Ram M et al. J Clin Immunol 200626299-307 2.Y
ong VW et al. J Neurol Sci 2007 Mar 22
Epub ahead of print 3. Dubois B et al. FEBS
Letter 1998427275-278. 4. Saja K et al. Mol
Cell Biochem 2007296185-192
63
Multiple Sclerosis Etiology Milk and Dairy

• Epidemiological studies have repeatedly
  associated MS prevalence with milk drinking 1- 4
• Milk drinking correlated highly to MS prevalence
  in 27 countries (Spearmans rho 0.84) 3
• Epidemiological experimental studies also link
  rheumatoid arthritis and type 1 diabetes to milk
  consumption

Milk and Dairy
1. Agranoff BW et al. Lancet 197421061-66 2.
Butcher PJ. Med Hypotheses 198619169-178 3.
Malosse D et al. Neuroepidemiol 199211
304-312 4. Lauer K. Neurology 1997 49s55-s61
64
Multiple Sclerosis and Milk Proposed Mechanisms
of Action

• In Dark Agouti Rats, immunization with the bovine
  milk protein, butyrophilin (BTN) triggers EAE 1
• Via a class II MHC restricted T cell response
  that cross reacts (molecular mimicry) with the
  autoantigen, myelin oligodendrocyte glycoprotein
  (MOG) peptide sequences 76-87 (IGEGKVALRIQN) 1
• Transmucosal (intranasal) administration of BTN
  or by I.V. suppresses disease activity 1
• In MS patients, MOG specific autoantibodies cross
  react with multiple epitopes of BTN 2
• Human Interpretation - Caveats 1) Will require
  permissive MHC haplotype, 2) BTN crosses gut
  barrier intact and induction of oral tolerance
  must be abrogated

Milk and Dairy
Dark Agouti Rat
1. Stefferl A et al. J Immunol 20001652859-2865
2. Guggenmos J et al. J Immunol 2004172661-68
65
Multiple Sclerosis and Milk Proposed Mechanisms
of Action (cont.)

• An additional bovine milk protein (BSA 193) was
  encephalitogenic to SJL mice subjected to
  EAE induction
• Structural homology (GLCHMYK) existed between
  exon 2 of the autoantigen myelin basic protein
  (MBP) and BSA 193
• However, T cell cross reactivity did not occur

plt0.0001
Proliferative T Cell Response (SI)

• Further, MS patients had significantly (plt
  0.0001) higher proliferative T cell responses to
  BSA 193 than healthy controls

SJL Mouse
1. Winer S et al. J Immunol 2001166 4751-56
66
Multiple Sclerosis and Milk Summary
Milk and Dairy

• Animal EAE and human tissue studies suggest that
  at least two bovine milk proteins (BTN and BSA
  193) may be involved in MS etiology by molecular
  mimicry 1-3
• Specific permissive human MHC haplotypes are
  required for cross reactivity 1
• Bovine proteins must cross the gut barrier and
  oral tolerance must be abrogated 1, 3
• Neonatal exposure to dietary antigens that cross
  react with CNS autoantigens may enhance disease
  susceptibility later in life 4

1. Stefferl A et al. J Immunol 20001652859-2865
2. Winer S et al. J Immunol 2001166 4751-56 3.
Guggenmos J et al. J Immunol 2004172661-683.
4. Miller AO et al. Eur J Immunol 1994241026-32
67
Influence of Diet on Disability in Four Multiple
Sclerosis Patients
Diets grain, legume and dairy free
Kurtzke Expanded Disability Scale
Years on diet
Years with MS
Anecdotal data provided by A Embry. Direct MS,
Canada http//www.direct-ms.org/
68
Thank You!