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Schizophrenia and Gluten: New Target and Precision MedicineDeanna L. Kelly, Pharm.D., BCPPProfessor of Psychiatry

Director, Treatment Research ProgramMaryland Psychiatric Research Program (MPRC)University of Maryland School of MedicineBaltimore, MD

Session resources at cpnp.org/TBD

@ProfDeannaKdlkelly@som.umaryland.edu

http://www.medschool.umaryland.edu/profiles/Kelly-Deanna/

Disclosures

• Dr. Deanna L. Kelly has served as a consultant for Lundbeck and Alkermes and has been an advisor for HLS Therapeutics. She has Dr. Kelly is currently funded by NIMH.

• No off-label indications of medications will be discussed. The investigational use of dietary products for a double-blind randomized diet will be discussed.

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Educational Need/Practice Gap

• Currently, treatments for schizophrenia remain inadequate and precision medicine is often not used.

• Needs are to move toward better precision medicine and identify treatments that best meet needs of individual patients.

• Education can help relay gaps in care and summarize new data emerging on potential future treatment targets.

Learning Objectives

Upon completion of this educational activity, you will be able to:

1. Describe the emerging role of inflammation in schizophrenia2. Discuss ongoing research to characterize a subgroup of schizophrenia

patients with immune response and inflammation to gluten3. Recognize potential pathophysiologic mechanisms related to an immune

response to gluten 4. Understand ongoing work for personalizing treatment in this subgroup of

schizophrenia with potential gluten sensitivity

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Inflammation and Schizophrenia

• Schizophrenia is linked to inflammation

• Prenatal maternal infection and immune activation associated with subsequent risk of schizophrenia in offspring

• Genome wide association studies (GWAS) suggest that the major histocompatibility (HLA) complex is linked to schizophrenia

• Large epidemiological studies show higher autoimmune diseases

• Increased levels of cytokines in periphery and cerebral spinal fluid (CSF) are present in schizophrenia, including drug naïve and first episode patients

Meyer U, et al Pharmacol Ther 2011;132(1):96-110; Fineberg AM, et al Biol Psychiatry, 2013;73(10):951-966; Moieni M., et al. Brain Behav Immun 2015;48:132-138; Potvin S, et al Biol Psychiatry 2008;63(8):801-808; Miller A., J Biol Chem 2012;287(6):3930-3945; Fillman SG, et al. Mol Psychiatry 2013;18(2):206-214. Hope TMH, et al. Neuroimage-Clinical, 2013;2:424-433; Hope TA, et al Mag Res Imaging 2013;31(9):1479-1484; van Berckel BN, et al. Biol Psychiatry 2008;64(9):820-822; Doorduin J of Nuclear Med 2009;50(11):1801-1807; Bloomfield PS, Am J of Psychiatry 2016;173(1):44-52; Bergen SE Mol Psychiatry 2012; 17(9):880-886; Sekar A, et al. Nature 2016;530(7589):177-183; Shi et al. Nature 2009;460(7256):753-757; Stefansson, et al, Nature 2009, 460(7256):744-747; Zhang et al. Curr Psychiatr 2013;12(3):24-33

Risk of Schizophrenia

45% increased risk for schizophrenia for persons with history of any autoimmune disease

1

Eaton WW et al, Am J Psychiatry. 2006;163(3):521-8 Benros ME, et al. Ann NY Acad Sci 2012;1262:56-66; permission granted for figure

Danish Registry Data:Schizophrenia related to 50% greater likelihood of

autoimmune disorder

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Caveats to Inflammatory Story

• Not all schizophrenia patients have similar symptoms• Not all have inflammation• Cytokine production is vastly different in studies• There may be subgroups in schizophrenia for which inflammation plays a significant

role

“Although it is possible that a single pathophysiologic process accounts for this diversity, the syndrome of schizophrenia probably comprises several disease processes, each with its own manifestations”

“If schizophrenia is a clinical syndrome rather than a single disease, the identification of specific diseases within the syndrome would facilitate the advance of knowledge and the development of more specific treatments”

Carpenter WT, Jr., Buchanan RW. NEJM 1994;330:681-90; Kirkpatrick B, Buchanan RW, Ross DE, Carpenter WT, Jr. Arch Gen Psychiatry 2001; 58:165-71

https://en.wikipedia.org/wiki/William_T._Carpenter

Deconstructing Schizophrenia

Leboyer M, et al. BMC Medicine 2016;14:173, with permission (open access)

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Wheat and Schizophrenia

Epidemiology and Wheat

• During World War II, as wheat consumption went down in Scandinavia, admissions for schizophrenia went down

• As wheat consumption in the United States went up, admissions for schizophrenia went up

Dohan FC, Acta Psychiatr Scand. 1966;42(1):1-2, with permission

Francis Curtis DohanMarch 24, 1907- Nov 9, 1991

https://en.wikipedia.org/wiki/F._Curtis_Dohan

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Epidemiology of Schizophrenia and Wheat

Is schizophrenia rare if grain is rare?

• Schizophrenia is almost nonexistent in populations consuming little or no grain• Three large examinations of native tribes in Papua New Guinea:

• found 2/65,000 having schizophrenia in 40s/50s• found 1% after westernization

• Tribes of Yap:• found 0/1955 with schizophrenia in 40s/50s • found 30/3086 (0.97%) in 1978/79 after westernization and

and wheat introduction• Tribes of Malaita in 1980s:

• found 0 cases in tribes with no grain• Found 70/32,000 (0.22%) in tribes with grain

Dohan FC, Harper EH, Clard NH, Rodriguez RB, Vigos V. Biological Psychiatry 1984;19:85-99

Removal of Wheat from Diet

• First relationship of wheat to schizophrenia noted in 1950s, Curtis Dohan

Bender 1953, Psychiatr Q; Sheldon 1959, Pediatrics; Dohan 1966, Am J of Clin Nutrition; Walsh and Walsh 1970, Irish Med Association; Dohan 1973, Am J of Psychiatry; Singh and Kay 1976, Science; Rice et al. 1978, Am J of Psychiatry; Vlissides et al. 1986, Br J of Psychiatry’ Potkin et al 1981, Am J Psychiatry; Storms et al 1981, Arch Gen Psychiatry, Osbourne et al 1982 Biol Psych; Levinta et al Adv Nutrition 2018.

Author Date Sample Results

Dohan 1966 102 62% discharged versus 32%

Dohan/Grasberger 1973 115 37% to open ward versus 16%

Singh and Kay 1976 14 Improvement on 30/39 measures

Rice 1978 16 Two improved

Potkin 1981 8 No change in functioning

Storms 1981 26 No change in functioning

Osborne 1982 4 No change in functioning

Vlissides 1986 24 Improvement in 5/12 measures

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What is Gluten?

• A protein composite found in foods containing wheat*, barley, rye and triticale (a cross between wheat and rye)

• Made up of many proteins, 1) gliadin, which gives bread the ability to rise during baking; and 2) glutenin, which is responsible for elasticity

• Glutenin is easily digested by enzymes because it is a long protein with much greater surface area for the enzymes to attach and degrade.

• Gliadin is a densely packed protein with a low surface area to volume ratio, making it difficult to digest.

• Due to the difficulty in digestion, gliadin is digested into long amino acid chains, called oligopeptides, rather than the di- or tri-amino acids

• wheatberries, durum, emmer, semolina, spelt, farina, farro, graham, KAMUT® khorasan wheat and einkornRafalski JA. Gene 1986;43:122129, Anderson OD, Greene FC. Theoretical Applied Gen 1997;95:59-65, Stern et al Gastroenterol Hepatol 2001;13:741-7

http://www.drschaer-institute.com/us/non-celiac-gluten-sensitivity/definition-1069.html; figure in public domain

Gluten Sensitivity often referred to as Non-Celiac Gluten Sensitivity

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•1% of population

•Autoimmune disorder (adaptive immune system) with genetic predispostion

•Damage to intestinal villi

•Gastrointestinal symptoms - bloating, constipation, diarrhea, vomiting, weight loss

•Some psychiatric and neurologic symptoms

•Triggered by ingestion of gluten

(wheat, rye, barley)

•Underdiagnosed

•Treated with gluten-free diet

•5-10% of population

•Involves innate immune system

•Some GI Side Effects

•Neurologic and psychiatric issues

•Eczema, joint pain, fogginess, headaches

Elli L et al. Dig Liver Dis 2017;49:138-46; Hill et al. J Pediatr Gastroenterology Nut 2016;63:156-65; Sapone et al. BMC Medicine 2012;10:13 Rubio-Tappio A, et al Am J Gastronenterology 2013;108:656-76, Bai JC, et al. World Gastroenterolgy Organisation, 2016; https://www.niddk.nih.gov/health-information/diagnostic-tests/celiac-disease-health-care-professionals; Volta U 2102 J Clin Gastroenterology 2012;46:680-5.

Gluten-Related Antibodies• Antibodies in Celiac Disease (CD):

• anti-tissue transglutaminase (tTg-IgA) antibodies (sensitivity 93% and specificity 98%)• endomysial antibodies (EMA) (more difficult to assay)• deaminated gliadin peptide (DGP) (antibody testing more expensive)• Intestinal biopsy can confirm villous atrophy even in absence of positive antibodies• guidelines in 2013 (also 2016 World Gastroenterology Organization)• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706994/pdf/nihms477392.pdf

• Antibodies in Gluten Sensitivity (GS):• native gliadin antibodies (AGA IgA) (low sensitivity or specificity)• native gliadin antibodies (AGA IgG) – (not considered gold standard, may aid in diagnosis when

CD is not diagnosed)• new consensus nomenclature in 2012

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292448/pdf/1741-7015-10-13.pdf

Sapone et al. BMC Medicine 2012;10:13 Rubio-Tappio A, et al Am J Gastronenterology 2013;108:656-76, Bai JC, et al. World Gastroenterolgy Organisation, 2016; https://www.niddk.nih.gov/health-information/diagnostic-tests/celiac-disease-health-care-professionals, Infantino M. Minerva Gastronenterol Diet 2017;63:14, Volta U 2102 J ClinGastroenterology 2012;46:680-5.

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Gluten-Related Antibodies in Schizophrenia

• One initial data suggested elevations in AGA IgA , not well replicated

• tTG similar, higher in some studies

• DGP, only expected to be higher in Celiac patients

• We’ve replicated these findings in two additional samples of no differences in these antibodies 0

2

4

6

8

10

12

14

16

AGA IgA DGP IgA DGP IgG tTG IgASchizophrenia (N=160) Healthy Controls (N=80)

Positivity was > 20 U for all tests with exception of tTG6 with positive cutoff >f 26U* DGP IgGand IgA was tested in 40 patients, while all other test were performed on the N=160 in the schizophrenia group. N=80 controls were tested for all laboratory tests

Cihakova D, et al. Schiz Res. 2018

IU

17.5% vs 15% positive

0 vs. 1.3% positive*

2.5 vs. 1.3% positive*

2.5 vs 0% positive

Gluten-Related AntibodiesGluten-Related Antibodies

AGA (IgG) in Schizophrenia vs. Healthy Controls

9%

30%

0%

5%

10%

15%

20%

25%

30%

35%

HC SZ

Sidhom, et al 2012

AGA IgG>20U

10%

31.9%

0%

5%

10%

15%

20%

25%

30%

35%

HC SZ

Cihakova, et al 2017

AGA IgG>20U

Cihakova D, et al, Schiz Res 2018; Sidhom OL, et al. Immunol Invest 2012;41(5):538-549, Dickerson et al. Biol Psychiatry. 2010:68(1):100-4; Okusga O, et al. World J Biol Psychiatry. 2013 Sep;14(7):509-15.

HC=healthy controlsSZ=schizophrenia

Using INOVA First Generation Kits for Native Gliadin

AGA IgG Mean Values 17.9 ± 21.4 SZAGA IgG Mean Values 9.2 ± 13.2 HC p<0.001

Two other published papers have shown increased OR of 2-5 for AGA IgG in schizophrenia

Of note all HC AGA IgG values were under30 with the exception of 4 high outliers who may have had autoimmune or other unknown reason for elevated values

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AGA IgG in Schizophrenia vs. Healthy Controls

Okusaga O, et al The World J of Biological Psychiatry 2013;14(7):509-515; Dickerson F, et al., Biological Psychiatry 2010;68(1):100-104; Yang et al Psychiatry Research 2018; McLean et al Translational Psychiatry 2018

0.52

0.81

00.10.20.30.40.50.60.70.80.9

HC SZ

Mean AGA IgG levels (U) P< 0.0001

Using kits from IBL International, Hamburg, Germany

Multi Episode vs. HC OR= 5.50; 95% CI 2.65-11.42Recent Onset vs. HC OR= 6.19; 95% CI 2.70-14.16

SZ vs. HC OR= 2.13; 95% CI 1.6-2.9

Mean AGA IgG Levels (U)P<0.05

2 papers: UK and China report gliadin derived peptide antigens, not replicating, but under study

What Does this Subgroup Look Like?

• Okusaga 2013 finds higher AGA IgG correlated to lower total PANSS score (r=-0.073, p=0.026)

• Our data shows significantly lower levels of BPRS positive symptoms in people who are AGA IgG positive compared to AGA IgG negative (4.11 ± 1.36 vs. 6.39 ±2.99, p = 0.020)

• Higher kynurenine levels associated with higher AGA IgG (p<0.05). Kynurenine is linked to schizophrenia pathophysiology

Okusga O, et al. World J Biol Psychiatry. 2013 Sep;14(7):509-15, Jackson J, et al Schizohr Res 2014 Nov;159(2-3):539-42, Okusaga O, et la. Psychosom Med 2016 Oct;78(8):931-939.

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AGA IgG and Inflammation in Schizophrenia

• We examined the relationship of the AGA IgG antibodies to inflammation• We completed two pilot studies which examined:

1) Peripheral cytokine measures (TNFα and IL-Iβ)• We recruited 100 people with DSM-IV diagnosis of schizophrenia or schizoaffective disorder• AGA IgG was assayed by the laboratory of Dr. Alessio Fasano (Harvard) using an automated enzyme-linked

immunosorbent assay (ELISA) method with kits from Phadia AB.

2) Proton Magnetic Resonance Spectroscopy (MRS) for central inflammation• We recruited 31 people with DSM-IV diagnosis of schizophrenia or schizoaffective disorder• AGA IgG (native gliadin) was assayed by the laboratory of Dr. Daniella Cihakova (Johns Hopkins) using

using ELISA

Correlation of AGA IgG to Cytokines

Kelly D, et al. Brain Behav Immun. 2018;69:57-59.

0

5

10

15

20

25

-1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5

AG

A-I

gG(U

)

TNF-α(Log10)

Correlation between AGA IgG and TNF-α (Log10), r=0.42, p<0.0001

-5

0

5

10

15

20

25

-1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4

AG

A-I

gG(U

)

IL-1β(Log10)

Correlation between AGA IgG and IL-1β (Log10), r=0.51, p<0.0001

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MRS Neurochemicals and Inflammation• MRS biochemicals such as total choline and myoinositol may serve as a proxy for inflammation • Myoinositol is localized primarily in glial cells and elevated in conditions characterized by central

nervous system inflammation (e.g., hepatitis C virus-associated encephalopathy, multiple sclerosis) • Increased myoinositol has also been reported in first-episode psychosis • Total choline reflects cellular membrane turnover, and increased levels are observed in

neuroinflammatory diseases such as multiple sclerosis and HIV

• Methods• Spectra were acquired from the anterior cingulate cortex (ACC) using

PR-STEAM: TR/TM/TE = 2000/10/6.5-ms, NEX=128, water reference (NEX=16) on a 3T Siemens Tim Trio. Spectra were analyzed with in-house Matlab and LCModel

• Correlations were run between AGA IgG and measures of MRS including: phosphocholine+glycerophosphocholine (total choline), myoinositol, glutamine, glutamine, glutamate/glutamine, glutathione, N-acetylaspartate, and creatine

Brand A, et al. Dev Neurosci 1993;15(3-5):289-298; Bokemeyer M, et al. Gut, 2011:60(3):370-377; Bagory M et al. IEEE Trans Biomed Eng 2012:59(10):2687-2694; Kirov I, et al. Neurology 2013;80(1):39-46 Plitman E, et al. Schizophr Bull 2016:42(2):415-424; Chang L, et al. Neuroimmune Pharmacol 2013;8(3):576-593

Correlation of AGA IgG andNeurochemicals

5

5.5

6

6.5

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7.5

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8.5

9

0 10 20 30 40 50 60 70 80

Myo

ino

sito

l (IU

)

AGA IgG (U)

Correlation between AGA IgG and Myoinositol, r=0.48, p=0.007

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

0 10 20 30 40 50 60 70 80

Tota

l Ch

olin

e (I

U)

AGA IgG (U)

Correlation between AGA IgG and Total Choline, r=0.36, p=0.045

Rowland, et al, Frontiers in Psychiatry 2017

Measure (N=31) Mean SD R Value P valueGlutamate 8.95 0.86 0.104 0.58Glutamine 2.07 0.36 -0.1048 0.58Glutamate/glutamine 11.02 1.14 0.006 0.97Glutathione 2.03 0.29 0.01 0.96N-acetylaspartate 10.00 1.11 0.006 0.97Creatine 9.23 0.48 0.25 0.18Myoinositol 7.22 0.70 0.48 0.007Total choline 1.83 0.36 0.36 0.045

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Summary

• Approximately 1/3 of people with schizophrenia have elevated AGA IgG• This subgroup is characterized by:

• Significantly lower positive symptoms than those without positivity• Higher levels of peripheral kynurenine and correlated to AGA IgG• Higher levels of peripheral inflammation measured by TNFα and IL-Iβ and correlated to

higher AGA IgG• Higher levels of myoinositol and total choline in the anterior cingulate cortex and

correlated to higher AGA IgG• Higher gut and BBB permeability?

• Strategies for treatment have all been completed in “all-comer” schizophrenia population

• Would a diet with removal of gluten improve psychiatric symptoms in a subgroup?

Severance EG, et al. Brain Behav Immun. 2015 Feb;44:148-58.

Case study of Schizophrenia with Celiac Disease

De Santis et al. Journal of Internal Medicine 1997; 242:421-423.

33 year old man with schizophrenia having severe diarrhea and weight loss.

Use of single photon emission computed tomography (SPECT) showed hypoperfusion in the left prefrontal cortex without evidence of structural cerebral abnormalities.

Jejunal biopsy showed villous atrophy and EMA antibodies present. Gluten free diet started.

Six months after a gluten-free diet, cerebral blood flow normalized, duodenal findings resolved and all psychiatric symptoms disappeared.

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Recent Case Reports• Longstanding schizophrenia symptoms resolve after starting low carb ketogenic diet in 70

year old, resolving in 19 days

• Our group recently published on complete resolution of psychotic symptoms with gluten free diet within weeks in a 16 year old male with autoimmune disorders and autoimmune disease frequently occurring in the family

• 20 year old male has improvement in psychiatric symptoms in one week

• 14 year old girl with 2 year psychosis history and complaints of gastrointestinal symptoms tests negative to all antibodies with exception of AGA IgG. Symptoms improve within one week of gluten free diet

• 23 year old female with auditory and visual hallucinations resolve within weeks of starting a gluten free diet

• 8 year old girl negative for tTG, resolving in days/weeks, rechallenge brings symptoms backKraft and Westman 2009, Nutrition and Metabolism, Eaton et al. 2015, Am J Psychiatry; Jansson N, Kristjánsson E, Nilsson L. Lakartidningen. 1984;81:448–44; Lionetti E, et al Nutrients 2015;7:5532-39; Biagi F, et al. Br J Nutrition 2009;102:882-7; Genius SJ, Lobo RA. Gastroenterology Research and Practice 2014; 293206

Open-Label Pilot StudyGoal: to examine the efficacy of a 2 week open label gluten free diet in AGA IgG positive patients on an inpatient unit

• Inclusion/Exclusion– Age 18-55 years– DSM-IV criteria for schizophrenia or schizoaffective disorder– Positive for AGA (>20 U)– Clinically stable on same antipsychotic for 2 months with an unchanged dose for prior 4 weeks– Ability to consent determined by a score of ≥ 10 on the Evaluation to Sign Consent – Pregnant women excluded

• Symptom ratings Brief Psychiatric Rating Scale (BPRS) Scale for the Assessment of Negative Symptoms (SANS)

• Side effect (neurological/extrapyramidal) ratings Simpson Angus Scale (SAS) Barnes Akathisia Rating Scale (BAS)

Jackson et al. Schiz Research 2012:140(1-3):262-3

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Pilot Study Results

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Change in TotalBPRS

Change in TotalSANS

Change in SAS Change in BAS

Patient 1Patient 2

Jackson et al. Schiz Research 2012:140(1-3):262-3

Randomized Double-Blind Gluten-Free Diet Study

• Double-blind randomized gluten-free inpatient feasibility study for 5 weeks

• All participants received a gluten free diet • Protein shake daily with 10 gm of Gluten flour or 10 gm of Rice flour

• Participants discharged on gluten free diet, evaluated for 8 weeks

• First gluten free diet study in schizophrenia to recruit a subgroup; feasibility and not powered to find an effect

• Clinical Trials.gov NCT#01927276 (NIMH funded R34)

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

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Inclusion/Exclusion Criteria

Inclusion• DSM-IV/DSM 5 diagnosis of schizophrenia or

schizoaffective disorder • Positive for gliadin antibodies (IgG ≥ 20 U)• Age 18- 64 years• BPRS total score >29• Same antipsychotic for at least 4 weeks• Ability to consent determined by a score of ≥ 10 on the Evaluation to Sign Consent

Exclusion• Already on gluten free diets• Positivity to tissue transglutaminase (tTg)

antibodies or known history of Celiac Disease• Pregnant or lactating females• Meets DSM-5 criteria for alcohol or substance use

disorder (other than nicotine) within last month • Gluten ataxia (Brief Ataxia Rating Scale)

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

375 screened for Antigliadin Antibodies (AGA)

26 completed eligibility screening for inpatient study

100/370 positive for AGA (27%)64 recruited for inpatient study

19 randomized

Gluten Free Diet (N=7)

Gluten Containing Diet (N=9)

2 excluded <20 U AGA IgG

1 excluded: <20 U AGA IgG

2 Withdrawals:1 Beginning of week 4

Personal choice1 End of Week 4 for Housing Reasons

N=7 completed N=7 completed

8 Post Discharge Randomized Open-Label Follow-Up of Gluten Free Diet

N=5 assessed at 8 weeks

N=3 assessed at 8 weeks

5 excluded for tTG positivity (1.3%) (referred for treatment)

PARTICIPANT FLOW

DIAGRAM GLUTEN-FREE DIET STUDY

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Demographic and Clinical Information

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

Gluten-Containing Diet (N=9) Gluten-Free Diet (N=7) Statistics

Mean age (Years) 42.0 ± 14.6 32.5 ± 9.7 p=0.22Sex (Male) N=5 (56%) N=4 (57%) p=1.00Race (African American) N=7 (78%) N=5 (71%) p=1.00Age of Illness onset (Years) 16.9 ± 3.4 18.2 ± 2.4 p=0.61Level of Education (Years) 11.8 ± 1.3 12.4 ± 2.1 p=0.29Smokers N=5 (56%) N=6 (86%) p=0.20Body Mass Index (kg/m2) 28.5 ± 4.7 31.4 ± 8.9 p=0.71Baseline AGA IgG (U) 55.8 ± 28.6 43.8 ± 12.2 p=0.56Baseline AGA IgA (U) 23.6 ± 21.1 32.9 ± 28.3 p=0.49Antipsychotics

First-Generation Antipsychotics (FGA)Second-Generation Antipsychotics (SGA)ClozapineSGA + FGA

N=2 (22%)N=2 (22%)N=2 (22%)N=3 (33%)

N=2 (29%)N=2(29%)N=1 (14%)N=2 (29%)

p=0.96

AntidepressantsAnticholinergic medications

N=6 (67%)N=7 (78%)

N=3 (43%)N=6 (86%)

p=0.34p=0.69

Meals per day with gluten 1.7 ± 1.1 2.1 ± 1.2 p=0.52Gastrointestinal DisorderDermatologic Disorder

N=4 (44%)N=1 (11%)

N=4 (57%) N=3 (43%)

p=0.53P=0.13

Change in SANS Total Score

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Week0 Week1 Week2 Week3 Week4 Week5

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Change of SANS Total Score by Group (LSMeans)

Gluten-Containing Diet (N=9) Gluten-Free Diet (N=7)

Effect Size: Cohen’s D= -0.53 Total SANS t=2.30, df=1,13.3, p=0.15

ES= -0.24 anhedonia; -0.43 avolition; -0.71 blunting; alogia < -0.12

≥30% decrease in SANS total: 57% (4/7) vs 11% (1/9); Chi-Square=3.88, df=1, p=0.048Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

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Results

• CGI Improvement noted in GFD group (ES=-0.75)

• No improvement in positive or depressive symptoms

• No differences in side effects, extrapyramidal symptom ratings or BMI

• Fasting Blood Glucose slightly decreased in GFD group (ES=-0.36)

-0.3

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MATRICS Consensus Cognitive BatteryEffect Sizes

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

Gluten Free Diet on Kynurenic Acid and Cytokines

KYNA IL-23Tnf-α

Cohen’s D=1.36 Cohen’s D=0.93 Cohen’s D=1.65

Gluten Free Diet N=7Gluten Containing Diet N=9

“Inappropriate activation of the interleukin (IL)-23 signaling pathway causes chronic inflammation through the induction of immunopathological Th17 cells in the intestine; adequate Th17 responses are essential for host defense against harmful organisms. IL-23 is primarily produced by innate myeloid cells including dendritic cells (DCs) and macrophages (M�s)”. Kayama et al. Int Immunol. 2019

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ES=-0.34

34% Decrease

16% Decrease

Baseline and Endpoint AGA IgG

0

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30

40

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60

Gluten-Containing Diet (N=9) Gluten-Free Diet (N=7)

Baseline Endpoint

16% Decrease 34% Decrease

AGA

IgG

(U)

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

Gastrointestinal Symptom Rating Scale (GSRS)

-14.00

-12.00

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-4.00

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0.00

2.00

GSRS Total Reflux Abdominal Pain Constipation Diarrhea Indigestion

Change in GSRS over 5 Weeks

Gluten-Containing Diet Gluten Free Diet

Kelly D, Eaton WW, et al. Journal of Psychiatry and Neuroscience, In press, 2019

ES= -0.46ES= -0.59ES= -0.40ES= -0.86ES= -0.81

Baseline mean ~30 ES = Effect size15 items scored 1-7; (range 15-105)

GSRS

Sco

re

ES= 0.18

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Confirmatory Double Blind Trial• Recently funded for a randomized double-blind trial of gluten-free vs. gluten-

containing diet in people with AGA IgG positivity and enriched with negative symptoms N=40

• Screening approximately 500-800 people at University of Maryland and Johns Hopkins, positive for AGA IgG

• Gluten or Rice flour 15 grams BID (30 gram total)• 5 weeks admitted to inpatient unit followed by 8 weeks outpatient• Outcome measures include:

• Psychiatric Symptoms• SANS, BPRS, CGI, CDS, GSRS, MCCB

• MRS• Gut permeability• Cytokines• Microbiome composition

NCT03183609 NIMH R01MH112617-01 Kelly and Eaton PIs

Mechanisms

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Gut Permeability

• Several studies in people with schizophrenia find some patients have permeability of the blood brain barrier (BBB)

• Abnormalities in blood flowing to the brain or passage of substances into the brain may lead to altered neuronal-glial function

• Post mortem ultrastructural abnormalities of capillaries and of pericapillary cellular environment suggest that BBB dysfunction might contribute to the pathogenesis of cortical lesions in schizophrenia

• Recent data shows that IgG antibodies cross into the CNS in schizophrenia but not controls

Severance EG, et al. Brain Behav Immun, 2015;44: 148-158; figure provided and used with permissionAxelsson et al. 1982; Torrey et al. 1985; Bauer and Kornhuber 1987; Kirch et al. 1992). Uronova et al. 2010; Hanson and Gottesman 2005

02

46

8

0 2 4 6 8 0 2 4 6 8

Correlation of AGA IgG:Serum and CSF

AGA IgG - CSF

Controls n=61

Schizophrenia n=105

R2=0.34,p<0.0001 ns

AGA

IgG

-Se

rum

Results independent of Antipsychotic treatment (N=75 naïve)

Zonulin

Human zonulin, discovered in 2000 as regulating tight junction permeability Same as protein formerly referred to as prehaptoglobin-2◦ Haptoglobin binds to hemoglobin inhibiting oxidative activity◦ Prehaptoglobin elevated in schizophrenia

Haptoglobin has been known to be elevated in schizophrenia relative to controls for decades

Haptoglobin gene has been implicated as a significant genetic risk factor in a meta-analysis of genetic association studies

Pursuing zonulin serum levels and BBB permeability markers in schizophrenia

Seal US and Eist H: Clin Chem 12(10): 709-16, 1966; Tripathi et al, PNAS 2009; Allen et al 2008, Wan 2007

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Celiac Disease (CD); Autoimmune Hepatitis (AIH), Primary Biliary Cirrhosis (PBC), Type I Diabetes (T1D), Autoimmune Polyendocrine

Syndrome (APS-1), Multiple Sclerosis (MS)

Figure printed with permission, unpublished data

Recent data from our group (N=100);

Mean zonulin levels in schizophrenia were 2.38 (SD 1.23)

0

5

10

15

20

25

30

35

Genotype1-1

Genotype1-2

Genotype2-2

Mean AGA IgG (U) in schizophrenia group by

Prehaptoglobin genotype

*P<0.05

AGA

IgG

Gluten and the Microbiome

• What if it’s related to the gut microbiota?• Pathologic underpinnings may be due to a particular dysbiotic profile

characterized by decreased butyrate-producing-bacteria (e.g., Firmicutes, Bifidobacteria)

• Low intestinal butyrate may lead to other events such as modulation of microbial lipopolysaccharide (LPS), intestinal alkaline phosphatase (IAP) and wheat α-amylase trypsin inhibitors (ATIs)

• Bacteria could modulate gluten degrading enzymes• Bacteria and gluten may change tryptophan metabolism• GS could be a multi-factor-onset disorder, transient and preventable, related

to diet and gut bacteria composition, and not from gluten itself

Leccioli V, et al. Nutrients 2017 Nov 2;9(11). Gutierrez S. Mol Metab 2017;6(7):693-702.

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Microbiome and Diet

Kelly, Fraser, Ament, Beurmann, unpublished data

Principal Coordinate Analysis (PCoA, multidimensional scaling), a way to explore similarities or dissimilarities of data (Bray-Curtis Distribution)

Analyzed by UMB IGS using 16S rRNA for bacterial identification

N=2 GFDN=4 GCD

Gluten containing all have similar endpoint variability

Gluten Containing

Gluten Free

Less variability in microbiota from 5 weeks similar GCD

After 5 weeks GFD have higher variability than at baseline

Mimicry a Possibility?• The human Glutamate Ionotropic Receptor NMDA type Subunit Associated with

protein 1 (GRINA1) has recently been shown using BLASTP analysis to be a molecular mimic to the 33-mer gliadin fragment, a part of gliadin thought to be responsible for the adaptive immune response against gluten

• Thus, AGA IgG may lead to targeting of GRINA (also known as TMBIM3) which is known to be mainly expressed in the central nervous system

• GRINA IgG levels were higher in schizophrenia patients than in healthy controls (0.43 ± 0.30 vs. 0.22 ± 0.24, p < 0.001). Logistic regressions showed an association between AGA IgG and GRINA IgG in schizophrenia (p = 0.016 for the estimated regression coefficient) but not for the controls (p = 0.471)

• Possible that mimicry from cross-reactivity between gliadin and GRINA might disrupt the functions of the glutamate system and relate to illness pathophysiology in those with schizophrenia and elevated AGA IgG?

BLASTP basic local alignment search tool) is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of proteins or the nucleotides of DNA and/or RNA sequences Čiháková D, Schizophrenia Research 2019; Garcia-Quintanilla, A and Miranzo-Navarro, D. Bioessays 2016;38:427-439;Rojas-Rivera, D, Armisen, R, Colombo, A, et al. Cell Death Differ 2012;19:1013-1026;Lisak, DA, Schacht, T, Enders, V, et al. Biochim Biophys Acta 2015;1853:2104-2114

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Future Potential Pharmacologic Strategies?• Gluten-free diet• Anti inflammatory treatments

• E.g., blocking cytokines such as IL-15 (HuMaxIL15) in Celiac Disease• Blocking absorption of gluten or increasing gluten digestion

• E.g., prolyl endopeptidases• Inhibition of zonulin

• E.g., larazotide, completed IIb studies for Celiac Disease• Blocking gliadin antibodies

• E.g., KCC009, tTG antibody• Silencing gluten-reactive T cells• Intestinal flora modulation

• E.g., pre or probiotics• Gluten vaccineYokayama S, et al. PNAS 2009;15:15849-54, Rodrigo L, et al, Expert Opin Invest Drugs 2009;18(12):1865-73

Gliadin Testing and Gluten Food Lists• Test for Native Gliadin (AGA IgG)LabCorp has is a Gluten Sensitivity Screen (Test Code 164125, CPT 83516) (Base price ~$60.00)1. This test first assays deamidated gliadin peptide

(DGP IgA and IgG) and tissue transglutaminase (tTG). If the patient is positive for tTG or DGP testing stops. A gluten free diet is standard of care, likely CD. 2. If negative for these antibodies, the lab will next test for AGA IgG (native). The additional cost is ~$20.3. If the DGP/tTG and AGA IgG is negative the test will examine for wheat allergen-specific IgE. This additional cost of $5.25 will be added if completed. AGA IgG only is Lab Corp number 164247 but is NOT orderable outside of the 164125 panel.

Gluten in contained in many foods:https://celiac.org/live-gluten-free/glutenfreediet/what-is-

gluten/#RJwuUOeHGuArT3EE.99

http://www.the-gluten-free-diet.com/foods-containing-gluten.html

FDA considers GF < 20 ppm

GFD is expensive, often not covered by insurance

https://www.labcorp.com/test-menu/26126/gluten-sensitivity-antibodies-cascade Gleed G, BMJ 2017;356:119

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Summary

• Elevated AGA IgG may be present in about 30% of all people with schizophrenia• AGA IgG is not related to tTG and DGP, antibodies seen in Celiac Disease• AGA IgG elevations represent a potential subgroup with high peripheral and

central inflammation - Maybe its “gluten sensitivity,” maybe elevated AGA IgG, maybe more expansive to dietary antigens

• Removing gliadin may improve negative symptoms in schizophrenia• Gut permeability and microbiotia may play role in mechanism • Confirmatory blinded study underway, other work should focus on

understanding mechanisms• Novel treatment targets can result if found to be effective

As researchers and clinicians we are on a journey, to help each person we treat find their best lives; everyone deserves optimized and personalized treatments.

We must know our best options for treatment, but we must also strive to find better treatments than what we have today.

= Attempting precision medicine

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With Many Thanks!MPRC• Haley Demyanovich• Laura M. Rowland• Heidi J. Wehring• Ann Marie Kearns• Megan Powell• Fang Liu• S. Andrea Wijtenburg • Stephanie Feldman• William T. Carpenter Jr.• Frank Blatt• Frank Gaston• Ana Pocivavsek• L. Elliot Hong• Robert P. McMahon• Robert W. Buchanan• Spring Grove Clinical Team

Johns Hopkins University• William W. Eaton– Co-PI• Katrina Rodriguez• Daniela Cihakova • Monica V. Talor• Nicola Cascella• Bruce Patterson• Emily Severance• Robert YolkenHarvard Medical School• Alessio Fasano• Craig SturgeonUniversity of Maryland• Debbie Santora• Greg BarberOther Trainees and Staff• Kelli Sullivan• Jessica Jackson

The work was supported by NIMH R34 MH100776-01 and

NIMH R01MH112617-01 (Kelly and Eaton MPIs)

And Brain & Behavior Research Foundation (BBRF) for the Maltz Prize for Innovative and Promising Schizophrenia

Research

https://www.youtube.com/watch?v=y-9lOpPUdg0