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Celiac-like Disorder in Developmentally-delayed Children from Bacteria Surface Proteins

June 16, 2011

jeffc3497 at gmail dot com

Bottom Line Up Front

B. pertussis pertactin and S. pneumoniae surface proteins contain regions with a strong homology to the deamidated wheat gluten fragments that cause the immunogenic response in celiac disease. These regions appear to have a strong binding affinity for APC HLA-DQ2 and would also appear to trigger T-cell recognition/stimulation. This is caused by the repeating proline/glutamate patterns found within. This is the same mechanism that leads to autoimmunity toward small bowel tissue in celiac. These surface proteins are found as a component of the acellular pertussis vaccine (DTaP) and as contaminants in the pneumococcal vaccine (PCV7, PCV13, and PPSV). DTaP and PCV7/PCV13 are routinely and repeatedly administered as part of the pediatric vaccine schedule. The aluminum salt adjuvants used in these vaccines forms an antigen depot within the muscle after injection. These depots cause a prolonged, continuous antigen persistence that has been shown to last months. In those genetically predisposed, it is hypothesized that homology toward gluten peptides and antigen persistence produce a continuous autoimmune response toward small bowel tissue. Repeated vaccine boosters result in a constant inflamed bowel state present for years. Malnutrition materializes as the inflamed small bowel impedes nutrient absorption. Destruction of the intestine mucosa reduces peptidase secretion required for protein digestion. It may also reduce secretin and cholecystokinin mucosa secretion needed to stimulate bile and pancreatic enzyme release. Malnutrition leads to neurological manifestations and developmental delay. It is conceivable that the bowel inflammation seen in celiac disease is actually intended to be a transitory state that purges the digestive tract of these pathogens. Gluten reactivity inadvertently results from its homology toward these pathogens after the fortuitous deamidation by tissue transglutaminase. As gluten is continually present, the inflammatory state becomes chronic. 2

Celiac-like symptoms in developmentally-delayed children Review of celiac disease Alpha-gliadin, 33-mer, and immunogenic epitopes Homologs in pertussis pertactin and pneumococcal surface proteins The bacteria surface protein hypothesis Review of published literature regarding bacteria surface proteins and celiac disease Bacteria surface proteins and vaccines Conclusions References

This presentation puts forth a hypothesis that some developmentallydelayed children children suffer from an autoimmune disorder closely related to celiac disease. It is suspected that triggers for this disorder are surface membrane proteins found in respiratory tract bacteria and used in vaccines. Regions of these proteins have a strong homology and potential stimulatory equivalence to immunogenic peptides found in wheat gliadin. Findings supporting this hypothesis are presented. This conclusion was reached after reviewing celiac-disease related studies, researching case reports, reading numerous parental descriptions of their childrens symptoms, personal interviews with parents, and the authors own experience with his sons regression into autism spectrum disorder (ASD) and subsequent dramatic recovery. The author has a sibling with celiac disease. It is thought that in this distinct ASD phenotype, this celiac-like disorder sets off a catastrophic nutritional downward spiral. The neurological impact is amplified by the immature state of the childs neurological system
Inflammation continuous adaptive and innate immune response Maldigestion leading to protein energy malnutrition Malabsorption particularly essential fatty acids and fat soluble vitamins Bacterial overgrowth due to the constant presence of maldigested food Food protein allergies from increased intestinal permeability

The author strongly believes many affected children can improve significantly and some can recover completely using diet restriction, targeted nutritional assistance, and supplemental digestive support

Celiac-like Symptoms in ASD

As early as the 1960s, there have been observations in medical literature noting gastro-intestinal (GI) and stool similarities between those with autism and untreated celiac disease [1,2] Parents and clinicians have long reported that many ASD children exhibit GI symptoms and manifestations that appear to mimic those seen in celiac disease [3-5].
Diarrhea often with alternating constipation Extremely malodorous stools Maldigestion undigested food in stool Evidence of malnutrition
Protein malnutrition mild kwashiorkor-like appearance (muscle wasting, distended abdomen, skin de-pigmentation, etc.) Essential Fatty Acid and fat soluble vitamin malnutrition elevated markers for lipid peroxidation and oxidative stress

Parents also report autistic symptoms often improve with a gluten-free diet. Unlike celiac disease, symptoms rarely resolve completely.
The gluten-free, casein-free diet is consistently one of the highest-rated treatment by parents in the Autism Research Institutes comprehensive survey [6]
Improvement in bowel function Neurological improvement (eye contact, alertness, tantrum frequency) Sleep improvement

However, these children typically dont have strongly elevated antibody markers seen in celiac (anti-gliadin, anti-tissue transglutaminase), as such they are not considered to have celiac disease. [7] Intestinal biopsies are rarely performed. The incidence rate of villous atrophy and brush border damage is unknown.

The Distinct Phenotype

Although not applicable to all with ASD, there does appear to be a distinct phenotype that shares common symptoms and responds remarkably well to treatment [8-10]
Gastrointestinal - celiac-like symptoms as described on the previous page Neurological
High functioning able to talk, although speech may be delayed or difficult Minimal stereotypy unlike many children with autism, obsessive behavior and repetitive actions appears to be limited in this group Apathetic, not alert needs name called or questions repeated several times Attention difficulties cant focus on specific tasks as required, has trouble quickly shifting focus from one task to another when needed Irritable and irrational petulant, tantrums at minor annoyances, punishment makes tantrums worse, acts in the moment without considering consequences

Muscle wasting low amounts of muscle tissue, condition may be hidden from view by body fat at young ages Hypotonia low muscle tone, poor motor skills, lacks physical coordination Pallor extremely pale or ashen tone face despite a more typical skin tone on other parts of the body. Allergic shiners may be pronounced.

Many of the symptoms associated with this group are shared with advanced untreated celiac disease and also kwashiorkor, a relatively common third-world disease caused by protein deficiency [11] . This is not surprising as malnutrition appears to be the common element in all three.

Gluten peptide

Celiac Process
Innate immune response triggering peptides

Tissue destruction, increased permeability, malabsorption

Passage to serosa (intestinal permeability)

Deamidation by tTG

10 7a
Innate immune response adds to inflammation

Consumption by APC

Th1 response initiated, inflammation begins

12 8
Food proteins enter bloodstream, immune response, chronic inflammation, allergies

Presentation by DQ2 or DQ8 (epitope dependant)

Gluten-specific, DQ2 or DQ8-restricted T cells proliferate

Recognition by T cell (CD4)

Th2 response initiated, Gliadin specific antibodies produced role in inflammation uncertain

Illustration from Bethune and Khosla 2008 [12] Annotations added.

BIOMARKERS USED CURRENTLY presence of antibodies to gliadin/tTg

Th2 response initiated, tTG specific antibodies produced role in inflammation uncertain

Celiac Review (page 1 of 3)

To better understand the mechanism at work, a review of celiac disease is presented. The understanding of this process is evolving; the consensus view in described [13-15]. The chart on page 7 accompanies this discussion. In celiac disease, wheat gluten triggers a destructive response toward small bowel tissue resulting in tissue damage, malabsorption, and eventual malnutrition. Research suggests the tissue damage results from both adaptive and innate immune responses. To date, the adaptive immune response is better understood. The following sequence triggers the adaptive immune response
Gliadin and glutenin (wheat proteins from the diet) are partially digested leaving protein fragments (peptides) that are resistant to proteolytic digestion due to their high proline content Via intestinal permeability, these peptides reach the intestinal serosa (the lamina propria) and ultimately the bloodstream Some of these peptides are ideal substrates for the tissue transglutaminase enzyme (tTG). They are selectively deamidated; glutamine (Q) is converted to glutamate (E) at specific locations The deamidated peptides are consumed by antigen presenting cells (APCs).

1 2 3 4

Celiac Review (page 2 of 3)

The adaptive immune response to gluten (continued)

The peptides are presented on the APC surface bound to the HLADQ2 or HLA-DQ8 haplotype molecules
The epitopes (immunogenic fragments) of many gluten peptides have a high binding affinity for HLA-DQ2 or HLA-DQ8 More than 90% of celiac cases carry the DQ2 haplotype, most of the rest are DQ8 [19] Roughly a third of the population has the DQ2 and/or DQ8 haplotype [15], yet the incidence of celiac disease is far lower. Other genetic or environmental factors must be involved.

APCs with HLA-DQ2/DQ8 presenting gluten epitopes are recognized by T-cells. The presence of proline and glutamate in specific positions bound to the DQ2/DQ8 molecule appears to play a key role in T-cell recognition. T-cells initiate the adaptive immune response
Cell-mediated Th1 response cross reactive with small bowel tissue, causes inflammation and ultimately small bowel tissue destruction Humoral Th2 response production of antibodies It is unclear if these play a role in tissue destruction. If so, the role is secondary to the Th1 response Antibodies are produced against gliadin and tTG. These antibodies can be used as markers for celiac disease as they are easily measured without invasive procedures.


7b 7c

If gluten consumption persists, gluten-specific, HLA-DQ2 or HLADQ8-restricted T-cells proliferate

Celiac Review (page 3 of 3)

The inflammatory response from the innate immune response appears to be involved but the mechanism is not as clear
Undigested gluten fragments (different from those involved in the adaptive immune response) trigger activation of the innate immune response
Relationship between the adaptive and innate response is uncertain The p31-43/49 fragment of alpha-gliadin appears to be involved as it induces interleukin 15 production in celiac mucosa. This sets off a chain of events that increases stress on small bowel tissue


The innate immune response adds to tissue inflammation leading to further small bowel tissue destruction

Inflammation and tissue destruction lead to malabsorption, maldigestion, and increased intestinal permeability. A vicious downward cycle results as the bodys malnourished state leads to a further decline in overall health. Removal of gluten breaks the cycle halting activation of the adaptive and innate immune response. Unless the case is very advanced, full recovery usually results if a gluten-free diet is maintained.


33-mer in Alpha-gliadin (Shan 2002)

Alpha2-gliadin amino acid sequence (290 amino acid residues) Key: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Shan et al [16] discovered that the 33-mer portion (marked in red above) of gliadin wheat protein was highly resistant to digestion by proteolytic enzymes
Note high frequency of Proline (P) and Glutamine (Q) Proline bonds are difficult to hydrolyze due to its unusual cyclic structure of the side chain. Repeating proline residues also gives the peptide a unique helical shape.

They also discovered 33-mer contains six overlapping sequences of immunogenic epitopes that invariably produce a response in adults with celiac disease [17] Once deamidated by tTG (glutamine glutamate) at specific positions, these epitopes will tightly bind to the HLA-DQ2 presenting molecule of the APC and trigger strong T-cell recognition [18]
33-mer (Shan 2002) overlapping epitopes shown

Illustration from Sollid 2002 [13]

Red Green Blue

(1 place) (3 places) (2 places)


Alpha-I Alpha-II Alpha-III

Note Pattern Similarity


Illustration from Sollid 2002 [13]

HLA-DQ2 and DQ8 haplotypes

Table from Tollefson et al 2006 [19]

Epitope from 33-mer

Registers P1-P9 of HLA-DQ2 Glutamate (E) deamidated from glutamine by tTG prior to APC consumption of peptide Examples of epitope binding to HLA registers. Note pattern similarities and differences between DQ2 & DQ8

Although genetic factors are not fully understood, There is a strong association with the HLA-DQ2 and HLA-DQ8 haplotypes in those with celiac disease HLA-DQ2 and HLA-DQ8 are molecules used by the Antigen Presenting Cell (APC) to present the gliadin epitope on its surface to T-cells. Upon recognition, the T-cell initiates the adaptive immune response. The amino acid sequence of the gliadin epitopes are well matched to the DQ2 and DQ8 register structure by virtue of the proline (P) locations and glutamate presence. There is a high binding affinity of the epitopes for DQ2 and DQ8. The glutamate (E) residue (deamidated from glutamine by tTG) presence and location are also critical for T-cell recognition of the APC presenting the epitope. Evidence also suggests some proline locations are crucial for T-cell recognition. The 33-mer epitopes bind tightly to DQ2 as do epitopes from other gliadin peptides Epitopes from other gliadin and glutenin fragments bind tightly to DQ8

33-mer Epitope Example (Tollefson 2006)

Epitope in Blue P F P Q P Q L P Y P Q P Q
Alpha-gliadin peptide Selective deamidation from tTG (glutamine glutamate) Peptide with glutamate residue in position 4 of epitope Consumption by APC


P F P Q P E L P Y P Q P Q 1 2 3 4 5 6 7 8 9 HLA-DQ2 P Registers

Presentation on APC surface bound with DQ2 (see below) Recognition by T cell Adaptive immune response starts

Binding for epitopes with HLA-DQ2 and HLA-DQ8 (Tollefson et al 2006) [19]: Proline - DQ2 binds gluten peptides with the proline residues localized in P1, P3, P5, P6, and P8 but not in P2, P4, P7, or P9. This pattern is similar for DQ8, which bind peptides with proline residues in P3, P6, and P8, but not in P1, P2, P4, P7 or P9. Glutamate - For DQ2, glutamate in P4 or P6 is crucial for T cell recognition. For DQ8, glutamate is needed in P1 or P9 for T cell recognition. Due to the negative charge of its side chain, glutamate also increases binding strength.


HLA-DQ2 Binding and T-cell Recognition

Tollefson, Vader, Shan, Kim, Milea, Stepniak and others [16,17,19-23] have characterized the alpha I, alpha II, and alpha III gliadin epitopes seen in 33-mer that frequently cause a response in adult celiac cases For these reactive peptides, a distinct pattern emerges for the nine residue binding cores
An iterative proline sequence causing a type II polyproline helical character that results in strong HLA-DQ2 binding
Proline in the 1st, 3rd, {5th or 6th}, and 8th position

Residues in key positions also appear critical for T-cell recognition

Proline in the 1st (sometimes), 3rd (often), and 8th (regularly) positions Glutamate in the 4th or 6th position note that in gliadin, glutamate is deamidated from glutamine by tTG prior to binding with HLA-DQ2

A review of the literature found preferences, but not apparent criticality to the 2nd, 7th or 9th position (also {4th/5th} depending on proline sequence) with a few caveats
They can not be proline as this distorts the helical character Glutamine (Q), leucine (L), phenylalanine (F), and tyrosine (Y) are seen in these epitopes. However, this seems to be due to the nature of these gluten proteins. It does not appear that these residues are exclusively required or even optimum for HLA-DQ2 binding.

Thus with these reactive deamidated gliadin peptides, there is seen an overlapping of two nine-residue patterns


Where x is L, F, Y, Q or E (tTG-deamidated Q)

Binding and Recognition (Stepniak et al 2008) [17]

33-mer Alpha-II and Alpha-I examples

HLA-DQ2 Binding
Helical shape from P sequence matched to DQ2 pocket structure Glutamate negative charge strengthens bond, position 4 and 6 matched to DQ2 pocket Bulky side chains fit in large P9 pocket

DQ2 is the only known DQ allele to accept P in the first position

35% of gluten stimulatory peptides have P in position 1 > 50% of gluten stimulatory peptides have P in position 3

> 80% of gluten stimulatory peptides have E in position 4/6 85% of gluten stimulatory peptides have P in position 8

T-cell Recognition
The peptides found in alpha-gliadin are uniquely suited for both DQ2 binding and T-cell recognition. PxPxPExPx and PxPExPxPx tend to fill both criteria.

33-mer Homologs (Shan 2002)

Shan et al also discovered that the 33-mer peptide has two strong homologs among non-gluten proteins [16]
Pertactin - a highly immunogenic surface protein that Shan et al identified as being from Bordetella pertussis Tyrosine phosphatase a mammalian protein of unknown function

(sequence alignment from Shan 2002 Supplemental Information [24])

They characterized these homologs and noted both share unique characteristics with 33-mer
High frequency of occurrence of proline and glutamine along with a similar pattern structure Comparable to 33-mer, both are excellent substrates for tTG (high levels of specificity)
Pertactin Tyrosine Phosphatase kcat/KM = 121/min/mM kcat/KM = 37/min/mM

Both exhibit a strong type II polyproline helical character

typical of peptides bound to class II MHC proteins (i.e. APCs) likely to enhance their binding affinity to these proteins

Shan 2002 did not comment on the possible involvement of either homolog in the pathogenesis of celiac disease. They did briefly comment on their conceptual use in oral vaccines.


B. Bronchiseptica vs. B. Pertussis Pertactin

Shan 2002 appears to have some inconsistencies between the text of the published paper and the footnotes regarding pertactin. These inconsistencies extend to the supplemental information (SI) [24] . The homolog identified in the footnotes as pertactin is actually from B. bronchiseptica (P.68), not pertactin from B. pertussis (P.69) as identified in the text. This was confirmed via the BLASTP [25] database. B. bronchiseptica is closely related to B. pertussis, with some distinctions
B. bronchiseptica is rare in humans, but is found in small mammals such as cats, dogs, and rabbits B. bronchiseptica does not produce the toxin as seen in B. pertussis, despite having the genes required. This feature, along with other similarities, suggests common ancestry for both strains.

Despite the discrepancy, the BLASTP database also confirms pertactin from B. pertussis (P.69) is structurally similar to P.68 and is also a strong 33-mer homolog

Comparison of pertactin from B. bronchiseptica (P.68) and B. pertussis (P.69), both are strong homologs of 33-mer Pertactin fragment shown is from an area referred to as region 2 in pertussisrelated literature and is known to have highly immunogenic qualities [26]

Pertactin and HLA-DQ2 / T-cell Recognition

Upon closer inspection, pertussis pertactin is not only a homolog of 33-mer, but it also contains two overlapping sequences that appear will bind tightly with HLA-DQ2 and trigger T-cell recognition. LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (33-mer)
1 2 3 4 5 6




These sequences obey all the HLA-DQ2 binding and T-cell recognition guidelines (see page 14 and 15), they do not match HLA-DQ8
They share all proline and some glutamine locations with 33-mer epitopes They already include a glutamate (E) residue at the correct position. They do not need deamidation by tTG prior to binding with DQ2
Epitope 1 Epitope 3, 5 Epitope 2, 4, 6


Epitope A

Epitope A

Epitope B

Red indicates glutamine (Q) deamidated to glutamate (E) by tTG. Pertactin epitopes A an B do not require deamidation. They are already a good match for HLA-DQ2 and have proline and glutamate at the correct positions for T-cell recognition. Note sequence similarities between 33-mer and pertactin sequences.


Pertactin Epitope B HLA-DQ2 Binding Example

Epitope in Blue P Q P Q P E A P A P Q P P
Pertussis pertactin sequence B No Selective deamidation from tTG is needed glutamate residue in position 4 of epitope without needing tTG Consumption by APC


P Q P Q P E A P A P Q P P 1 2 3 4 5 6 7 8 9 HLA-DQ2 P Registers

Presentation on APC surface bound with DQ2 Recognition by T cell Adaptive immune response starts

Pertussis pertactin sequences A & B share a close similarity with those that are found in 33-mer Prolines are located in the exact same positions as 33-mer epitopes Glutamate resides in the position where glutamine glutamate via tTG in 33-mer The residues that are different between the two have very similar properties
Ala (A), leu (L), phe (F) are all non-polar, and neutrally charged. Tyr (Y) and Gln (Q) are polar and neutrally charged. However, polar Y and Q appear to exchange freely with nonpolar F and L in 33-mer. 19

Pneumococcal Surface Protein A (PspA)

Illustration from Moreno et al 2010 [28]

Similar to pertussis pertactin, Streptococcus pneumoniae also contains proline-rich surface proteins. These are found in Pneumococcal surface protein A (PspA) and surface protein C (PspC). PspA contains proline-rich blocks (P above) separated by non-proline blocks (N). These proline-rich blocks are highly-reactive and have been shown to produce an antibody response [28, 29]. The structure of these proline-rich blocks are notable in their similarity to the epitopes of alpha-gliadin 33-mer. They contain multiple overlapping sequences of the patterns PxPxPExPx and PxPExPxPx

PspA and HLA-DQ2 / T-cell recognition (1 of 2)

Beall et al 2000 [30] did extensive work sequencing the clade defining region and the proline-rich region of PspA for pneumococcal serotypes 6B, 9V, 14, 19A, 19F, and 23F. These serotypes cause the majority of pneumonia cases and are the basis of the PCV and PPSV vaccines [31-33]. Beall 2000 listed 33 distinct accession number variants spread across the sequenced serotypes listed above. All were retrieved from BLAST and checked for pattern matches to PxPxPExPx and PxPExPxPx; the patterns seen in 33-mer. Matches were found in every serotype/strain. The number of matching sequences ranged from a low of three to an astonishingly high of fourteen overlapping sequences in two variants of serotype 9V.
Number of PxPxPExPx or PxPExPxPx matches (accession number from Beall et al 2000 in parenthenses) Pneumococcal Serotype 6B 9V 14 19A 19F 23F (1 to 7) 23F (8 to 14) variant 1 10

variant 2 8

variant 3 3

variant 4 5

variant 5 5

variant 6 3

variant 7 7




























PspA and HLA-DQ2 / T-cell recognition (2 of 2)

A Sampling of PspA proline-rich sequences from various serotypes
Red lettering match for PxPxPExPx or PxPExPxPx Blue lettering non-proline blocks 33-mer included for comparison
Accession ID
33-mer Serotype 6B PspA Serotype 6B PspA Serotype 9V PspA Serotype 14 PspA Serotype 19A PspA Serotype 19F PspA Serotype 23F PspA Serotype 23F PspA ETPAPAPQPEKPAPAPKPEQPAPAPKPEQPTPAPKPEQPTPAPKPGWKQENGM


6 sequences 8 sequences



7 sequences


14 sequences


10 sequences


6 sequences


9 sequences


4 sequences


3 sequences

Note the variety of arrangements and overlapping patterns seen. It is similar to the overlapping structure seen in 33-mer and other gliadin sequences.

PspA Epitopes
All sequence matches found in PspA from the Beall sequenced serotypes [30] are shown Most appear multiple times, PKPEQPAPA appears 21 times All have exact proline matches to the 33-mer epitopes. All have glutamate (E) correctly located in position 4 or 6 and would not required deamidation from tTG Other positions are from a small group of amino acids
Alanine (A) Threonine (T) Glutamine (Q) Valine (V) Lysine (K) (positive charge)

Pattern 33-mer


All except lysine have similar properties to the residues seen in the same positions in 33-mer


Pneumococcal Surface Protein C and HLA-DQ2

Although fewer strains have been sequenced than PspA, a check of the BLASTP database revealed that Streptococcus pneumoniae surface protein C (PspC) also exhibited pattern matches to 33-mer. LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (33-mer)
1 2 3 4 5 6




Sequences A, B, and C all have exact proline matches and glutamate (E) at the same position where glutamine is deamidated by tTG in 33mer. They overlap in the same manner as 33-mer epitopes 1, 2, and 3.
Epitope 1 Epitope 3, 5 Epitope 2, 4, 6 Epitope 3,5

33-mer PspC

Epitope A

Epitope A

Epitope B

Epitope C

Also as with pertactin, the amino acid substitutes from 33-mer have similar properties to those they replace (uncharged). Again, lysine (K) appears to be the exception with its positively-charged side chain. Many more matches almost certainly exist as the pattern is very similar to that seen in PspA. To date, PspC has been sequenced in fewer strains.


Other Interesting Sequences

Pattern searches using BLAST reveal that PxPxPExPx and PxPExPxPx appear in other pathogenic microorganisms, usually in surface membrane proteins. Many more probably exist.
Bacillus cereus BDRD-ST26 Collagen adhesion protein (PQPEQPKPQPEKP)
A soil-dwelling, Gram-positive beta hemolytic bacterium. Some strains are harmful to humans and cause food borne illness

Campylobacter rectus RM3267 Surface-layer-RTX protein (PQPQPEQPKPNPE)

A species of Campylobacter implicated as a pathogen in chronic periodontitis

Corynebacterium amycolatum SK46 hypoth. protein (PAPAPEAPAPAPEAP)

Causes of endocarditis in patients who have underlying structural heart disease or are immunocompromised, as well as of prosthetic-valve endocarditis.

Trichomonas vaginalis surface antigen BspA-like (PNPTPETPNPAPETP)

An anaerobic, flagellated protozoan, the causative agent of trichomoniasis, and is the most common pathogenic protozoan infection of humans in industrialized countries.

Corn (Zea mays) has an interesting pattern match that appears multiple times in a cell-wall protein [35]
Vegetative cell wall protein gp1 precursor (PKPEVPHPVPELPKPE) Further research is needed as the documentation is unclear if this protein is found in the consumable portion of corn This is an interesting finding as some with celiac disease report sensitivity to corn and at least one study has shown heightened antibody responses to corn protein in celiac patients [36]

Bacteria Surface Protein-mediated Process 9

Gluten peptide

Innate immune response gluten triggering peptides Intestinal permeability not required

Tissue destruction, increased permeability, malabsorption

Deamidation by tTG not required

8 5a
Innate immune response adds to inflammation

Pertactin from bloodstream

Th1 response initiated, inflammation begins


10 6
Food proteins enter bloodstream, immune response, chronic inflammation, allergies

Consumption by APC

Presentation by DQ2 only (not DQ8)

Recognition by T cell (CD4)

Pertactin-specific, DQ2-restricted T cells proliferate

Pertactin Pertactin

Th2 response initiated, Pertactin specific antibodies produced No gluten-specific antibodies

Illustration from Bethune and Khosla 2008 [12] Annotations added.

CELIAC BIOMARKERS NEGATIVE no antibodies to gliadin or tTg

No tTG specific antibodies produced


Bacteria Surface Proteins Hypothesis (1 of 2)

The chart on page 26 accompanies this hypothesis It is hypothesized that early exposure to bacteria surface proteins can trigger a Th1 autoimmune response toward small bowel tissue in those genetically predisposed. Exposure can be from vaccines or illness. Damage is inflicted via comparable mechanism to that seen in celiac disease. This results from the remarkable structurally similarity of certain gluten (i.e. 33-mer) and bacteria surface protein sequences. This genetic disposition overlaps those criteria that predispose an individual for celiac disease
HLA-DQ2 haplotype, HLA-DQ8 does not appear susceptible Other unknown genetic factors

Repeated vaccine administration (i.e. primary series and boosters) causes surface protein-specific DQ2-restricted T cells to proliferate
Use of aluminum compound adjuvants causes a muscular antigen deposition and long-term persistence that can last several months [37]

This specificity to surface proteins may act analogously to a vaccination for 33-mer. The HLA-DQ2 positive individual is less likely to develop an adaptive immune response to 33-mer as the offending APCs and T-cells are now more specific to surface proteins.
Few anti-gliadin antibodies present Would expect a negative correlation between anti-33-mer and anti-pertactin or pneumococcal surface protein antibodies Consistent with observation that children with celiac (more likely to be recently vaccinated) are less reactive to 33-mer epitopes than adults [17,20]

Bacteria Surface Proteins Hypothesis (2 of 2)

Vaccine-mediated autoimmunity has a faster onset and higher rate of incidence than classic celiac disease in those genetically predisposed
The immunogenic epitope has direct access to the bloodstream Time and additional triggers are not required to develop intestinal permeability and possibly required protein maldigestion

As the individual does not need a gluten reaction, and deamidation with tTG is not required for surface protein epitopes, neither antigliadin or anti-tTG antibodies are present in large quantities
Negative result from standard celiac disease biomarker tests

Protein maldigestion may result as tissue destruction reduces brush border peptidases. Partially digested gluten fragments lead to further tissue destruction as the innate immune response is activated. Removing gluten from the diet stops the innate immune response, but not necessarily the adaptive immune response The manufacturers could potentially make a safer and more effective pertussis vaccine if they were motivated to do so.
A study by Hinjen et al [38] intriguingly showed that immunization using an engineered pertactin without region 2 (includes the homologs to the 33-mer epitopes) had the highest protection level of multiple vaccine variations. This could indicate that some pertactin is being sidetracked from its intended immune response target in genetically predisposed individuals (i.e. HLA-DQ2). If this hypothesis is born out, Hinjens observation indicates a safer and more effective pertussis vaccine without PRN region 2 is possible

Pertactin Celiac Link Studies

An exhaustive search of the scientific literature turned up only one paper pursuing the 33-mer/pertactin homology noted in Shan 2002 Citing Shan 2002, He et al (Vaccine [23] 2005) [39] conducted a study to determine if there was evidence of pertussis or pertussis vaccination contributing to celiac disease He 2005 looked for cross-reactivity between anti-pertactin and anti-33mer antibodies in two populations
Those exposed to pertussis
Infants and toddlers vaccinated with the aP vaccine Individuals with pertussis Those with the clinical diagnoses of celiac disease as determined by lab tests Those with celiac symptoms but negative lab tests Healthy controls

Those with or suspected of having celiac disease

He et all concluded, We found no cross-reactivity between human antibodies to the two different components, suggesting that neither pertussis immunization nor disease contributes to the pathogenesis of CD (celiac disease) In light of the novel findings presented in this summary, it is reasonable to conclude that He et al may have misinterpreted their findings.



33-mer / Pertactin Cross-reactivity (He 2005)

= -0.127 = -0.001 = -0.088


Plots from He et al 2005 [39]

= -0.019 = -0.142 = 0.016

33-mer/pertactin (Prn) cross reactivity data from He et al 2005. Charts are X-Y scatter plots of 33-mer IgA or IgG antibodies vs. pertactin IgG antibodies in various populations. The population He et al calls non-CD patients actually were suspected of having celiac disease but were not confirmed serologically. Note large scale changes from plot to plot making visual comparison difficult Note weak negative correlation in some plots. Further examination of the data raises interesting questions regarding the conclusions of He et al


Data from [39] replotted using [40]

Another Look at He et al 2005

33mer IgA 33mer IgG

PRN IgG vs. 33-mer IgA/IgG - Celiac Confirmed

320 280 240
33-mer IgA/IgG

PRN IgG vs. 33-mer IgA/IgG - Celiac Not Confirmed


33 mer IgA

33 mer IgG

Celiac Confirmed
280 240
33-mer IgA/IgG

Celiac Suspected, But Not Confirmed

200 160 120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80

200 160 120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80

He 2005 scatter plots (previous page) did not appear to show a correlation between Pertactin IgG (x axis) and 33-mer IgA or IgG (yaxis) The scatter plots were digitized and reproduced here using identical scaling between plots and also placing the 33-mer IgA and IgG data on the same graphs [40]. Using identical scaling, it becomes clearer that the healthy control group has a pronounced clustering of data points in the lower left corner of the graph See next page for expanded view

PRN IgG vs. 33-mer IgA/IgG - Healty Controls

320 280 240
33-mer IgA/IgG

33 mer IgA

33 mer IgG

Healthy Controls

200 160

Data Cluster
120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80


He 2005 Healthy Controls (40 subjects)

Data from [39] replotted using [40]

PRN IgG vs. 33-mer IgA/IgG - Healty Controls


33 mer IgA

33 mer IgG

Healthy Controls
280 240
33-mer IgA/IgG

200 160 120 80 40 0 0 10 20

Data Cluster Those that do not exhibit an exaggerated response to either 33-mer or pertactin


40 PRN IgG





33-mer and pertactin (PRN) are both specific to the HLA-DQ2 haplotype only. It is suspected that those inside the rectangle (84% of the healthy controls) do not carry the HLA-DQ2 haplotype or if they do, have not have had APCs exposed to the immunogenic portions of gluten or pertactin. Presumably, this is the normal response in an unaffected individual.


He 2005 Celiac Cases (19 Subjects)

Data from [39] replotted using [40]

PRN IgG vs. 33-mer IgA/IgG - Celiac Confirmed


33mer IgA

33mer IgG

Celiac Confirmed
PRN IgG above normal range of controls

33-mer IgA/IgG

200 160 120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80

He 2005s confirmed celiac cases plotted on the same scale as controls. Note that 5 of 19 cases (26%) have 33-mer IgA/IgG and pertactin (PRN) IgG entirely within the normal range of controls (red box). Since 33-mer and PRN are both HLA -DQ2 specific, it is suspected these 5 cases are HLA-DQ8 or may be susceptible to epitopes other than those in 33-mer. Also note that 8 of 19 cases (42%) have PRN IgG above the normal range. Controls were 4 of 40 cases (10%) 33

He 2005 Non-Celiac Cases (16 Subjects)

Data from [39] replotted using [40]

PRN IgG vs. 33-mer IgA/IgG - Celiac Not Confirmed

320 280 240

33 mer IgA

33 mer IgG

Celiac Suspected, But Not Confirmed

PRN IgG above normal range of controls

33-mer IgA/IgG

200 160 120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80

He 2005s non celiac cases plotted on the same scale as controls. Note that 9 of 16 cases (56%) have 33-mer IgA/IgG and pertactin (PRN) IgG entirely within the normal range of controls (red box). From He 2005 - non-celiac cases include those who were suspected to have CD but not serologically confirmed at the department. Suspected celiac is more appropriate. Also note that 5 of 16 cases (31%) have PRN IgG above the normal range. Controls were 4 of 40 cases (10%) 34

He 2005 All Cases Combined (75 Subjects)

Data from [39] replotted using [40]

PRN IgG vs. 33-mer IgA/IgG - All Cases

320 280

33 mer IgA

33 mer IgG

PRN IgG above normal range of controls

33-mer IgA/IgG

200 160 120 80 40 0 0 10 20 30 40 PRN IgG

Both 33-mer IgA and PRN IgG high 6 cases Both 33-mer IgG and PRN IgG high 1 case

33-mer IgA or IgG above normal range of controls





He 2005s all subject case categories plotted together. Of those that fall outside the normal range (red box) there is a distribution that appears non-random.
15 (IgA) and 7(IgG) cases have a 33-mer exaggerated immune response 17 cases have a pertactin (PRN) IgG exaggerated immune response 6 (IgA) and 1 (IgG) case(s) have exaggerated immune responses to both 33-mer and PRN IgG. This group appears to be strongly under represented if the two variables were truly independent.


He 2005 Celiac Cases Reanalyzed (14 Subjects)

Data from [39] replotted using [40]

PRN IgG vs. 33-mer IgA/IgG - Confirmed Celiac Those with "normal" PRN and 33-mer removed
320 280 240

33 mer IgA 33 mer IgG Linear (33 mer IgA) Linear (33 mer IgG)

Correlation with PRN IgG 33-mer IgA = -0.672 33-mer IgG = -0.345

33-mer IgA/IgG

200 160 120 80 40 0 0 10 20 30 40 PRN IgG 50 60 70 80

He 2005s confirmed cases after removing 5 cases with normal 33-mer IgA, 33-mer IgG, and pertactin (PRN) IgG (the suspected HLA-DQ8 haplotype or 33-mer nonresponders). Linear trend lines calculated using remaining data from 14 cases. Note strong negative 33-mer IgA to PRN IgG correlation. 33-mer IgG to PRN IgG exhibits weaker negative correlation.


He et al 2005 Review Summary (1 of 2)

He et al concluded that they found no evidence of cross reactivity between 33-mer and pertactin antibodies. What they missed is that in affected individuals the pattern is not random, there is a negative correlation between 33-mer and pertactin antibodies. This is consistent with the hypothesis that APCs and T-cells will be selective toward either 33-mer or pertactin depending upon exposure.
The under representation of cross-reactors suggests a dependence between variables
Pertactin exaggerated response 33-mer exaggerated response Cross exaggerated response 17 IgG cases 7 (IgG) 15 (IgA) cases 1 (IgG) 6 (IgA) cases

There was a strong negative correlation in confirmed celiac cases with exaggerated immune responses to either 33-mer or pertactin
Correlation between pertactin IgG and 33-mer Correlation between pertactin IgG and 33-mer IgA = - 0.672 IgG = - 0.345

They also failed to note that the confirmed celiac and suspected but not confirmed celiac groups exhibit an exaggerated pertactin antibody response at a much higher rate than the healthy control group.
Confirmed Celiac Suspected Celiac but not confirmed Healthy Controls 8 of 19 cases 5 of 16 cases 4 of 40 cases 42% 31% 10%

He et al 2005 Review Summary (2 of 2)

He 2005s conclusions appear to have blunted further investigations into pertactin-celiac links as there is nothing in the literature after it [41] Their methods of data presentation and analysis may have led subsequent reviewers to overlook important trends
Failure to note the lack of cross-reactors to 33-mer and pertactin despite it being statistically unlikely Plot auto-scaling used when presenting side-by-side study population comparisons gives the impression of data randomness that is not born out under more detailed scrutiny Misleading labeling of non-celiac cases on plots when the papers text states those who were suspected to have CD but not serologically confirmed at the department in 2002. Suspected celiac but not confirmed is a far more appropriate description and could alter conclusions in light of the findings presented here.

Far from closing the book on pertactin, the raw data from He 2005 supports the hypothesis that APCs and T-cells can be selective toward either pertactin or 33-mer peptides. It also indicates pertactin antibodies are higher in confirmed and suspected celiac cases than in healthy controls. Although there could be many explanations for this, the conclusion that some celiac cases are actually pertactin-mediated cant be easily dismissed. To facilitate further analysis, the published paper, raw data, and processing algorithms should placed online with full open-access to independent researchers.

Pneumococcal Celiac Link Studies

A review of the scientific literature turned up no publications studying the relationship between immunogenic gluten peptides and Streptococcus pneumoniae.
There are many publications regarding the high incidence and serious nature of pneumococcal sepsis among those with celiac disease. This phenomena is attributed to hyposplenia (splenic atrophy) and is welldocumented in studies and case reports [42-44]. While hyposplenia is undoubtedly a major factor, it is an interesting coincidence that the surface protein sequence of a pathogen that plagues celiac patients so closely match that of 33-mer.

The negative correlation seen between pertussis pertactin and 33-mer antibodies in the re-analyzed He 2005 data raises an interesting question. Could the proliferation of APCs and T-cells selective toward 33-mer reduce the quantity of pneumococcal antibodies? If so, this could be a contributing factor beyond hyposplenia in explaining the high rate of pneumococcal infection in those with celiac disease. A single small study by McKinley et al 1995 [45] appears to contradict this theory. They found 10 celiac patients all demonstrated appropriate acute antibody responses to a polyvalent pneumococcal vaccine. More investigation is warranted as their raw data has not been reviewed in detail.

Selectivity Toward 33-mer or Surface Proteins

Stepniak [17] and Vader [20] have noted the differences in epitope reactivity patterns between adults and children with celiac disease
Adults routinely produce a response to alpha I, alpha II, and alpha III; the three epitopes found in 33-mer. Although many other epitopes have been identified, response to these is less certain (see list next page) Children tend to exhibit a much more varied response, with reactivity to the three epitopes in 33-mer being far less frequent than seen in adults

They have hypothesized that at the start, celiac disease produces a response to a wide range of epitopes. As the disease progresses, response becomes more focused on the immunodominant epitopes found in 33-mer. The findings presented here regarding the homology between 33-mer epitopes and bacteria surface proteins suggest a different explanation
Children are far more likely to have received a recent vaccination that includes pertactin or pneumococcal surface proteins as these are included in the routine pediatric vaccine schedule APC and T-cell selectivity toward bacterial surface proteins would diminish reactivity toward the epitopes in 33-mer. This is consistent with the negative 33-mer/pertactin reactivity correlation found in the reanalyzed He 2005 data.

The observed reactivity patterns support the hypothesis that recent vaccination will drive APC/T-cell selectivity away from 33-mer and toward the bacteria surface proteins

Gluten Stimulatory Epitopes

Table from Stepniak et al 2008 [17]

Found in 33-mer

Gluten stimulatory epitopes, 33-mer sequences boxed in red at the top of the list. In adults with celiac disease, responses to these three sequences are invariably present, less so for other sequences. Interestingly, responses to the others are seen more often in children with celiac disease, responses to the 33-mer sequences are often not found [17,20].


Pertussis Pertactin and Vaccines

Pertactin is a highly immunogenic virulence factor of Bordetella pertussis, the bacteria that causes whooping cough. It is a surface membrane protein that promotes adhesion to the trachea.
Pertactin is purified from Bordetella pertussis and is used in production of the acellular pertussis vaccine [46] The acellular pertussis (aP) vaccine replaced the whole-cell pertussis (wP) vaccine used previously due to adverse reaction concerns with wP.

The pertussis vaccine is administered simultaneously with diphtheria and tetanus toxins in a tri-valent combination known as the DTaP vaccine (acellular version) or the DTP vaccine (whole-cell version) [47] Timeline of the pertussis vaccine introduction in the United States
In the late 1940s, the whole cell pertussis vaccine (DTP) was introduced to widespread use in the U. S. It has since been almost entirely replaced by the acellular vaccine (DTaP) in the developed world. The acellular pertussis vaccine (DTaP) was recommended for use for only the 4th and 5th doses in the pediatric series in February 1992 [48] DTaP was recommended for all doses in the pediatric series in March 1997 [49]

The pertactin motif that contains the alpha-gliadin 33-mer pattern matches is known to be highly immunogenic and exhibits virtually no variation among pertussis strains
Known as region 2 in pertussis literature, it has been extensively studied and is well characterized [26,38]. Pertactin-based vaccines would contain the 33-mer pattern match region

Pneumococcal Surface Proteins and Vaccines

Pneumococcal surface membrane polysaccharides are used in the production of the pneumococcal conjugate vaccine (PCV) [31,32]
Saccharides of the capsular antigens from multiple serotypes are individually conjugated to diphtheria CRM197 protein (used as a carrier)

Timeline of the pneumococcal vaccine introduction in the United States

PPSV (23 serotypes, non-conjugate) was recommended for high-risk groups over two years of age in March 1997 [50] PCV7 (7 serotypes) was recommended for routine pediatric use in June 2000, and added to the CDC schedule in 2001 [51] PCV13 (13 serotypes) was recommended for pediatric use in February 2010 [52]

Yu et al 2003 [53] studied surface protein PspA contamination of the PCV7 and PPSV pneumococcal vaccines. They concluded these vaccines are contaminated with PspA though lot variability appears to be high.
Adults given a single dose of PPSV or PCV7 produced a significant increase in anti-PspA antibodies. PPSV response appeared manufacturer/lot dependant. They noted that the pediatric schedule frequency and dosage may produce higher anti-PspA responses than seen in this study.

The serotypes sequenced by Beall et al 2000 are found in these vaccines. Vaccine recipients would receive PspA contaminants that contain the sequence pattern matches to alpha-gliadin 33-mer.
Pnuemococcal Serotypes
6A 4 1 1 2 3 3 4 4 5 5 6A 6B 6B 6B 6B 7F 7F 8 9V 9V 9V 9V 10A 11A 12F 14 14 14 14 18C 19A 19F 19F 23F 23F 23F 20 22F 23F 33F

Sequenced by Beall et al 2000 PCV7 [31] PCV13 [32] PPSV [33]

18C 19A 19F 15B 17F 18C 19A 19F


Haemophilus influenzae B and Vaccines

The Haemophilus influenzae B (Hib) conjugate vaccine components were checked for pattern matches to the gliadin 33-mer sequence. A review of the relevant sequences in the BLAST database found no matches.
No matches among the Hib capsular antigens No matches for the tetanus, diphtheria, or Neisseria meningitidis proteins used as carriers for the conjugate

However, Hib has a unique interaction with pneumococcal strains in eliciting an immune system response against Streptococcus pneumoniae. The mechanism is not well understood [54].
The presence of both Hib and pneumococcal strains together provokes a vigorous immune system response against the pneumococcal strains. Hib is not affected. The immune response is not elicited if Hib is absent

Due to this interaction, widespread use of the Hib vaccine in infants may have led to a proliferation of the pneumococcal strains
The Hib vaccine was recommended for routine pediatric use in 1991 in the U.S., a decade prior to the pneumococcal conjugate vaccine [55] Although findings in studies have been inconsistent, Baer et al 1995 [56] did find that pneumococcal infections in children increased after widespread introduction of the Hib vaccine in Finland

The potential change in the mucosal flora brought on by the Hib vaccine could be a contributing factor in this hypothesis. A surge in pneumococcal proliferation after Hib vaccine administration may trigger the autoimmune response toward small bowel tissue in those genetically susceptible.


Use of Aluminum Compound Adjuvants

The vaccines discussed use aluminum compound adjuvants intended to amplify and prolong the adaptive immune response
DTaP - aluminum hydroxide [46] PCV7/PCV13 - aluminum phosphate [31,32] Hib - aluminum hydroxide [57] (manufacturer dependant)

The aluminum adjuvant serves to form a depot within the muscle providing a long-duration persistence of the antigen [37]. This is key to the surface protein hypothesis as it explains the chronic nature of the small bowel tissue destruction.
Unlike a pathogenic acute infection that leads to either rapid antigen clearance (via the immune system/antibiotics) or death, there is low-level, continuous persistence of the antigen Analogous to the continued presence of gluten in the diet, the antigen persistence causes a chronic inflammatory state of the small bowel

Although data is sparse, studies have shown this antigen persistence from aluminum depots continues for several months
Using an aluminum compound adjuvant, Harrison 1935 [58] demonstrated diphtheria antigen persistence in guinea pigs lasted at least seven weeks, the duration of this study. Verdier et al 2005 [59] demonstrated persistence at the injection site of up to 3 months with aluminum phosphate and at least 6 months with aluminum hydroxide using a tetanus/diphtheria vaccine in monkeys

Despite use for over 80 years, the mechanisms at work with aluminum compound adjuvants are not well understood

CDC Pediatric Vaccine Schedule

Table from CDC 2011 [47]

As can be seen in the CDC schedule [47], genetically-susceptible infants and toddlers would be repeatedly injected with vaccines that contain pertactin (DTaP) or pneumococcal surface protein A (PCV) contaminants. The repeated administration of the Hib vaccine may also play a role. The long duration persistence from the aluminum adjuvant deposition may appear to the immature pediatric immune system as a nearly continuous exposure.


Bacteria Surface Proteins and Vaccines

In developed countries, DTaP is routinely administered to infants, toddlers, and children PCV and Hib are also routinely administered to infants and toddlers under 2 years of age in the United States and other countries All are often administered together and with other vaccinations as the CDC schedule combines vaccinations at single age milestones [47]
2 months DTaP, PCV plus vaccinations for Hib, polio, rotavirus, and hepatitis B 4 months DTaP, PCV plus Hib, polio, rotavirus, and hepatitis B 6 months DTaP, PCV plus Hib, polio, rotavirus, hepatitis B, and influenza 12-15 months PCV plus Hib, polio, measles, mumps, rubella, varicella, hepatitis A, and hepatitis B 18 months - DTaP plus hepatitis A and influenza ~ 5 years (prior to kindergarten admission) DTaP, plus polio, measles, mumps, rubella, varicella, and influenza

Although anecdotal, many parents report autistic regression shortly after vaccination. The temporal relationship between the introduction of the DTaP and Hib vaccines and the increase in autism rates deserves further review.
DTaP was introduced for the 4th dose (at 18 months) and 5th dose (at 5 years) in the series in 1992. These correspond to 1990-1991 and 1987 birth year cohorts, respectively. Hib was introduced in 1991 for ages 2 to 15 months. Although it is not presumed to be the sole trigger of autism and developmental delays, increases in autism rates do appear to be correlated with the DTaP and Hib vaccine introduction when plotted by birth year cohort (see next page).

Bacteria Surface Proteins and Vaccines

California Autism Rate by Birth Year Cohort Source: and California Health and Human Services Agency 120
PCV7 Birth Year Cohort Introduction DTaP 1st to 3rd dose (2 to 6 mos) Birth Year Cohort Introduction

ASD rate per 10,000 births

DTaP 4th dose (18 mos) Birth Year Cohort Introduction DTaP 5th dose (5 years) Birth Year Cohort Introduction



Latest Data Shown (2009) Drop in ASD rates after 2003 is due to age of birth year cohort (< 6 years old)

Hib Birth Year Cohort Introduction

0 1975



1990 Birth Year




California data shown. Due to the Lanterman Act of 1969, California has the most comprehensive autism data prior to 1990.

Conclusion - Tying It All Together

Celiac disease may have its roots in an entirely appropriate evolutionary response to surface proteins in pathogenic microorganisms. During exposure to a pathogen, the immune system may induce a temporary state of inflammation and malabsorption to quickly purge infectious bacteria from the digestive tract. Once the acute stage passes, normal digestive function returns. In those genetically predisposed, the immune system is fooled by partially digested gluten peptides selectively deamidated by tTG. As wheat is continually present, malabsorption continues to the point of malnutrition. Celiac disease may take decades to manifest itself, probably due to the need for an additional environmental trigger or prior health deterioration. Intestinal permeability and possibly protein maldigestion need to develop first to allow undigested gluten fragments access to the APCs. In the case of pediatric vaccinations, bacterial surface membrane proteins have direct and immediate access to the bloodstream and APCs without any need for intestinal permeability, protein maldigestion, or deamidation by tTG. The vaccine regimens high concentration of surface membrane proteins, the simultaneous introduction of multiple strains, the presence of powerful aluminum adjuvants that produce antigen persistence, and the reintroduction over the course of multiple boosters may trigger the celiac-like disorder in those genetically disposed. Analogous to gluten, the immature immune system interprets the unorthodox, subclinical, and persistent re-exposure to the epitopes as a continuous infection. The use of a one-size-fits-all pediatric vaccination schedule should be reexamined. Results from genetic screening and a review of family history may warrant a more cautious approach to vaccination. An individual risk-benefit analysis seems reasonable, rather than a presumption of low risk/high benefit.

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1. the purpose and character of the use, including whether such use is of a commercial nature or is for nonprofit educational purposes; 2. the nature of the copyrighted work; 3. the amount and substantiality of the portion used in relation to the copyrighted work as a whole; and 4. the effect of the use upon the potential market for or value of the copyrighted work.

The fact that a work is unpublished shall not itself bar a finding of fair use if such finding is made upon consideration of all the above factors.