Mast cells disrupt the gut-blood-brain barriers and contribute to autism
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Autism, Blood-brain-barrier and Mast cells
T. C. Theoharides, M.S., Ph.D., M.D.
Professor of Pharmacology, Internal Medicine and Biochemistry; Associate Professor of Psychiatry Director, Molecular Immunopharmacology and Drug Discovery Laboratory Tufts University School of Medicine Tufts Medical Center, Boston, MA, USA Clinical Pharmacologist, Massachusetts Drug Formulary Commission (1986-2010)
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Pervasive Developmental Disorders
DSMIV TR Diagnostic Criteria
Pervasive Developmental Disorders
Rett's Disorder Childhood Disintegrative Disorder Autistic Disorder Asperger's Disorder PDD-NOS
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Autism Spectrum Disorders
Nomenclature
sm uti A od Kanner’s Autism High Functioning Autis o m NVLD ldh i me Ch Early Infa Syndro ntile Autism er’s Asperg
tism Au
m utis A ca l i typ A
Autism Spectrum Disorders
High-Functioning ASD
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(intact intellectual capacity)
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Autism spectrum disorders: concurrent clinical disorders
Xue Ming , Brimacombe M, Chaaban J, Zimmerman-Bier B, Wagner GC. J Child Neurol. 2008 Jan;23(1):6-13. Epub 2007 Dec 3. • Individuals with autism spectrum disorders are heterogeneous in clinical presentation, concurrent disorders, and developmental outcomes. This study characterized the clinical co-occurrences and potential subgroups in 160 children with autism spectrum disorders who presented to The Autism Center between 1999 and 2003. • Medical and psychiatric co-occurrences included sleep disorders, epilepsy, food intolerance, gastrointestinal dysfunction, mood disorder, and aggressive and self-injurious behaviors. Sleep disorders were associated with gastrointestinal dysfunction (P < .05) and mood disorders (P < .01). Food intolerance was associated with gastrointestinal dysfunction (P = .001).
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Immunologic Dysregulation in Autism
• • • Plasma from autistic patients (and their mothers) contains autoantibodies to brain antigens, suggesting BBB disruption. Brain of autistic patients have higher expression of IL-6 Maternal infection in mice increases circulating IL-6 levels that cross the placenta at midgestation and induce the release of fetal stress hormones resulting in fetal injury. Viral poly(I:C) injection increases IL-6 in the placenta and brain of mice and, like IL-6 injection, induces autistic-like behavior in mice. Acute stress increases serum IL-6, absent in mast cell deficient mice. Autistic patient CSF has high levels of macrophage chemoattractant protein1 (MCP-1), which is chemotactic for mast cells Autistic patient plasma has reduced transforming growth factor-beta1 (TGFbeta1), which inhibis for mast cell maturation and activation.
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• • • •
Mast Cell Activation Seen With Nomarski Optics
Triggers
Allergen/IgE
Mediators
Histamine Chymase Tryptase Leukotrienes Prostaglandins Chemokines IL-1, IL-6, IL-8 GM-CSF, TNF-α VEGF
Neuropeptides Normal mast cells
Activated mast cell
Mast cell degranulation leads to the release of mediators with potent2009 May 1, vasodilatory, nociceptive, and inflammatory properties Copyright-Dr. T.C. Theoharides 7
Differential release of mast cell mediators and the pathogenesis of inflammation.
Theoharides TC, Kempuraj D, Tagen M, Conti P, Kalogeromitros D. Immunol Rev. 2007 Jun;217:65-78.
•
Mast cells can be activated by bacterial or viral antigens, cytokines, growth factors, neuropeptides and stress hormones, leading to selective release of distinct mediators without degranulation.
– Trigger CRH IL-1 IL-33 LPS SCF TLR-9 Mediator Implication in autism VEGF IL-6 High brain and serum levels IL-13 TNF-α High CSF levels IL-6 High brain and serum levels IL-6 High brain and serum levels
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Evidence of “allergic” symptomatology in autism
• In a nested control study, there was over 2-fold higher risk of children with ASD (n=407) born to mothers with diagnosis of allergies or asthma during 2nd trimester of pregnancy as compared to controls (n=2095). • ASD children had a significant family history of atopy (30%) as compared to controls (2.5%). • ASD children had at least one manifestation of allergy and 50% of ASD children may have two or more symptoms. • Non-IgE-mediated food intolerance was significantly more common in ASD children (n=133) than controls (n=81). • Serum levels of IgG4, associated with atopic phenotype, were increased in children with autism disorder • A study of 362 ASD patients in Italy showed that the only statistically significant association was with history of allergies • ASDs and mastocytosis children share common symptoms such as, anxiety, atopic dermatitis,“brain fog”, and food intolerance
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www.tmsforacure.org
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Proposed Role of Mast Cells in Autism
CNS originating mast cell activation triggers: Adenylate cyclase activating peptide, corticotropin-releasing hormone, myelin basic protein neurotensin, substance P Mast cell
Intestine-originating mast cell triggers Butyrophilin, casein, gliadin, gluten, IL-1, IL-33, reactive oxygen species, rotavirus, vasoactive intestinal peptide
Blood vessel
Mast cell-derived cytokines and other neurotoxic mediators IL-1, IL-6, IL-8, IL-13, IL-32, MCP-1, prostaglandin D2, serotonin, tumor necrosis factor
Mast cell-derived vasoactive mediators Bradykinin, histamine, IL-6, nitric oxide, tryptase, vascular endothelial growth factor, vasoactive intestinal peptide
Gut
Increased Gut-blood-brain barrier permeability ASD
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Mast cells are located perivascularly close to nerve endings
Rozniecki, Dimitriadou, Lambracht-Hall et al. (1999) Brain Res 849: 1.
May 1, Bv=blood vessel; white arrowhead=nerve endings; dark arrow=mast cells; g=granule; e=endothelial 2009 Copyright-Dr. T.C. Theoharides 12 Cell; er=erythrocyte; n=nucleus; p=pericyte; vl=blood vessel lumen
Mast cell are located close to blood vessels and sensory neurons
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Preterm birth, perinatal stress, corticotropinreleasing hormone and autism
• A retrospective study of preterm children born in Atlanta (198193) who survived to 3 years of age showed that preterm birth at <33 weeks gestation was associated with a two-fold higher risk of autism in all infants identified through the Metropolitan Atlanta Developmental Disabilities Surveillance Program • A prospective follow-up assessment on 91 ex-preterm infants (<1500 g) found 26% of these (mean age=22 months) to have a positive Modified Checklist for Autism in the Toddlers (M-CHAT) • Another study found that 21% of infants (212/988) born before 28 weeks gestation screened positive using M-CHAT as compared to 5.7% of healthy children 16-30 months old • A statistically significant association was reported between extremely low birth weight and higher rates of autistic and Asperger disorders
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Prematurity
Maternal CRH Neonatal stress Newborn CRH Genetics BBB Disruption Delivery (Neonatal ICU) BBB Disruption Infancy (Positive M-CHAT) Allergies/food intolerance Infections
Environmental toxins
Brain Inflammation
Oxidative stress
Autism (Positive ADOS-G, ADI-R)
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Immunocytochemistry of CRH-R in human mast cells
A
A
R1 no IL-4
4
R1 + IL-4
C
D
R2 no IL-4
E
R2 + IL-4
BD
B
B E F F G
B
C
No R1 primary
E F
DAPI
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CRH Induces Selective VEGF Release from Human Mast Cells
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Mast cells were stimulated with CRH at increasing concentration for 6 hours in culture medium with A) or without B) 10% fetal bovine serum (FBS). Secretion of VEGF in the samples were measured with a VEGF-specific ELISA kit. VEGF release was compared to unstimulated control samples. The values are given as mean±SD of five independent experiments.
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VEGF (pg/106cells)
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99
Tc-G Extravasation Study— Experimental Design
30 minutes restraint stress
Plasma leak -99Tc-G extravasation
mouse
HPA axis activation -Blood corticosterone
tv 99Tc-G C57BL +/+ W/Wv mast cell-deficient Neurokinin-1 receptor -/Substance P -/May 1, 2009 Copyright-Dr. T.C. Theoharides
Mast cell activation -Toluidine blue
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Stress-induced brain vascular permeability is dependent on mast cells
99-Technetium-gluceptate extravasation
200 600
HPA axis activation
control stress
* % change from control
150
corticosterone (ng/ml)
500 400 300 200 100 0
*p<0.05 *p<0.05
100
50
0
+/+
W/W v
+/+
W/W v
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Corticotropin-releasing hormone and the blood-brain-barrier
Theoharides TC, Konstantinidou AD. Front Biosci. 2007 Jan 1;12:1615-28.
• Increased BBB permeability precedes any clinical or pathologic signs in various brain inflammatory disorders. • Mast cell specific tryptase is elevated in the CSF of MS patients, induces microvascular leakage and stimulates protease-activated receptors (PAR), leading to widespread inflammation. • BBB permeability, appears to worsen in response to acute stress that leads to the local release of corticotropin-releasing hormone (CRH), which activates brain mast cells to selectively release IL-6, IL-8 and VEGF. • Acute stress increases BBB permeability that is dependent on CRH and mast cells.
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Summary
• Mast cells regulate gut-blood-brain barrier permeability, disrupted by acute stress through corticotropin-releasing hormone (CRH). CRH can activate skin mast cells and increase vascular permeability in rodents and humans, through activation of CRH receptor-1. Similar effects have been shown in the bladder and the intestines, where CRH was shown to be involved in intestinal inflammation and motility. CRH could increase permeability in normal human colonic biopsies through activation of subepithelial mast cells. Neonatal maternal deprivation stress induced long-term alterations of colonic nerve-mast cell interactions, while a recent review indicated that prenatal stress could contribute to the development of autism in humans.
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•
•
•
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Interleukin-6 and mast cells
Conti P, Kempuraj D, Di Gioacchino M, Boucher W, Letourneau R, Kandere K, Barbacane RC, Reale M, Felaco M, Frydas S, Theoharides TC. Allergy Asthma Proc. 2002 Sep-Oct;23(5):331-5.
• IL-6 is a pleiotropic cytokine that was originally named interferon beta 2 or B cell-stimulating factor or differentiating B cell factor inducing immunoglobulin production. • IL-6 mediates immune responses and inflammation • IL-6 also is a crucial cytokine for mast cell maturation • IL-6 also up-regulates histamine production and induces the expression of immunoglobulin E (IgE) receptors on the surface of mast cells
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Stress-induced IL-6 release in mice is mast cell-dependent
Huang M, Pang X, Karalis K, Theoharides TC. Cardiovasc Res. 2003 Jul 1;59(1):241-9
.
• Acute stress increases serum IL-6 in mice • Stress-induced IL-6 release is absent in W/Wv mast cell-deficient mice • Both normal and W/Wv mast cell-deficient mice respond similarly to stress
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Effect of acute stress on serum IL-6 levels in (A) stressed W/Wv mice and their +/+ controls, as well as in (B) stressed CRH k/o mice and their controls. May 1, 2009 Copyright-Dr. T.C. Theoharides 33
IL-1 induces vesicular secretion of IL-6 without degranulation from human mast cells
Kandere-Grzybowska K, Letourneau R, Kempuraj D, Donelan J, Poplawski S, Boucher W, Athanassiou A, Theoharides TC. J Immunol. 2003 Nov 1;171(9):4830-6. • IL-1 stimulates secretion of IL-6 without release of the granuleassociated protease tryptase in normal human umbilical cord blood-derived mast cells. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures.
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•
•
IL-1 stimulates differential release of IL-6 without degranulation
8-16 wk hCBMCs + IL-1 6 hourscell free supernatants assay IL-6 (ELISA) or tryptase (fluoroimmunoenzyme assay).
MC)
1000 750 500 250 0
A
MC)
spont stim Tryptase *
125 100 75 50 25 0
B
*
IL-6
*
5
Tryptase (ng/5x10
IL-6 (pg/5x10
5
IL-1
test
anti-IgE
IL-1
anti-IgE
35
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*p<0.05 paired tCopyright-Dr. T.C. Theoharides
Ultrastructural cryoimmunocytochemistry of IL-6 molecules released from a secretory vesicle, 1/10th the diameter of that of 140 nm secretory granules, of a human mast cell 140 stimulated with nm IL-1
nm
190 nm
140 nm
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190 nm
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70 nm
140 nm
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Serum IL-6 reflects disease severity and osteoporosis in mastocytosis patients
Theoharides TC, Boucher W, Spear K. Int Arch Allergy Immunol. 2002 Aug;128(4):344-50.
•
Mastocytosis is characterized by an increased number of mast cells in the bone marrow or in skin areas known as urticaria pigmentosa (UP) Patients may present with flushing, itching, food intolerance, but also anxiety, headaches, lack of concentration (“brain fog”) Serum IL-6 is elevated in mastocytosis patients and correlates with severity of symptoms and the presence of osteoporosis. High serum IL-6 may not only signify disease progression, but may also participate in the pathophysiology of mastocytosis
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•
•
•
Double Immunocytochemistry for c-kit and CRH-R
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Novel therapeutic targets for autism
Theoharides TC, Doyle R, Francis K, Conti P, Kalogeromitros D. Trends Pharmacol Sci. 2008 Aug;29(8):375-82. Epub 2008 Jul 6.
• Children with mastocytosis often have ADD, learning difficulties and “brain fog” • Data were obtained in response to the question listed below that was sent by the Mastocytosis Society to a database of 400 patients • “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g.Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made”
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Mastocytosis patients with ASD*
Patient 1 (BN) 2 (SH) 3 (RC) 4 (BL) 5 (?) 6 (EC) 7 (ESC) 8 (?) 26 (BN) 10 (CJP) 11 (EA) 12 (NP) 13 (MH) 14 (MH) 15 (MH) 16 (AB) 17(SMMcC) 18 (DA) 19(JDPH) 20 (ME) 21 (AC) Age ‘08 4 15 11 18 44 21 21 18 7.5 4 9 3.5 46 (father) 10 (daughter) 8 (son) 2.5 17 9 15 23 4 Sex M M M M+ M+ F F M M M M M M# F# M# F F M M F M ASD Stuttering, high functioning ASD, poor speech Asperger’s ASD, dyslexia Asperger’s SID SID Asperger’s Autism ASD, ADHD, SID Asperger’s PDD-NOS Asperger’s Asperger’s Mood problems, ADD Autism spectrum, PDD-NOS Asperger’s Asperger’s Asperger’s Mild autism, ADD Asperger’s Mast cell diagnosis Mastocytoma, solitary UP, asthma UP, NF1 SM Mastocytosis, UP? UP Mastocytosis Mastocytosis Mastocytoma, solitary Mastocytosis, UP?, flushing, GI UP, hives, rash, GI (brother with masto) Mastocytosis Hives, asthma Diarrhea, hives, itching Mastocytosis UP, SM? Mast cell disease, food intolerance UP Mast cell disease, asthma Mast cell disease Mastocytosis
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Mastocytosis patients with ASD*
Patient 22 (ND) 23 (AS) 24 (GC) 25 (AL) 26 (SS) 27 (EAIS) 28 (JT) Age ‘08 7 2 21 4.5 8 5 2.5 Sex M M F M F F M ASD Asperger’s (mild) ASD, SID Asperger’s ASD, moderate Asperger’s PDD-NOS Regressive autism Mast cell diagnosis UP Mastocytosis, Celiac disease SM Mastocytoma, solitary UP Mast cell disease, flushing, milk intolerance, anxiety Mast cell disease, flushing, food intolerance, acute lymphocytic anemia UP, GI symptoms
29 (CJM)
3.5
M&
PDD-NOS
30 (TDM) 31 (JRM)
6.6 10.5
M& M&
DID ADD, Asperger’s ?
Food hypersensitivities Food hypersensitivities
*Data obtained in response to the question listed below that was sent by the Mastocytosis Society (www.tmsforacure.org) to a database of 400 patients. + Same family (uncle); #Same family; &Same family (brothers) “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g. Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made.” ADD=Attention Deficit Disorder; PDD-NOS=Pervasive developmental disorder-not otherwise specified; NF1=Neurofibromatosis-1; SID=Sensory Integration Dysfunction; SM=systemic mastocytosis; UP=Urticaria Pigmentosa
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Mastocytosis parents with children with ASD
Patient 1 (CB) 2 (LA) 3 (?) 4 (SH) 5 (LK) 6 (CH) 7 (RK) 8 (CS) 9 (MH) 10 (SS) 12 (RDK) Age Sex Mast cell diagnosis 48 43 45 ? 45 ? ? ? 42 ? 49 F F F F F F F F F F F M Systemic mastocytosis Mast cell disease Systemic mastocytosis UP, Systemic mastocytosis Systemic mastocytosis Systemic mastocytosis Mastocytosis Systemic mastocytosis Mast cell disease UP Mast cell disease Indolent mastocytosis Child with ASD-Sex, Age at Dx ADD, Asperger’s-M, 5 y/o Autism, FCAS-F triplets, 2 y/o Asperger’s, Mast cell disease Asperger’s, ADD, asthma, M Mild autism, ADD, Mast cell disease, F Asperger’s, ADD, M Asperger’s, 15 y/o Niece with Asperger’s, F Asperger’s F 10 y/o, ADD M 8 y/o Rett syndrome, F Asperger’s, F, 5 y/o Asperger’s, M, 14 y/o
11 (LLMcC) 56
*Data obtained in response to the question listed below that was sent by the Mastocytosis Society (www.tmsforacure.org) to a database of 400 patients. + Same family (uncle); #Same family; “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g. Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made.” ADD=Attention Deficit Disorder; PDD-NOS=Pervasive developmental disorder-not otherwise specified; May 1, 2009 Copyright-Dr. T.C. Theoharides 42 NF1=Neurofibromatosis-1; SID=Sensory Integration Dysfunction; SM=systemic mastocytosis; UP=Urticaria Pigmentosa
Serum levels of a mast cell trigger and a mast cell mediator are increased in young children with autism
• Serum Neurotensin is significantly elevated in autistic children (n=28, <4 years old) as compared to healthy, normally maturing controls (n=8, <4 years old) • Serum IL-6 is significantly elevated in autistic children (n=29, <4 years old) as compared to healthy, normally maturing controls (n=7, <5 years old)
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Serum levels of neurotensin
p=0.0356
2500 2500
Serum NT (pg/ml)
2000 2000 1500 1500 1000 1000 500 500 00
Control n=8 Autism n=28
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Serum levels of IL-6
p=0.0128
1200 Serum IL-6 (pg/ml) 1000 800 600 400 200 0 Control n=7 Autism n=29
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Effectof of mercuryonintracellular calcium ion of mercury on histamine release from Effect mercury on intracellular calcium ion Effect LAD2 human mast cells levelin LAD 22mast cells in LAD mast cells level
*
Histamine release (%)
100 90 80 70 60 50 40 30 20 10 0
*
Spontaneous
SP (0.1 µM)
SP (2 µM)
HgCl2(0.1 µM)
HgCl2 ( µM) 1
HgCl2(10 µM)
SP (0.1M) + HgCl2(0.1 µM) µ
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SP (2 M) + HgCl2(0.1 µM) µ
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Effect of mercury on intracellular calcium ion level in LAD2 mast cells
2.5 2 1.5 1 0.5 0 1 3 5 7 9 11 13 15 17 19 21 Time (min)
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Intracellular calcium (arbitrary unit)
Control HgCl2
Effect of mercury on VEGF release from LAD2 human mast cells
cells)
1400 1200 1000
*
* *
VEGF release (pg/10
6
800 600 400 200 HgCl 2 (1 µ M) HgCl 2 (0.1 µ M) HgCl 2 ( 1 0 µ M) HgCl 2 ( 1 0 0 µ M) Control 0
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VEGF release (pg/10 cells)
1000 250 500 750 0 Spontaneous SP (0.1 M) µ SP (2 M) µ HgCl (0.1µM) 2 HgCl (1 µ M) 2 HgCl (10µM) 2 HgCl (0.1µ M) + SP (0.1M) µ 2
6
* * *
Effect of mercury and SP on VEGF release from LAD2 human mast cells
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HgCl (0.1µ M) + SP (2M) µ 2 HgCl (1µ M) + SP (0 µM) .1 2 HgCl (1µ M) + SP (2M) µ 2 HgCl (1 µ M) + SP (2M) µ 2 0
50
Effect of thimerosal on VEGF release from Umbilical cord human mast cells
hCBMCs - VEGF release (pg/106 cells)
1200 1000 800 600 400 200 Control Thimerosal (1 M) µ Thimerosal (10 M) µ Thimerosal (100 M) µ 0
*
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Cell viability (%)
100 20 40 60 80 0 Control HgCl (0.1 M) µ 2 HgCl(1µ M) 2 HgCl(10µ M) 2 HgCl(100 µ M) 2
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Thimerosal (0.1 M) µ Thimerosalµ (1 M) Thimerosal (10 M) µ
*
Effect of mercury and thimerosal on LAD2 human mast cell viability
Thimerosal (100 M) µ
52
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Brain Inflammation
Autism and Oxidative Stress
• One study of 305 autistic children and 205 controls showed that the ratio of S-adenosylhomocysteine, used as an indicator of methylation ability, was significantly reduced in autistic children • Reduced plasma levels of S-adenosylmethionine (SAMe), the main endogenous antioxidant. Both of these studies and a recent review imply that autistic patients may have excessive free radical production • Reactive oxygen species are generated during mast cell activation and further stimulate mast cells
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Beneficial Effect of Flavonoids in Brain Inflammation
Middleton E Jr, Kandaswami C, Theoharides TC. Pharmacol Rev. 2000 52(4):673-751. Review.
• Flavonoids are potent anti-oxidant and antiinflammatory compounds. • Select flavonoids inhibit mast cell activation. • Flavonoids inhibit glia IL-6 release. • The most potent flavonoids are luteolin and quercetin, but need to be given in a liposomal formulation to increase absorption.
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Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells
Kempuraj D, Madhappan B, Christodoulou S, Boucher W, Cao J, Papadopoulou N, Cetrulo CL, Theoharides TC. Br J Pharmacol. 2005 Aug;145(7):934-44.
•Allergic stimulation of human mast cells results in secretion of histamine, the proteolytic enzyme tryptase, and the pro-inflammatory cytokines IL-6, IL-8 and TNF-α •Quercetin inhibits histamine release by 52-77%, tryptase release by 79-96%, and cytokines release by 82-93%
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Inhibition of inflammatory molecules by quercetin from human mast cells
100 90 80 Pe rce nt inhibition 70 60 50 40 30 20 10 0 Histamine Tryptase IL-6 IL-8 TNF-alpha * * * * *
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Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin
Kandere-Grzybowska K, Kempuraj D, Cao J, Cetrulo CL, Theoharides TC. Br J Pharmacol. 2006 May;148(2):208-15.
•IL-1-stimulates IL-6 production from human mast cells •Quercetin inhibits IL-6 secretion induced by IL-1 in a dose-dependent manner
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Inhibitory effect of quercetin on tryptase and MCP-1 chemokine release, and histidine decarboxylase mRNA transcription by human mast cell-1 cell line
Castellani ML, Kempuraj D, Frydas S, Theoharides TC, Simeonidou I, Conti P, Vecchiet J. Neuroimmunomodulation. 2006;13(3):179-86. Epub 2006 Dec 21.
• Quercetin, is a potent antioxidant, cytoprotective and anti-inflammatory compound capable of inhibiting histamine and cytokines release from mast cells. In this study quercetin inhibits, in a doseresponse manner, tryptase and MCP-1 • Quercetin inhibited the transcription of histidine decarboxylase using RT-PCR
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Luteolin inhibits myelin basic protein-induced human mast cell activation and mast celldependent stimulation of Jurkat T cells
Kempuraj D, Tagen M, Iliopoulou BP, Clemons A, Vasiadi M, Boucher W, House M, Wolfberg A, Theoharides TC. Br J Pharmacol. 2008 Sep 22. [Epub ahead of print]
• Addition of mast cells to Jurkat T cells activated by anti-CD3/anti-CD28 increases IL-2 release by 30-fold (n=3, P<0.05). SP-stimulated mast cells and their supernatant fluid further increase Jurkat cell IL-2 release (n=3, P<0.05) • Luteolin pretreatment inhibits mast cell activation (n=3-6, P<0.05), Jurkat cell activation and mast celldependent Jurkat cell stimulation (n=3, P<0.05)
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Quercetin protects against acute immobilization stress-induced behaviors and biochemical alterations in mice
Kumar A, Goyal R. J Med Food. 2008 Sep;11(3):469-73.
• Acute immobilization stress for 6 hours causes severe anxiety, analgesia, and impaired motor activity in mice, accompanied by partial depletion of brain levels of reduced glutathione and catalase activity • Pretreatment with quercetin reversed immobilized stress-induced anxiety and analgesia and reduced locomotor activity; it also restored the depleted brain glutathione and catalase activity
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Conclusions
• It is important to investigate food intolerance and introduce appropriate diets • The flavonoids quercetin and luteolin inhibit IL-6 and TNF-α release from mast cells • Luteolin inhibits IL-6 release from microglia, as well as IL-1 mediated release of IL-6, IL-8 and MCP-1 from astrocytes • The dietary supplement NeuroProtek contains quercetin and luteolin, along with other antioxidants
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NeuroProtek Label
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Autism, Blood-brain-barrier and Mast cells
T. C. Theoharides, M.S., Ph.D., M.D.
Professor of Pharmacology, Internal Medicine and Biochemistry; Associate Professor of Psychiatry Director, Molecular Immunopharmacology and Drug Discovery Laboratory Tufts University School of Medicine Tufts Medical Center, Boston, MA, USA Clinical Pharmacologist, Massachusetts Drug Formulary Commission (1986-2010)
May 1, 2009 Copyright-Dr. T.C. Theoharides 1
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Pervasive Developmental Disorders
DSMIV TR Diagnostic Criteria
Pervasive Developmental Disorders
Rett's Disorder Childhood Disintegrative Disorder Autistic Disorder Asperger's Disorder PDD-NOS
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Autism Spectrum Disorders
Nomenclature
sm uti A od Kanner’s Autism High Functioning Autis o m NVLD ldh i me Ch Early Infa Syndro ntile Autism er’s Asperg
tism Au
m utis A ca l i typ A
Autism Spectrum Disorders
High-Functioning ASD
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(intact intellectual capacity)
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Autism spectrum disorders: concurrent clinical disorders
Xue Ming , Brimacombe M, Chaaban J, Zimmerman-Bier B, Wagner GC. J Child Neurol. 2008 Jan;23(1):6-13. Epub 2007 Dec 3. • Individuals with autism spectrum disorders are heterogeneous in clinical presentation, concurrent disorders, and developmental outcomes. This study characterized the clinical co-occurrences and potential subgroups in 160 children with autism spectrum disorders who presented to The Autism Center between 1999 and 2003. • Medical and psychiatric co-occurrences included sleep disorders, epilepsy, food intolerance, gastrointestinal dysfunction, mood disorder, and aggressive and self-injurious behaviors. Sleep disorders were associated with gastrointestinal dysfunction (P < .05) and mood disorders (P < .01). Food intolerance was associated with gastrointestinal dysfunction (P = .001).
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Immunologic Dysregulation in Autism
• • • Plasma from autistic patients (and their mothers) contains autoantibodies to brain antigens, suggesting BBB disruption. Brain of autistic patients have higher expression of IL-6 Maternal infection in mice increases circulating IL-6 levels that cross the placenta at midgestation and induce the release of fetal stress hormones resulting in fetal injury. Viral poly(I:C) injection increases IL-6 in the placenta and brain of mice and, like IL-6 injection, induces autistic-like behavior in mice. Acute stress increases serum IL-6, absent in mast cell deficient mice. Autistic patient CSF has high levels of macrophage chemoattractant protein1 (MCP-1), which is chemotactic for mast cells Autistic patient plasma has reduced transforming growth factor-beta1 (TGFbeta1), which inhibis for mast cell maturation and activation.
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• • • •
Mast Cell Activation Seen With Nomarski Optics
Triggers
Allergen/IgE
Mediators
Histamine Chymase Tryptase Leukotrienes Prostaglandins Chemokines IL-1, IL-6, IL-8 GM-CSF, TNF-α VEGF
Neuropeptides Normal mast cells
Activated mast cell
Mast cell degranulation leads to the release of mediators with potent2009 May 1, vasodilatory, nociceptive, and inflammatory properties Copyright-Dr. T.C. Theoharides 7
Differential release of mast cell mediators and the pathogenesis of inflammation.
Theoharides TC, Kempuraj D, Tagen M, Conti P, Kalogeromitros D. Immunol Rev. 2007 Jun;217:65-78.
•
Mast cells can be activated by bacterial or viral antigens, cytokines, growth factors, neuropeptides and stress hormones, leading to selective release of distinct mediators without degranulation.
– Trigger CRH IL-1 IL-33 LPS SCF TLR-9 Mediator Implication in autism VEGF IL-6 High brain and serum levels IL-13 TNF-α High CSF levels IL-6 High brain and serum levels IL-6 High brain and serum levels
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Evidence of “allergic” symptomatology in autism
• In a nested control study, there was over 2-fold higher risk of children with ASD (n=407) born to mothers with diagnosis of allergies or asthma during 2nd trimester of pregnancy as compared to controls (n=2095). • ASD children had a significant family history of atopy (30%) as compared to controls (2.5%). • ASD children had at least one manifestation of allergy and 50% of ASD children may have two or more symptoms. • Non-IgE-mediated food intolerance was significantly more common in ASD children (n=133) than controls (n=81). • Serum levels of IgG4, associated with atopic phenotype, were increased in children with autism disorder • A study of 362 ASD patients in Italy showed that the only statistically significant association was with history of allergies • ASDs and mastocytosis children share common symptoms such as, anxiety, atopic dermatitis,“brain fog”, and food intolerance
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www.tmsforacure.org
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Proposed Role of Mast Cells in Autism
CNS originating mast cell activation triggers: Adenylate cyclase activating peptide, corticotropin-releasing hormone, myelin basic protein neurotensin, substance P Mast cell
Intestine-originating mast cell triggers Butyrophilin, casein, gliadin, gluten, IL-1, IL-33, reactive oxygen species, rotavirus, vasoactive intestinal peptide
Blood vessel
Mast cell-derived cytokines and other neurotoxic mediators IL-1, IL-6, IL-8, IL-13, IL-32, MCP-1, prostaglandin D2, serotonin, tumor necrosis factor
Mast cell-derived vasoactive mediators Bradykinin, histamine, IL-6, nitric oxide, tryptase, vascular endothelial growth factor, vasoactive intestinal peptide
Gut
Increased Gut-blood-brain barrier permeability ASD
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Mast cells are located perivascularly close to nerve endings
Rozniecki, Dimitriadou, Lambracht-Hall et al. (1999) Brain Res 849: 1.
May 1, Bv=blood vessel; white arrowhead=nerve endings; dark arrow=mast cells; g=granule; e=endothelial 2009 Copyright-Dr. T.C. Theoharides 12 Cell; er=erythrocyte; n=nucleus; p=pericyte; vl=blood vessel lumen
Mast cell are located close to blood vessels and sensory neurons
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Preterm birth, perinatal stress, corticotropinreleasing hormone and autism
• A retrospective study of preterm children born in Atlanta (198193) who survived to 3 years of age showed that preterm birth at <33 weeks gestation was associated with a two-fold higher risk of autism in all infants identified through the Metropolitan Atlanta Developmental Disabilities Surveillance Program • A prospective follow-up assessment on 91 ex-preterm infants (<1500 g) found 26% of these (mean age=22 months) to have a positive Modified Checklist for Autism in the Toddlers (M-CHAT) • Another study found that 21% of infants (212/988) born before 28 weeks gestation screened positive using M-CHAT as compared to 5.7% of healthy children 16-30 months old • A statistically significant association was reported between extremely low birth weight and higher rates of autistic and Asperger disorders
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Prematurity
Maternal CRH Neonatal stress Newborn CRH Genetics BBB Disruption Delivery (Neonatal ICU) BBB Disruption Infancy (Positive M-CHAT) Allergies/food intolerance Infections
Environmental toxins
Brain Inflammation
Oxidative stress
Autism (Positive ADOS-G, ADI-R)
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Immunocytochemistry of CRH-R in human mast cells
A
A
R1 no IL-4
4
R1 + IL-4
C
D
R2 no IL-4
E
R2 + IL-4
BD
B
B E F F G
B
C
No R1 primary
E F
DAPI
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CRH Induces Selective VEGF Release from Human Mast Cells
May 1, 2009
Mast cells were stimulated with CRH at increasing concentration for 6 hours in culture medium with A) or without B) 10% fetal bovine serum (FBS). Secretion of VEGF in the samples were measured with a VEGF-specific ELISA kit. VEGF release was compared to unstimulated control samples. The values are given as mean±SD of five independent experiments.
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VEGF (pg/106cells)
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99
Tc-G Extravasation Study— Experimental Design
30 minutes restraint stress
Plasma leak -99Tc-G extravasation
mouse
HPA axis activation -Blood corticosterone
tv 99Tc-G C57BL +/+ W/Wv mast cell-deficient Neurokinin-1 receptor -/Substance P -/May 1, 2009 Copyright-Dr. T.C. Theoharides
Mast cell activation -Toluidine blue
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Stress-induced brain vascular permeability is dependent on mast cells
99-Technetium-gluceptate extravasation
200 600
HPA axis activation
control stress
* % change from control
150
corticosterone (ng/ml)
500 400 300 200 100 0
*p<0.05 *p<0.05
100
50
0
+/+
W/W v
+/+
W/W v
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Corticotropin-releasing hormone and the blood-brain-barrier
Theoharides TC, Konstantinidou AD. Front Biosci. 2007 Jan 1;12:1615-28.
• Increased BBB permeability precedes any clinical or pathologic signs in various brain inflammatory disorders. • Mast cell specific tryptase is elevated in the CSF of MS patients, induces microvascular leakage and stimulates protease-activated receptors (PAR), leading to widespread inflammation. • BBB permeability, appears to worsen in response to acute stress that leads to the local release of corticotropin-releasing hormone (CRH), which activates brain mast cells to selectively release IL-6, IL-8 and VEGF. • Acute stress increases BBB permeability that is dependent on CRH and mast cells.
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Summary
• Mast cells regulate gut-blood-brain barrier permeability, disrupted by acute stress through corticotropin-releasing hormone (CRH). CRH can activate skin mast cells and increase vascular permeability in rodents and humans, through activation of CRH receptor-1. Similar effects have been shown in the bladder and the intestines, where CRH was shown to be involved in intestinal inflammation and motility. CRH could increase permeability in normal human colonic biopsies through activation of subepithelial mast cells. Neonatal maternal deprivation stress induced long-term alterations of colonic nerve-mast cell interactions, while a recent review indicated that prenatal stress could contribute to the development of autism in humans.
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•
•
•
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Interleukin-6 and mast cells
Conti P, Kempuraj D, Di Gioacchino M, Boucher W, Letourneau R, Kandere K, Barbacane RC, Reale M, Felaco M, Frydas S, Theoharides TC. Allergy Asthma Proc. 2002 Sep-Oct;23(5):331-5.
• IL-6 is a pleiotropic cytokine that was originally named interferon beta 2 or B cell-stimulating factor or differentiating B cell factor inducing immunoglobulin production. • IL-6 mediates immune responses and inflammation • IL-6 also is a crucial cytokine for mast cell maturation • IL-6 also up-regulates histamine production and induces the expression of immunoglobulin E (IgE) receptors on the surface of mast cells
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Stress-induced IL-6 release in mice is mast cell-dependent
Huang M, Pang X, Karalis K, Theoharides TC. Cardiovasc Res. 2003 Jul 1;59(1):241-9
.
• Acute stress increases serum IL-6 in mice • Stress-induced IL-6 release is absent in W/Wv mast cell-deficient mice • Both normal and W/Wv mast cell-deficient mice respond similarly to stress
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Effect of acute stress on serum IL-6 levels in (A) stressed W/Wv mice and their +/+ controls, as well as in (B) stressed CRH k/o mice and their controls. May 1, 2009 Copyright-Dr. T.C. Theoharides 33
IL-1 induces vesicular secretion of IL-6 without degranulation from human mast cells
Kandere-Grzybowska K, Letourneau R, Kempuraj D, Donelan J, Poplawski S, Boucher W, Athanassiou A, Theoharides TC. J Immunol. 2003 Nov 1;171(9):4830-6. • IL-1 stimulates secretion of IL-6 without release of the granuleassociated protease tryptase in normal human umbilical cord blood-derived mast cells. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures.
May 1, 2009 Copyright-Dr. T.C. Theoharides 34
•
•
IL-1 stimulates differential release of IL-6 without degranulation
8-16 wk hCBMCs + IL-1 6 hourscell free supernatants assay IL-6 (ELISA) or tryptase (fluoroimmunoenzyme assay).
MC)
1000 750 500 250 0
A
MC)
spont stim Tryptase *
125 100 75 50 25 0
B
*
IL-6
*
5
Tryptase (ng/5x10
IL-6 (pg/5x10
5
IL-1
test
anti-IgE
IL-1
anti-IgE
35
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*p<0.05 paired tCopyright-Dr. T.C. Theoharides
Ultrastructural cryoimmunocytochemistry of IL-6 molecules released from a secretory vesicle, 1/10th the diameter of that of 140 nm secretory granules, of a human mast cell 140 stimulated with nm IL-1
nm
190 nm
140 nm
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190 nm
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70 nm
140 nm
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Serum IL-6 reflects disease severity and osteoporosis in mastocytosis patients
Theoharides TC, Boucher W, Spear K. Int Arch Allergy Immunol. 2002 Aug;128(4):344-50.
•
Mastocytosis is characterized by an increased number of mast cells in the bone marrow or in skin areas known as urticaria pigmentosa (UP) Patients may present with flushing, itching, food intolerance, but also anxiety, headaches, lack of concentration (“brain fog”) Serum IL-6 is elevated in mastocytosis patients and correlates with severity of symptoms and the presence of osteoporosis. High serum IL-6 may not only signify disease progression, but may also participate in the pathophysiology of mastocytosis
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•
•
•
Double Immunocytochemistry for c-kit and CRH-R
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Novel therapeutic targets for autism
Theoharides TC, Doyle R, Francis K, Conti P, Kalogeromitros D. Trends Pharmacol Sci. 2008 Aug;29(8):375-82. Epub 2008 Jul 6.
• Children with mastocytosis often have ADD, learning difficulties and “brain fog” • Data were obtained in response to the question listed below that was sent by the Mastocytosis Society to a database of 400 patients • “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g.Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made”
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Mastocytosis patients with ASD*
Patient 1 (BN) 2 (SH) 3 (RC) 4 (BL) 5 (?) 6 (EC) 7 (ESC) 8 (?) 26 (BN) 10 (CJP) 11 (EA) 12 (NP) 13 (MH) 14 (MH) 15 (MH) 16 (AB) 17(SMMcC) 18 (DA) 19(JDPH) 20 (ME) 21 (AC) Age ‘08 4 15 11 18 44 21 21 18 7.5 4 9 3.5 46 (father) 10 (daughter) 8 (son) 2.5 17 9 15 23 4 Sex M M M M+ M+ F F M M M M M M# F# M# F F M M F M ASD Stuttering, high functioning ASD, poor speech Asperger’s ASD, dyslexia Asperger’s SID SID Asperger’s Autism ASD, ADHD, SID Asperger’s PDD-NOS Asperger’s Asperger’s Mood problems, ADD Autism spectrum, PDD-NOS Asperger’s Asperger’s Asperger’s Mild autism, ADD Asperger’s Mast cell diagnosis Mastocytoma, solitary UP, asthma UP, NF1 SM Mastocytosis, UP? UP Mastocytosis Mastocytosis Mastocytoma, solitary Mastocytosis, UP?, flushing, GI UP, hives, rash, GI (brother with masto) Mastocytosis Hives, asthma Diarrhea, hives, itching Mastocytosis UP, SM? Mast cell disease, food intolerance UP Mast cell disease, asthma Mast cell disease Mastocytosis
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Mastocytosis patients with ASD*
Patient 22 (ND) 23 (AS) 24 (GC) 25 (AL) 26 (SS) 27 (EAIS) 28 (JT) Age ‘08 7 2 21 4.5 8 5 2.5 Sex M M F M F F M ASD Asperger’s (mild) ASD, SID Asperger’s ASD, moderate Asperger’s PDD-NOS Regressive autism Mast cell diagnosis UP Mastocytosis, Celiac disease SM Mastocytoma, solitary UP Mast cell disease, flushing, milk intolerance, anxiety Mast cell disease, flushing, food intolerance, acute lymphocytic anemia UP, GI symptoms
29 (CJM)
3.5
M&
PDD-NOS
30 (TDM) 31 (JRM)
6.6 10.5
M& M&
DID ADD, Asperger’s ?
Food hypersensitivities Food hypersensitivities
*Data obtained in response to the question listed below that was sent by the Mastocytosis Society (www.tmsforacure.org) to a database of 400 patients. + Same family (uncle); #Same family; &Same family (brothers) “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g. Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made.” ADD=Attention Deficit Disorder; PDD-NOS=Pervasive developmental disorder-not otherwise specified; NF1=Neurofibromatosis-1; SID=Sensory Integration Dysfunction; SM=systemic mastocytosis; UP=Urticaria Pigmentosa
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Mastocytosis parents with children with ASD
Patient 1 (CB) 2 (LA) 3 (?) 4 (SH) 5 (LK) 6 (CH) 7 (RK) 8 (CS) 9 (MH) 10 (SS) 12 (RDK) Age Sex Mast cell diagnosis 48 43 45 ? 45 ? ? ? 42 ? 49 F F F F F F F F F F F M Systemic mastocytosis Mast cell disease Systemic mastocytosis UP, Systemic mastocytosis Systemic mastocytosis Systemic mastocytosis Mastocytosis Systemic mastocytosis Mast cell disease UP Mast cell disease Indolent mastocytosis Child with ASD-Sex, Age at Dx ADD, Asperger’s-M, 5 y/o Autism, FCAS-F triplets, 2 y/o Asperger’s, Mast cell disease Asperger’s, ADD, asthma, M Mild autism, ADD, Mast cell disease, F Asperger’s, ADD, M Asperger’s, 15 y/o Niece with Asperger’s, F Asperger’s F 10 y/o, ADD M 8 y/o Rett syndrome, F Asperger’s, F, 5 y/o Asperger’s, M, 14 y/o
11 (LLMcC) 56
*Data obtained in response to the question listed below that was sent by the Mastocytosis Society (www.tmsforacure.org) to a database of 400 patients. + Same family (uncle); #Same family; “Could you please let us know if you or any of your children have been diagnosed with Autism or an Autistic Spectrum Disorder (e.g. Asperger’s disorder). Please include yours and your child’s sex, current age, age at time of diagnosis, and how/where diagnosis was made.” ADD=Attention Deficit Disorder; PDD-NOS=Pervasive developmental disorder-not otherwise specified; May 1, 2009 Copyright-Dr. T.C. Theoharides 42 NF1=Neurofibromatosis-1; SID=Sensory Integration Dysfunction; SM=systemic mastocytosis; UP=Urticaria Pigmentosa
Serum levels of a mast cell trigger and a mast cell mediator are increased in young children with autism
• Serum Neurotensin is significantly elevated in autistic children (n=28, <4 years old) as compared to healthy, normally maturing controls (n=8, <4 years old) • Serum IL-6 is significantly elevated in autistic children (n=29, <4 years old) as compared to healthy, normally maturing controls (n=7, <5 years old)
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Serum levels of neurotensin
p=0.0356
2500 2500
Serum NT (pg/ml)
2000 2000 1500 1500 1000 1000 500 500 00
Control n=8 Autism n=28
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Serum levels of IL-6
p=0.0128
1200 Serum IL-6 (pg/ml) 1000 800 600 400 200 0 Control n=7 Autism n=29
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Effectof of mercuryonintracellular calcium ion of mercury on histamine release from Effect mercury on intracellular calcium ion Effect LAD2 human mast cells levelin LAD 22mast cells in LAD mast cells level
*
Histamine release (%)
100 90 80 70 60 50 40 30 20 10 0
*
Spontaneous
SP (0.1 µM)
SP (2 µM)
HgCl2(0.1 µM)
HgCl2 ( µM) 1
HgCl2(10 µM)
SP (0.1M) + HgCl2(0.1 µM) µ
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SP (2 M) + HgCl2(0.1 µM) µ
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Effect of mercury on intracellular calcium ion level in LAD2 mast cells
2.5 2 1.5 1 0.5 0 1 3 5 7 9 11 13 15 17 19 21 Time (min)
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Intracellular calcium (arbitrary unit)
Control HgCl2
Effect of mercury on VEGF release from LAD2 human mast cells
cells)
1400 1200 1000
*
* *
VEGF release (pg/10
6
800 600 400 200 HgCl 2 (1 µ M) HgCl 2 (0.1 µ M) HgCl 2 ( 1 0 µ M) HgCl 2 ( 1 0 0 µ M) Control 0
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VEGF release (pg/10 cells)
1000 250 500 750 0 Spontaneous SP (0.1 M) µ SP (2 M) µ HgCl (0.1µM) 2 HgCl (1 µ M) 2 HgCl (10µM) 2 HgCl (0.1µ M) + SP (0.1M) µ 2
6
* * *
Effect of mercury and SP on VEGF release from LAD2 human mast cells
Copyright-Dr. T.C. Theoharides
HgCl (0.1µ M) + SP (2M) µ 2 HgCl (1µ M) + SP (0 µM) .1 2 HgCl (1µ M) + SP (2M) µ 2 HgCl (1 µ M) + SP (2M) µ 2 0
50
Effect of thimerosal on VEGF release from Umbilical cord human mast cells
hCBMCs - VEGF release (pg/106 cells)
1200 1000 800 600 400 200 Control Thimerosal (1 M) µ Thimerosal (10 M) µ Thimerosal (100 M) µ 0
*
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Cell viability (%)
100 20 40 60 80 0 Control HgCl (0.1 M) µ 2 HgCl(1µ M) 2 HgCl(10µ M) 2 HgCl(100 µ M) 2
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Thimerosal (0.1 M) µ Thimerosalµ (1 M) Thimerosal (10 M) µ
*
Effect of mercury and thimerosal on LAD2 human mast cell viability
Thimerosal (100 M) µ
52
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Brain Inflammation
Autism and Oxidative Stress
• One study of 305 autistic children and 205 controls showed that the ratio of S-adenosylhomocysteine, used as an indicator of methylation ability, was significantly reduced in autistic children • Reduced plasma levels of S-adenosylmethionine (SAMe), the main endogenous antioxidant. Both of these studies and a recent review imply that autistic patients may have excessive free radical production • Reactive oxygen species are generated during mast cell activation and further stimulate mast cells
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Beneficial Effect of Flavonoids in Brain Inflammation
Middleton E Jr, Kandaswami C, Theoharides TC. Pharmacol Rev. 2000 52(4):673-751. Review.
• Flavonoids are potent anti-oxidant and antiinflammatory compounds. • Select flavonoids inhibit mast cell activation. • Flavonoids inhibit glia IL-6 release. • The most potent flavonoids are luteolin and quercetin, but need to be given in a liposomal formulation to increase absorption.
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Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells
Kempuraj D, Madhappan B, Christodoulou S, Boucher W, Cao J, Papadopoulou N, Cetrulo CL, Theoharides TC. Br J Pharmacol. 2005 Aug;145(7):934-44.
•Allergic stimulation of human mast cells results in secretion of histamine, the proteolytic enzyme tryptase, and the pro-inflammatory cytokines IL-6, IL-8 and TNF-α •Quercetin inhibits histamine release by 52-77%, tryptase release by 79-96%, and cytokines release by 82-93%
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Inhibition of inflammatory molecules by quercetin from human mast cells
100 90 80 Pe rce nt inhibition 70 60 50 40 30 20 10 0 Histamine Tryptase IL-6 IL-8 TNF-alpha * * * * *
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Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin
Kandere-Grzybowska K, Kempuraj D, Cao J, Cetrulo CL, Theoharides TC. Br J Pharmacol. 2006 May;148(2):208-15.
•IL-1-stimulates IL-6 production from human mast cells •Quercetin inhibits IL-6 secretion induced by IL-1 in a dose-dependent manner
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Inhibitory effect of quercetin on tryptase and MCP-1 chemokine release, and histidine decarboxylase mRNA transcription by human mast cell-1 cell line
Castellani ML, Kempuraj D, Frydas S, Theoharides TC, Simeonidou I, Conti P, Vecchiet J. Neuroimmunomodulation. 2006;13(3):179-86. Epub 2006 Dec 21.
• Quercetin, is a potent antioxidant, cytoprotective and anti-inflammatory compound capable of inhibiting histamine and cytokines release from mast cells. In this study quercetin inhibits, in a doseresponse manner, tryptase and MCP-1 • Quercetin inhibited the transcription of histidine decarboxylase using RT-PCR
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Luteolin inhibits myelin basic protein-induced human mast cell activation and mast celldependent stimulation of Jurkat T cells
Kempuraj D, Tagen M, Iliopoulou BP, Clemons A, Vasiadi M, Boucher W, House M, Wolfberg A, Theoharides TC. Br J Pharmacol. 2008 Sep 22. [Epub ahead of print]
• Addition of mast cells to Jurkat T cells activated by anti-CD3/anti-CD28 increases IL-2 release by 30-fold (n=3, P<0.05). SP-stimulated mast cells and their supernatant fluid further increase Jurkat cell IL-2 release (n=3, P<0.05) • Luteolin pretreatment inhibits mast cell activation (n=3-6, P<0.05), Jurkat cell activation and mast celldependent Jurkat cell stimulation (n=3, P<0.05)
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Quercetin protects against acute immobilization stress-induced behaviors and biochemical alterations in mice
Kumar A, Goyal R. J Med Food. 2008 Sep;11(3):469-73.
• Acute immobilization stress for 6 hours causes severe anxiety, analgesia, and impaired motor activity in mice, accompanied by partial depletion of brain levels of reduced glutathione and catalase activity • Pretreatment with quercetin reversed immobilized stress-induced anxiety and analgesia and reduced locomotor activity; it also restored the depleted brain glutathione and catalase activity
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Conclusions
• It is important to investigate food intolerance and introduce appropriate diets • The flavonoids quercetin and luteolin inhibit IL-6 and TNF-α release from mast cells • Luteolin inhibits IL-6 release from microglia, as well as IL-1 mediated release of IL-6, IL-8 and MCP-1 from astrocytes • The dietary supplement NeuroProtek contains quercetin and luteolin, along with other antioxidants
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NeuroProtek Label
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