Recently, researchers at Harvard University, Alessio Fasano et. al., and the National Institutes of Health (laboratories of immunology and cellular and molecular biology), reported real-time microscopic observations of gluten-induced neutrophil activation. According to authors, " To what extent neutrophil function adds to, or protects against, gluten intolerance is currently under vigorous investigation." This presentation will shed light on this question. It will also review the Fasano study and examine the role of neutrophil function in multiple disease conditions, as well as explore how neutrophil function may also play a dual role in protecting the body from the untoward effects of dietary and environmental agents.

1. Understanding the Pathogenesis
of Gluten Spectrum Disorders

2. Founder and CEO of Cell Science Systems,
Co-developer, Alcat technology
Roger Deutsch

3. Innate immunity in gluten spectrum
and other disorders
• Coeliac vs. NCGS
• Innate immune disorders in the gut and auto-immunity
• Other food triggers
• Neutrophil activation
• Q&A
Topic Overview

4. Gliadin induces Neutrophil Migration via
Engagement of the Formyl Peptide Receptor,
FPR1
Results
In vivo intestinal luminal injection of PT-gliadin induces an
immediate and substantial recruitment of neutrophils.
Increased intestinal permeability favors the access of gliadin
and other macromolecular antigens from the intestinal lumen
into the lamina propria where the host, by interpreting them as
danger signals, will respond with first-line defense mechanisms
such as neutrophil recruitment to the site of exposure.
RESEARCH
ARTICLE
Karen M. Lammers, Marcello Chieppa, Lunhua Liu, Song Liu, Tatsushi Omatsu, Mirkka Janka-Junttila,
Vincenzo Casolaro, Hans-Christian Reinecker, Carole A. Parent, Alessio Fasano
PLOS ONE | DOI:10.1371/journal.pone.0138338 September 17, 2015

5. Figure 4
Human neutrophils migrate to
fMet-Leu-Phe and PT-gliadin.
(E) PT-gliadin induced neutrophil
migration in
a dose-dependent manner in the
EZ-TAXIScan assay. As a positive
control, fMet-Leu-Phe was used.
Data are obtained from three
independent experiments.
(D) PT-gliadin induced neutrophil
migration in a dose-dependent
manner in a Transwell chemotaxis
assay (P<0.01). Data are obtained
from six independent experiments.

7. Table 1. Blocking of FPR1 abrogates neutrophil migration to fMet-Leu-Phe and
gliadin.
Pretreatment of neutrophils with cyclosporine H, a specific inhibitor of FPR1,
completely abrogated the neutrophil migration induced by PT-gliadin and fMet-
Leu-Phe. As expected, LTB4 induced neutrophil chemotaxis was not affected by
cyclosporine H.

8. • The classical paradigm of inflammatory pathogenesis
involving specific genetic makeup and exposure to
environmental triggers has been challenged recently by
the addition of a third element, the loss of intestinal
barrier function.
• Fassano, Zonulin and its Regulation of Intestinal Barrier
Function: the Biological Door to Inflammation,
Autoimmunity and Cancer Physiological Revs. 1 Jan 2011 V 91

9. …the activation of the zonulin pathway by food-derived
environmental triggers or changes in gut microbiota all
seem to be key ingredients involved in the pathogenesis of
inflammation, autoimmunity, and cancer.
Fassano, Zonulin and its Regulation of Intestinal Barrier
Function: the Biological Door to Inflammation,
Autoimmunity and Cancer Physiological Revs. 1 Jan 2011 V 91

10. This new theory implies that once the pathological
process is activated, it is not auto-perpetuating.
Fassano, Zonulin and its Regulation of Intestinal Barrier Function: the
Biological Door to Inflammation, Autoimmunity and Cancer Physiological
Revs. 1 Jan 2011 V 91

11. Divergence of gut permeability and mucosal immune
gene expression in two gluten associated conditions:
celiac disease and gluten sensitivity

12. Divergence of gut permeability and mucosal immune
gene expression in two gluten associated conditions:
celiac disease and gluten sensitivity
“These findings might indicate that GS is an inflammatory
condition mostly supported by innate immune mechanisms.
Among these, TLR’s represent a family of evolutionary
conserved receptors able to detect microbial invasion via
pattern recognition and mediate a rapid inflammatory
response which may or may not progress into an antigen-
dependent adpative response.”
Fasano et al. BMC Medicine 2011, 9:23

13. Divergence of gut permeability and mucosal immune
gene expression in two gluten associated conditions:
celiac disease and gluten sensitivity

14. Divergence of gut permeability and mucosal immune
gene expression in two gluten associated conditions:
celiac disease and gluten sensitivity

18. Conclusion
“To what extent neutrophil function adds to, or protects
against, gluten intolerance is currently under vigorous
investigation.”

19. Regardless of whether food induced
activation of neutrophils is
protective or pathogenic, do other
foods besides Gluten cause
intestinal permeability and
activation of neutrophils?

20. Mucosal reactivity to cow’s milk protein in
coeliac disease
G. Kristjansson, et. al,
Clin & Exp. Imm., 147, 2007
In 18 of 20 patients gluten challenge induced neutrophil
activation defined as increased MPO release and increased
NO synthesis. Ten of these 20 patients showed a similarly
inflammatory reaction to CM challenge….
A mucosal response similar to that elicited by gluten was
produced by CM protein in about 50% of the patients with
coeliac disease. Casein, in particular, seems to be involved
in this reaction.

21. Gut mucosal granulocyte activation precedes nitric
oxide production: studies in coeliac patients
challenged with gluten and corn
G. Kristjansson, et. al, Gut, 2005 June 54(6)
Conclusion
Mucosal activation of neutrophils and eosinophils precedes
pronounced enhancement of mucosal NO production after
rectal wheat gluten challenge in patients with coeliac
disease. Some of our patients displayed signs of an
inflammatory reaction, as measured by NO and
granulocyte markers, after rectal corn gluten challenge.

22. High Correlation of the Alcat Test Results With
Double-Blind Challenge (DBC) in Food Sensitivity
For the 58 Alcat positive foods selected from the 19
subjects, 46 were positive on DBC (79.3%) and 12 were
negative. Of the 56 Alcat negative foods, 49 were also
negative by DBC (87.5%) and 7 were positive. Overall
correlation between Alcat and DBC was 83.4%.
Peter J. Fell, MD, Director, Oxford Allergy Centre, London
Jonathon Brostoff, MA DM DSc FRCP FRCPath, Dept. of Immunology,University College & Middlesex School of Med, London
Presented at the 45th Annual Congress of the American College of Allergy and Immunology, Los Angeles, CA: November
12-16, 1988. Published in Cof. Preceedings -Annals of Allergy.

23. POLYMORPHONUCLEAR CELLS (PMN) REACTIVITY
WHEN EXPOSED TO ALCAT TEST AGENT
Following are images showing some examples of the changes seen for PMN cells when exposed to an Alcat Test agent. (Control,
Barley and Lamb). All cell images were taken at the same magnification.
Work Performed at: NIS Labs, 1437 Esplanade Ave, Klamath Falls, OR 97601 by Kathy Benson
In the Control well (above), the cells stayed round, non-reactive and were sized and
counted to formulate the CONTROL GRAPH – CON3
PMN Cell observed in the
Control Well by CytoViva
Microscopy
CONTROL GRAPH (Analyzed with the
Robocat II)
PMN Cells in the Control Well
analyzed by FACSCalibur flow
cytometry

24. POLYMORPHONUCLEAR CELLS (PMN) REACTIVITY
WHEN EXPOSED TO ALCAT TEST AGENT
In the Barley test well (above), the PMN cells became enlarged and
adherent to the slide. There was significant movement of granules
in the PMN cells exposed to antigen.
PMN Cells observed in the
Barley Well using CytoViva
microscopy
PMN Cells in the Barley Well
analyzed by FACSCalibur flow
cytometry
BARLEY RX GRAPH (Analyzed with the
Robocat II) plotted against the CONTROL
GRAPH

25. POLYMORPHONUCLEAR CELLS (PMN) REACTIVITY
WHEN EXPOSED TO ALCAT TEST AGENT
In the Lamb test well (above), the PMN cells also became enlarged and
adherent to the slide. There was significant movement of granules in the PMN
cells exposed to antigen. The Lamb Rx was so extreme that it appears that all
the cells have lysed and granules are spread all across the slide.
PMN Cells observed in the Lamb
Well using CytoViva microscopy
LAMB RX GRAPH (Analyzed with the
Robocat II) plotted against the CONTROL
GRAPH
PMN Cells in the Lamb Well
analyzed by FACSCalibur flow
cytometry

26. The Short Term Efficacy of the Alcat Test of Food Sensitivities to
Facilitate Changes in Body Composition and Self-Reported
Disease Symptoms:
A Randomized Controlled Study

27. Conclusion
“To what extent neutrophil function adds to, or protects
against, gluten intolerance is currently under vigorous
investigation.”

29. 552 - Alcat Test results in the treatment of gastrointestinal symptoms
Berardi L.^, De Amici M.°, Castello M.^, Torre C.°, Giunta V.°, Legoratto S.^, Vignini M.^
^ Dermatology Clinic- University of Pavia- Foundation IRCCS Policlinic San Matteo; Pavia, Italy
° Hospital Pediatric Clinic – Foundation IRCCS Policlinic San Matteo; Pavia, Italy
Presented at the 30th Congress of the European Academy of Allergy and Clinical Immunology, 11-15 June 2011 - Istanbul, Turkey
48 patients (pts) (37 females, 11 males, median age 38.5 years) affected by GI symptoms were
tested by ALCAT. Pts also answered a survey regarding a tailored (diet based on ALCAT results) two
month elimination diet.
Clinical evaluation was also performed, considering the symptom score before and after initiating the
diet (0-10), everyday conditions (score 0-10), dietary changes (0-10) and symptom improvement after
the diet (1=nothing; 2=mild; 3=very important).
The symptom score before starting the diet
(p=0.0042), dietary changes (p=0.0003) and
everyday conditions score (p=0.0015) significantly
changed with regard to symptom improvement
score. In particular, higher values in dietary
changes score, everyday conditions score and
symptom score were associated with large
improvements. Finally, the difference between
symptom score before (md: 6.5; IQR: 6-8) and after
the diet (md: 4; IQR: 3-5) was significant
(p<0.0001).
The study provides evidence that an elimination diet based on Alcat Test results improves
symptoms in 71% of patients. In particular, symptom improvements were most evident in
patients with higher symptom scores, dietary changes and everyday conditions.

30. From: Bach JF. The effect of infections on
susceptibility to autoimmune and allergic diseases. N
Engl J Med. Sep 2002;347(12):911–920.

31. Oxidative stress levels in circulating neutrophils is
linked to neurodegenerative diseases.
“Oxidative lesions are a hallmark of Alzheimer’s & Parkinson’s.
Circulating neutrophils are the most powerful sources of
reactive oxygen species…Significantly increased oxidative
stress levels were observed in patients' group...”
Vitte J, Michel BF, Bongrand P, Gastaut JL. Laboratoire d'Immunologie, INSERM Hopital Sainte-Marguerite, Marseille. J Clin
Immunol. 2004 Nov;24(6):683-92.

32. Phagocytes: mechanisms of inflammation
and destruction
Further, the dysregulation of the phagocytosis may be
critical to the pathogenesis of a variety of autoimmune
diseases, including RA and systemic lupus erythematosus
(SLE).
Hongrao Liu, MD, PhD, Richard M. Pope, MD, Div. Of Rheumatology, Dept. of Medicine, Feinberg School of Medicine. The Veteran’s Affairs
Med. Center, Chicago, IL Rheumatic Diseases Clinics of N. America, Vol. 30, No. 1, Feb 2004.

33. Phagocytes: mechanisms of inflammation
and destruction
In acute inflammation, the initial leukocytes that are
infiltrated are mainly neutrophils….neutrophils die in situ
by apoptosis…. The apopototoic neutrophils are cleaned
by macrophage phagocytes.
Hongrao Liu, MD, PhD, Richard M. Pope, MD, Div. Of Rheumatology, Dept. of Medicine, Feinberg School of Medicine. The Veteran’s Affairs
Med. Center, Chicago, IL Rheumatic Diseases Clinics of N. America, Vol. 30, No. 1, Feb 2004.

34. Ligation of receptors initiates formation of
Neutrophil Extracellular Traps (NETs)

38. Chronic activation of the innate immune system
may underlie the metabolic syndrome
Most importantly, activation of this system leads to
fundamental changes in body metabolism. In this regard, it
is notable that pro-inflammatory cytokines are major
stimulants of the hypothalamic-pituitary-adrenal axis,
leading to increased cortisol secretion and inhibition of sex
hormone production.
Sao Paulo Med. J. Vol. 119 no.3 Sao Paulo May 2001
Social Medicine Department, School of Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

39. Macrophage activity in semen is significantly
correlated with sperm quality in infertile men.
“The presence of leucocytes within semen has the
potential to impair sperm function. Neutrophils and
macrophages make up 95% of seminal leucocytes, with
both having the ability to damage sperm via the generation
of reactive oxygen species, proteases and the induction of
apoptosis…..We were able to confirm for the first time
that seminal plasma does indeed contain neopterin and
that the levels of this macrophage activity marker are
threefold higher in infertile than fertile men.”
Tremellen K, Tunc O. Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health,
University of Adelaide, Adelaide, SA, Australia. Int J Androl. 2010 Jan 28.

40. Aging through oxidative stress,
inflammation, and progression to CVD
In younger (but not older) participants, each shortened
kilobase pair of TRF corresponded with a threefold
increased risk of myocardial infarction… telomere
attrition may be related to diseases of aging through
mechanisms involving oxidative stress, inflammation, and
progression to CVD.
Annette L. Fitzpatrick, et al. American Journal of Epidemiology 2007 165(1):14-21.

41. Leukocyte Telomere Length and CVD in the
Cardiovascular Health Study
Significant or borderline inverse associations were found
between TRF length and diabetes, glucose, insulin,
diastolic blood pressure, carotid intima-media thickness,
and interleukin-6.

42. Increase in fragmented phosphatidylcholine in
blood plasma by oxidative stress.
Oxidatively modified phospholipids have attracted much
interest because of their pro-inflammatory activity and
their potential involvement in atherosclerosis…The
increase coincided with a surge of circulating neutrophils.
Frey B, Haupt R, Alms S, Holzmann G, Konig T, Kern H, Kox W, Rustow B, Schlame M. Department of Anesthesiology
and Intensive Care Medicine, University Hospital Charite, Humboldt University, Berlin, Germany. J Lipid Res. 2000
Jul;41(7):1145-53

43. Relative Risk of Future Myocardial Infarction as
a Function of CRP and Cholesterol
Libby P, Ridker PM, and Maseri A. in Circulation, Vol. 105, No. 9, Pages 1135-1143, March 5, 2002
0
2
4
6
8
10
1 2 3 4 5
1
3
5
CRP
Quintiles of Cholesterol
Relative
Risk

44. Changes of neutrophil myeloperoxidase in coronary
circulation among patients with acute coronary
syndrome
“MPO is a better marker for inflammation of the local
plaques. It may be one of the mechanisms that MPO induces
the transforming from LDL to ox-LDL in plaques
vulnerability.”
Li L, Zhang Y, Chen YG, Li GS, Wang Y, Ma X, Li JF, Zhong M, Zhang W. Key Laboratory of Cardiovascular Remodeling
and Function Research, Chinese Ministry of Education and Public Health, Department of Cardiology, Qilu Hospital of
Shandong University, Jinan 250012, China.

45. alpha(4)-integrin mediates neutrophil-induced
free radical injury to cardiac myocytes
…circulating neutrophils adhere to cardiac myocytes and
cause cellular injury… emigrated PMNs have the capacity
to injure cardiac myocytes
Poon BY, Ward CA, Cooper CB, Giles WR, Burns AR, Kubes P.
Immunology Research Group, University of Calgary, Calgary, Alberta T2N 1N4, Canada. J Cell Biol. 2001 Mar 5;152(5):857-66.

46. Serum zonulin levels in
subjects affected by
different types of MS.
Alessio Fasano Physiol Rev 2011;91:151-175
Serum zonulin levels were
assessed in MS subjects
affected by different subtypes:
relapsing-remitting (RRMS)
during exacerbation of the
disease (n = 30), RRMS in
remission (n = 14), seconda...

47. Increase in Type 1 diabetes incidence by
genotype risk
Medium risk genotype
Low risk genotype
Very low risk genotype
2x
3x
7x
(Fourlanos et al. 2008b)
Odegaard and Chawla.
Department of Pathology, Stanford University School of Medicine
Cardiovascular Research Institute, Department of Physiology, and
Department of Medicine, University of California, San Francisco

48. Priming of NADPH oxidase by tumor necrosis factor
alpha in patients with inflammatory and autoimmune
rheumatic diseases.
“Oxidative damage caused by oxygen free radicals from
activated phagocytes contributes to the pathology of arthritis.
There was a two- to eightfold increase in phagocytic
superoxide production in rheumatic patients, when compared
to healthy subjects [p < 0.005].”
Miesel R, Hartung R, Kroeger H. University of Otago, Department of Biochemistry Dunedin, New Zealand. Inflammation.
1996 Aug;20(4):427-38.

49. TLR Signaling in the Gut in Health and Disease
Maria T. Abreu, et. al., The J. of Immunology, April 2005 174 No. 8
• “Secretion of various anti-microbial peptides by Paneth cells and
enterocytes is likely to be regulated by TLR-mediated recognition
of PAMPs”
• Exposure of colonic epithelial cells to bacteria results on sealing
tight junctures
• “Depending on the scenario, TLR signaling may aid in certain
aspects of barrier function but not others.”
• “TLR signaling in response to the presence of bacteria in the
lamina propria following epithelia injury is critical for healing of
the gut.”
• “Intestinal bacteria are essential for the normal development of
GALT. In the absence of luminal bacteria, B cells and T cells do
not home to the lamina propria…IgA is not secreted.”

50. TLR Signaling in the Gut in Health and Disease
Maria T. Abreu, et. al., The J. of Immunology, April 2005 174 No. 8
• “ …the gut environment uses signals from luminal bacteria to
develop tolerance by a mechanism involving regulatory T
cells”
• “…polymorphisms in TLR9 have been linked to Chrohn’s
disease.”
• “Mice with a myeloid cell-specific deletion of Stat3 are
deficient in IL10 production and develop enterocolitis.”
• “…TLR4-/- and MyD88-/- have increased rectal bleeding in
response to DSS….and, a dramatic paucity of neutrophils…”

51. TLR Signaling in the Gut in Health and Disease
Maria T. Abreu, et. al., The J. of Immunology, April 2005 174 No. 8
• “however, the balance between the requirement of
TLR4 or other TLRs to initiate and sustain acute and
chronic inflammation vs its role in healing of the
epithelium is probably contextual and depends on other
host factors…”
• Conclusion: “In response to recognition of PAMPs
through TLRs, both inflammatory and homeostatic
pathways are activated. In this way, there is clearance
of bacteria and resolution of normalcy.”

52. Summary
• Manifestations of gastrointestinal disease depends upon; genetics,
microbial and host interactions, environmental exposures and other
psycho-physiological factors.
• The molecular and cellular pathways associated with GI disorders are being
elucidated.
• The immune system normally confers balance and homeostasis but may tip
into imbalance and promote pathology
• Acute activation of the innate immune system precedes adaptive immune
responses
• Regardless of whether the innate response is protective or pathogenic it
may be a reliable indicator of pathogenic food and environmental triggers

53. CellScienceSystems.com