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Immunotoxicity:Contribution of Chemicals to Allergic Disease Assesment, Examples, & Mechanisms. MaryJane Selgrade National Health & Environmental Effects Research Laboratory, U.S, EPA, Research Triangle Pk, NC [email protected] September 18,2006. Lecture Outline.

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Immunotoxicity:Contribution of Chemicals to Allergic Disease Assesment, Examples, & Mechanisms

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Immunotoxicity contribution of chemicals to allergic disease assesment examples mechanisms l.jpg

Immunotoxicity:Contribution of Chemicals to Allergic DiseaseAssesment, Examples, & Mechanisms

MaryJane Selgrade

National Health & Environmental Effects Research Laboratory, U.S, EPA, Research Triangle Pk, NC

[email protected]

September 18,2006


Lecture outline l.jpg

Lecture Outline

  • Mechanisms of immune mediated injury

  • Chemicals which elicit immune responses

    • Contact sensitivity

    • Respiratory sensitivity

    • Food Allergy

  • Chemicals which modulate responses to other antigens


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Hypersensitivity

Definition: Excessive humoral or cellular response to an antigen which can lead to tissue damage


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Hypersensitivity: Classification

  • Type 1: IgE mediated (Immediate type)

  • Type 2:IgM, IgG, cytolysis of cells

  • Type 3: IgM, IgG Immune complex mediated

  • Type 4: T-cell mediated (delayed-type)

(Gel & Coombs classification)


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Two Stages(Distinguishes from irritation)

Induction

Sensitization

(1st exposure)

Elicitation

Challenge

(subsequent exposure)


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Sensitization

Mediators (as such Histamine)

Bronchoconstriction

Elicitation

Mast Cell

Degranulation

Type I (Immediate)

(Atopy)

Example:

Bee Sting


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Elicitation

Ab bind to cell bound antigen

Cell lysis

Type 2 (Cytotoxic)

Ag Ab (or autoantibody)

Ag binds to cell surface

Sensitization

Examples:

Goodpastures syndrome

Hemolytic anemia


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Sensitization

Ag Ab

Elicitation

Formation of

Ag/Ab complexes;

Deposition in tissues

Activation of macrophages

And Complement

Influx of PMN’s, Eosinophils,

Lymphocytes (Inflammation)

Activated

macrophage

Polymorphonuclear

leukocyte

Type 3 (Arthus)

Examples: Late onset

asthmatic response,

fibrosis, serum sickness


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Elicitation

Antigen

Ag presenting cell

Activated T cell

Sensitization

Antigen

Ag presenting cell

Clonal expansion

Cytokines

Activated

macrophage

Inflammatory activity

Type 4 (Cell Mediated)

Example:

Contact

Sensitivity


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Antigen Presented to T Cells Via MHC


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TH1

TH2

IL-2 TGFßIFN

IL-5 IL-13 IL-6

IL-4 IL-10

Suppresses TH2

Suppresses TH1

IgG2a, CF Antibody

DTH, Cytotoxicity

IgE, IgG2b, IgG1

Mast Cells + Eosinophils

Defense Against

Intracellular Pathogens

Inflammatory Diseases

Defense Against Parasitic

Infection

Immediate-type Hypersensitivity


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Type IV Responses


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Potential Effects of Chemicals on Allergic Disease

  • Can themselves act as antigens

    • Haptens

      • Contact sensitizers

      • Respiratory sensitizers

      • Systemic hypersensitivity (drugs)

    • Proteins

      • Respiratory allergens

      • Food Allergens

      • Systemic hypersensitivity (drugs)

  • Can enhance development or expression of allergic reactions


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Potential Contact Sensitizers

  • Cosmetics and Fragrances

  • Dyes

  • Preservatives (formaldehyde

  • Metals (Ni, Co, Be, Cr)

  • Pesticides


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Contact Hypersensitivity

Allergic Contact Dermatitis (ACD

INDUCTION PHASE

ELICITATION PHASE

EDEMA AND ERYTHEMA

ALLERGEN

NONSPECIFIC INFLAMMATORY MEDIATORS

IL-1B, IL-6, IL-12

LANGERHANS CELL (LC)

Migration to Local Lymph Node

CELLULAR INFLUX

“PRIMED” LYMPHOCYTES

SPECIFIC RESPONSE

LC AND LYMPHOCYTE INTERACTION

LYMPHOCYTE PROLIFERATION


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GUINEA PIG MODELS

Guinea Pig Maximization Test

Buehler Assay

20 animals/ group

Topical application - closed patch:

Days 0, 6-8, and 13-15

Day 27-28 topical challenge

of the untreated flank for 6 h

Read: 21, 24, 48 h after removing patch

> 15% positive

ID injection w/ and without FCA plus topical application:

Days 5-8

Day 20-22 topical challenge

Read: 48,72 h after challenge

>30% positive

Induction

Challenge

Endpoint erythema

Criteria


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Mouse Ear Swelling Test

Application of

Agent or Vehicle

to Back (local)

or Abdomen

(systemic)

Sensitization

Induction Period

Challenge

Application of

Agent

Measure Ears: 24-48 hours post challenge


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The Local Lymph Node Assay

Agent applied to ears Days 1,2,3

Proliferation measure:

isotope incorporation scintillation counting

5 hour after IV injection

IV 3H-thymidine injection Day 6

Endpoint expressed as dpm


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Contact Hypersensitivity Models and Endpoint Mechanisms

PHASE

Induction

LLNA

proliferation

GPMT and BA

Elicitation

MEST

erythema

edema


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Structure Activity Relationship (SAR) Models

  • Computer modeling using chemical structure to predict ability to induce CHS

  • Based on following concepts:

    • Biologic mechanism of chemical effect is related to structure

    • Chemicals with related structures have similar mechanisms & hence effect

  • How do SAR & QSAR models work

    • Chemical structure added into program

    • Structure compared to structures in data base of know contact sensitizers

    • Structures associated with sensitizers are flagged


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Skin

penetration/

metabolism

Protein

reactivity

Metabolic

Transform

Immune

reactivity

Using SAR to Predict ACD

Predict

ACD

(Allergic Contact

Dermatitis)

Chemical

See attached


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Prevalence of Asthma, United States

0- 4 yr

80

5-14

15-34

70

35-64

>65

60

Total

50

Rate (per thousand)

40

30

20

10

1980

1981-

1984-

1987-

1990-

1993-

1983

1986

1989

1992

1994


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Immediate Response

Bronchoconstriction

IgE mediated (IL-4)

Late phase

Hyperresponsive to non-specific stimuli (methacholine)

Eosinophilic Inflammation

Th2 mediated (Il-5, IL-13)

Characteristics of Allergic Asthma


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Principal Asthma/Respiratory Allergy Concerns for Toxicologist

  • Proteins - e.g detergent enzymes, biotech

  • Haptens (low molecular wt) - isocyanates & anhydrides, platinum

  • Adjuvants - air pollution


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oscillograph

Differential

Pressure transducer

Measures pulmonary

response

exhaust

Ag

Temperature

transmitter

air

Method used to Assess for Potential

Pulmonary Hypersensitivity

Criteria for Positive Response

Respiratory rate 35%

Temp  0.6%


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Protein allergens

  • Detergent Enzymes

  • Molds and spores

  • Latex

  • microbial pesticides

  • animal dander

*Not all proteins are equally allergenic; as yet no particular amino acid sequences has been associated with allergenicity


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Guinea Pig Intratracheal Test & Detergent Enzymes Assessment

  • IT dose response cytophilic Ab response to unknown

  • IT dose response to Alcalase (subtilisin B)

  • Test protein/Reference protein = relative potency

  • Relative potency <1 set exposure level same as alcalase

  • Relative potency > 1 adjust by appropriate factor


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Low Molecular Wt (<3000 molecular weight) Compounds

  • Toluene diisocyanate

  • Diphenylmethane diisocyanate

  • Phthalic anhydride

  • Trimellitic anhydride

  • Platinum salts

  • Reactive dyes

  • 50 or so known allergens (asthmagens)


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Assessing Low Molecular Wt (<3000) as Potential Respiratory Allergens -Asthmagens

  • Karol test - Inhalation exposure, respiratory effects, Guinea pig

  • Sarlo approach - Guinea Pigs

    • Structure activity

    • In vitro reactivity with protein

    • In vivo antibody response (IT exposure)

    • In vivo reactivity (Karol test)


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Assessing Low Molecular Wt: Potential Screening Hazard ID Approaches

  • Mouse IgE test-

    • exposure variation of LLNA

    • assess total serum IgE

  • Cytokine profiling

    • exposure variation on LLNA

    • assess cytokine profiles in draining lymph node, look for differences in expression of Th1, Th2 profiles

  • LLNA


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Air Pollutants That Appear to Be Adjuvants

  • Rodent models

    • Nitrogen dioxide (NO2)

    • Residual oil fly ash (ROFA

  • & Human

    • Diesel exhaust

  • & Rhesus Monkey

    • Ozone


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TH1

TH2

IL-2 TGFßIFN

IL-5 IL-13 IL-6

IL-4 IL-10

Suppresses TH2

Suppresses TH1

IgG2a, CF Antibody

DTH, Cytotoxicity

IgE, IgG2b, IgG1

Mast Cells + Eosinophils

Defense Against

Intracellular Pathogens

Inflammatory Diseases

Defense Against Parasitic

Infection

Immediate-type Hypersensitivity


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Conditions That Influence Th1/Th2 Polarization

  • Nature or Dose of Antigen

  • Local Micro Environment

    • cytokines & other mediators produced by macrophages & epithelial cells

  • Genetics of Host

  • Route of Exposure


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Food allergens

  • Around 5% of children and 2-3 % or adults have food allergies

  • Manifestations can range from life threatening anaphylaxis, to respiratory or gut irritation

  • Peanut classic example

  • Toxicologist involved because of GMOs

    • acid resistant

    • homology with known allergens


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Impact

Goal –Relate Potency of GMO Protein to Other Food Proteins


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