Dietert_Frontmatter.pdf
Dietert_Part-I.pdf
Dietert_Ch01.pdf
Chapter 1
Immunotoxicology Testing: Past and Future
1. Introduction
2. Immunosup-pression
3. Skin Sensitization Testing
4. Needs in Immunotoxicity Screening Testing
5. Conclusion
References
Dietert_Part-II.pdf
Dietert_Ch02.pdf
Chapter 2
Developmental Immunotoxicity (DIT): The Why, When, and How of DIT Testing
1. Introduction
2. The “Why” of DIT Testing
3. The “When” of DIT Testing
4. The “How” of DIT Testing
5. Summary
References
Dietert_Ch03.pdf
Chapter 3
An In Vivo Tiered Approach to Test Immunosensitization by Low Molecular Weight Compounds
1. Introduction
2. Materials
2.1. Subcutaneous Exposure
2.2. Oral Exposure
2.3. Delayed Type Hypersensitivity Assay
2.4. Preparation of Single Cell Suspension of Lymph Nodes
2.5. Serum Ig ELISA
2.6. ELISpot
3. Methods
3.1. Subcutane Exposure and Experimental Setup RA–PLNA
3.2. Oral Exposure and Experimental Setup
3.2.1. Collecting Blood
3.2.2. Delayed Type Hypersensitivity Response
3.2.3. Isolation of the Auricular Lymph Node
3.3. Preparation of Cell Suspensions
3.4. RA-IgG1 and IgG2a ELISA for Serum Samples
3.5. ELISPOT Assay
3.6. Flow Cytometry
3.7. Cell Culture
4. Notes
References
Dietert_Ch04.pdf
Chapter 4
Risk of Autoimmune Disease: Challenges for Immunotoxicity Testing
1. Introduction
2. The Multifactorial Nature of Autoimmune Disease
3. Environmental Toxicant Exposure and Skewed Host Responses to Microbes as a Problem
4. Sex-Specific Issues in Autoimmunity and Testing
5. The Challenge of Identifying Immunotoxic Risk Factors for Autoimmune Disease
6. Role of Autoimmune-Prone Strains in Immunotoxicity Testing?
7. Tiered Approach
8. Conclusions
References
Dietert_Ch05.pdf
Chapter 5
Markers of Inflammation
1. Introduction
2. General Considerations from Standard Toxicology Studies
2.1. Cells of the Inflammatory Response
2.1.1. Neutrophils
2.1.2. Basophils
2.1.3. Eosinophils
2.1.4. Platelets
2.1.5. Lymphocytes
2.1.6. Macrophages
2.1.7. Red Blood Cells
2.1.8. Summary of Inflammatory Cell Evaluation
2.2. Cell Surface Receptors and Adhesion Molecules
2.2.1. Selectins
2.2.2. Integrins
2.3. Soluble Mediators of the Inflammatory Response
2.3.1. Cytokines
2.3.2. Nuclear Factor-kB
2.3.3. Chemokines
2.3.4. Acute Phase Proteins
3. Conclusions
References
Dietert_Ch06.pdf
Chapter 6
Evaluating Macrophages in Immunotoxicity Testing
Introduction
2. Hematopoietic Origin of Macrophages
3. Types of Macrophages
4. Macrophage Functional Assays
4.1. Antimicrobial Killing/Phagocytosis
4.2. Autophagy
4.3. Antigen Presentation Assays
4.4. Biochemical Assays
4.5. Calcium Influx
4.6. Colony Forming Assays
4.7. Confocal Microscopy/Immunofluorescence
4.8. Enzyme-Linked Immunosorbent Assay (ELISA)
4.9. Flow Cytometry
4.10. Protein Phosphorylation
4.11. Transcriptional Activation/RNA Quantification
4.12. Transcriptome Expression Analysis
References
Dietert_Part-III.pdf
Dietert_Ch07.pdf
Chapter 7
Host Resistance Assays Including Bacterial Challenge Models
1. Introduction
2. Host Resistance Assays
2.1. Comprehensive Host Resistance Assay
2.1.1. Influenza Virus Host Resistance Assay
2.2. Targeted Host Resistance Assays
2.2.1. Latent Virus Reactivation Model
2.2.2. Fungal HR Model
2.2.3. Parasite HR Models
2.2.4. Tumor HR Models
2.3.1. Evaluation of Innate Immunity
2.3.2. Evaluation of Therapeutics Affecting Neutrophils and/or Macrophages
2.3.3. Evaluation of Inflammatory Therapeutics
2.3.4. Evaluation of Therapeutics Targeting TNF-a
2.3.5. Marginal Zone B Cell HR Evaluation
2.3.6. Neutrophil/Gram Negative Bacterial HR Model
2.3.7. Intracellular Bacterial HR Model for Evaluation of Liver and Splenic Macrophages and Neutrophils
2.3. Bacterial HR Assays
3. Summary
References
Dietert_Ch08.pdf
Chapter 8
Viral Host Resistance Studies
1. Introduction
2. Influenza: A Viral Host Resistance Model
2.1. General Study Design
2.2. Viral Clearance Assessment
2.3. Additional Assessments
2.3.1. Elicitation of Cytokines
2.3.2. Natural Killer Cell Activity
2.3.3. Alveolar Macrophages
2.3.4. Cytotoxic T-Lymphocyte Activation
2.3.5. Influenza Specific T-Cell Dependent Antibody Response
3. Additional Viral Models
3.1. Reovirus Gastrointestinal Rodent Model
3.2. Latent Viral Rodent Models
3.3. Serendipitous MMTV Host Resistance Model
3.4. Non-human Primate Viral Host Resistance Models
References
Dietert_Ch09.pdf
Chapter 9
Parasite Challenge as Host Resistance Models for Immunotoxicity Testing
1. Introduction
1.1. Background
1.2. Biology of the Parasite
1.3. Resistance to Infection
1.4. Trichinella spiralis Infection as a Host Resistance Model
1.5. Outline of Major Procedures
2. Materials
2.1. Isolation of Larvae
2.1.1. Equipment
2.1.2. Reagents
2.2. Infection of Experimental or Stock Animals
2.2.1. Equipment
2.2.2. Reagents and Supplies
2.3. Adult Parasite Counts
2.3.1. Equipment
2.3.2. Reagents and Supplies
2.4. Larvae Counts
2.4.1. Equipment
2.4.2. Reagents and Supplies
2.5. Parasite Fecundity
2.5.1. Equipment
2.5.2. Reagents and Supplies
2.6. Preparation of Tsp Antigen (T. spiralis extract, TsE)
2.6.1. Equipment
2.6.2. Reagents and Supplies
2.7. Determination of IgM and IgG Antibody Titers to Trichinella Antigens
2.7.1. Equipment
2.7.2. Reagents and Supplies
3. Methods
3.1. Maintenance of Stock Larvae Donors
3.2. Isolation of Infectious Larvae
3.3. Infection of Experimental or Stock Animals
3.4. Adult Parasite Counts
3.5. Collection of Blood for Antibody Titers
3.6. Larvae Counts
3.7. Counts of Larvae in Tissue Sections
3.8. Determination of Parasite Fecundity
3.9. Preparation of Trichinella Antigens (T. spiralis Extract, TsE)
3.10. Determination of IgM and IgG Antibody Titers to Trichinella antigens (see Note 3)
3.11. Determination of Total IgE Concentration
3.12. Parasite-Specific Lymphoproliferative Responses
4. Notes
References
Dietert_Ch10.pdf
Chapter 10
Tumor Challenges in Immunotoxicity Testing
1. Introduction
2. Materials
2.1. Maintenance of Tumor Cell Cultures
2.2. Preparation for In Vivo Challenge
2.3. Tumor Cell Injection
2.4. Measurements and Endpoints
3. Methods
3.1. Maintenance of Tumor Cell Cultures
3.1.1. EL4 Lymphoma Cells
3.1.2. B16F10 Melanoma Cells
3.2. Preparation for In Vivo Challenge
3.3. Tumor Cell Injection
3.3.1. Subcutaneous (sc) Injection
3.3.2. Intravenous (iv) Injection
3.4. Measurements and Endpoints
3.4.1. Subcutaneous (sc) Challenge
3.4.2. Intravenous (iv) Challenge
Pilot Study
3.4.3. Actual Tumor Challenge Study
4. Notes
References
Dietert_Part-IV.pdf
Dietert_Ch11.pdf
Chapter 11
The T-Dependent Antibody Response to Keyhole Limpet Hemocyanin in Rodents
1. Introduction
1.1. Rat KLH TDAR Assay- General Study Design Background
1.2. Mouse KLH TDAR Assay – General Study Design Background
2. Materials
2.1. Rat KLH TDAR
2.2. Mouse KLH TDAR
3. Methods
3.1. Rat KLH TDAR
3.1.1. In Vivo Dosing and KLH Administration
3.1.1. ELISA (see Note 7)
3.2. Mouse KLH TDAR
3.2.1. In Vivo Dosing and KLH Administration
ELISA (see Note 7)
4. Notes
References
Dietert_Ch12.pdf
Chapter 12
The Sheep Erythrocyte T-Dependent Antibody Response (TDAR)
1. Introduction
2. Materials
2.1. Solutions
2.2. Reagents
2.3. Plastic and Glassware
2.4. Equipment
3. Methods
3.1. Labeling of Tubes, Petri Dishes, Reagent Bottles, and Data Collection Forms
3.2. Preparation of Sheep Erythrocytes for Sensitization
3.3. Preparation of Animal for Injection of Sheep Erythrocytes
3.4. Preparation of Spleen Cells
3.5. Plaquing Procedures
4. Notes
References
Dietert_Ch13.pdf
Chapter 13
The Delayed Type Hypersensitivity Assay Using Protein and Xenogeneic Cell Antigens
1. Introduction
2. Materials
2.1. SRBCs Used in Mice
2.2. KLH Used in Rats Without Adjuvant
2.3. BSA Used in Rats and Mice with Adjuvant
2.4. BSA Used in Chickens Without Adjuvant
3. Methods
3.1. Method 1 SRBCs Used in Mice
3.2. Method 2 KLH in Rats Without Adjuvant
3.3. Method 3 BSA in Rats and Mice with Adjuvant
3.3.1. BSA with Adjuvant in Rats
3.3.2. BSA with Adjuvant in Mice
3.4. Method 4 BSA in Juvenile Chickens
4. Notes
References
Dietert_Ch14.pdf
Chapter 14
The Cytotoxic T Lymphocyte Assay for Evaluating Cell-Mediated Immune Function
1. Introduction
2. Materials
3. Methods
3.1. Animals, Viruses, and Target Cells
3.2. Prepare Effector Cells
3.3. Target Cell Infection
3.4. Labeling of Target Cells
3.5. CTL Assay
3.6. Calculate the Percent CTL Lysis (51Cr release)
4. Notes
References
Dietert_Ch15.pdf
Chapter 15
NK Cell Assays in Immunotoxicity Testing
1. Introduction
2. Materials
2.1. Cell Lines as Target Cells
2.2. Cell Culture and NK Activity Assay
2.3. Flow Cytometry
3. Methods
3.1. NK Activity Assay
3.1.1. Preparation for Murine Splenocytes as Effectors (3, 8, 11–13)
3.1.2. Preparation for Human Peripheral Blood Lymphocytes as Effectors (16–18)
3.1.3. Preparations for Target cells
3.1.4. NK Activity Determined by 51Cr-Release Assay
3.2. Determining the Number of NK Cells
3.2.1. Determining the Murine Splenic NK Cells by Flow Cytometry (12, 19)
3.2.2. Determining the Human NK Cells by Flow Cytometry (16–18, 20)
3.3. Intracellular Levels of Perforin, Granzymes, and Granulysin in NK Cells
3.3.1. Preparations for NK-92CI Cells
3.3.2. Treatment with Chemicals
3.3.3. Cell Staining and Flow Cytometric Analysis
4. Notes
References
Dietert_Ch16.pdf
Chapter 16
The Local Lymph Node Assay and Skin Sensitization Testing
1. Introduction
2. Materials
3. Methods
3.1. Topical Exposure to Chemical
3.2. Injection of Thymidine and Processing of Lymph Nodes
3.3. Calculation and Interpretation of Results
4. Notes
References
Dietert_Ch17.pdf
Chapter 17
Use of Contact Hypersensitivity in Immunotoxicity Testing
1. Introduction
1.1. Contact Hypersensitivity
1.2. General Aspects
1.3. Contact Hypersensitivity Testing in Mice
1.4. Contact Hypersensitivity Testing in the Guinea Pig
Conclusion
References
Dietert_Ch18.pdf
Chapter 18
Evaluation of Apoptosis in Immunotoxicity Testing
1. Introduction
2. Materials
2.1. Annexin V/Propidium Iodide Staining
2.2. TUNEL Assay
2.3. In situ TUNEL Assay
2.4. Gel Electrophoresis for Detection of DNA Fragmentation
2.5. Mitochondrial Membrane Potential
2.6. Detection of Caspase 3/7 Activity
2.7. Protein Assay
2.7.1.Western Blot Solutions
2.8. RT-PCR Analysis of Gene Expression
2.8.1. Extraction of Total RNA Using Trizol Reagent
2.8.2. Extraction of Total RNA Using RNeasy Mini Kit
2.8.3. RT-PCR for Gene Expression
3. Methods
3.1. Annexin V/Propidium Iodide Staining
3.1.1. Procedure
3.2. TUNEL to Detect DNA Fragmentation
3.2.1. Procedure
3.3. In Situ TUNEL Staining
3.3.1. Procedure
3.4. Gel Electrophoresis for Detection of DNA Fragmentation
3.5. Mitochondrial Membrane Potential
3.5.1. Procedure
3.6. Detection of Caspase Activity
3.6.1. Procedure
3.7. Western Blotting for Protein Estimation
3.7.1. Procedure
3.8. RT-PCR Analysis of Gene Expression
3.8.1. RNA Extraction Using Trizol Reagent
3.8.2. Extraction of Total RNA Using RNeasy Mini Kit
Procedure
3.8.3. RT-PCR
Procedure
4. Notes
References
Dietert_Ch19.pdf
Chapter 19
Dendritic Cells in Immunotoxicity Testing
1. Introduction
2. Materials
2.1. Mice
2.2. Generation of Bone Marrow DCs
2.3. Immunization
2.4. Blood Collection and Serum Preparation
2.5. Measurement of Antigen Specific IgG Isotype in Serum by ELISA
2.6. Total IgE Detection
2.7. ELISA for Antigen Specific IgE in Serum
2.8. Flow-Cytometric Analysis
2.9. Splenocyte Preparation
2.10. CD4+ T Cell Isolation
2.11. Cell Culture
2.12. Cytokine Analysis
2.13. Cell Proliferation
2.14. DTH Assay
2.15. Protein Assay
2.16. Phosphoprotein Assay
3. Methods
3.1. Generation of BM-DCs
3.2. Immunization
3.3. Blood Collection and Serum Preparation
3.4. Measurement of Antigen Specific IgG Isotype in Serum by ELISA
3.5. Total IgE Detection
3.6. ELISA for Antigen Specific IgE in Serum
3.7. Flow-Cytometric Analysis
3.8. Splenocyte Preparation
3.9. CD4+ T Cell Isolation
3.10. Cell Culture
3.11. Cytokine Analysis
3.12. Cell Proliferation
3.13. DTH Assay
3.14. Protein Assay
3.15. Phosphoprotein Assay
3.15.1. Cell Lysate Preparation
3.15.2. Phosphoprotein Detection
3.15.3. Statistical Analysis
4. Notes
References
Dietert_Ch20.pdf
Chapter 20
Evaluating Cytokines in Immunotoxicity Testing
1. Introduction
1.1. Sample Collection and Storage
1.1.1. Primary Sources
1.1.2. Secondary Sources
1.1.3. Clean-up and Storage of Samples
1.1.4. Cryopreservation of Cells
1.1.5. Immunoenzymatic Assay vs. Bioassay: Advantages and Disadvantages
2. Materials
2.1.1 Sandwich ELISA
2.1.2 Competitive ELISA
2.1.3 ELISpot
2.2 FACS
2.3 Bioassays
2.4 Gene Expression
3. Methods
3.1. Immunoassays
3.1.1. Typical ELISA (Sandwich Protocol)
3.1.2. Competitive ELISA
3.1.3. ELISpot
3.2 Fluorescene-Activated Cell Sorting (FACS)
3.3 Bioassays
3.4 Gene Expression (RT-PCR Protocol)
4. Concluding Comments
5. Notes
References
Dietert_Ch21.pdf
Chapter 21
Flow Cytometry in Preclinical Drug Development
1.Introduction
1.1. Regulatory Oversight
1.2. General Uses of Flow Cytometry in Discovery, Development and Clinical Research
1.2.1. Discovery
1.2.2. Development
1.2.3. Clinical
1.2.4. Flow Cytometry Method Qualification/Validation
1.2.5. Specific Flow Cytometry Applications
2. Materials (see Note 1)
2.1. Immunopheno-typing (Single Cell Suspensions from Body Fluids, Bone Marrow or Lymphoid Tissues): Non-human Primate and Rat
2.2. Peripheral Blood Cross-reactivity Evaluation: Multiple Species
2.3. Binding Activity and Stability Testing for Novel Test Antibody: Cultured Cell Lines (see Note 3)
2.4. Receptor Occupancy Assay: Multiple Species (see Note 6)
3. Methods
3.1. Immunopheno-typing: Non-human Primate and Rat
3.2. Cross-reactivity: Multiple Species (see Note 11)
3.3. Binding Activity and Stability Testing: Cultured Cell Lines (see Note 14)
3.4. Receptor Occupancy (see Note 14)
4. Notes
References
Dietert_Ch22.pdf
Chapter 22
Enhanced Histopathology Evaluation of Lymphoid Organs
1. Introduction
2. Basic Approach to Enhanced Histopathology
2.1. Required Information
2.2. Tissues to Evaluate
2.3. Determining Range of Normal
2.4. Preventing Diagnostic Drift
2.5. When to Use Blind Scoring
2.6. Knowledge of Normal Structure, Function and Histology
3. How to Evaluate Lymphoid Organs Using Enhanced Histopathology
3.1. Enhanced Histopathology of the Thymus
3.2. Enhanced Histopathology of the Spleen
3.3. Enhanced Histopathology of the Lymph Nodes
3.4. Enhanced Histopathology of the MALT
3.5. Enhanced Histopathology of the Bone Marrow
References
Dietert_Ch23.pdf
Chapter 23
Immunotoxicity Testing in Nonhuman Primates
1. Introduction
2. Materials
2.1. Immunophenotyping
2.2. Determination of Cytokines
2.3. Functional Natural Killer (NK) Cell Testing
2.4. T-Cell Dependent Antibody Response
2.5. Bone Marrow Examination
2.6. Immunhistochemical Staining of Tissue
2.7. Lymphocyte Proliferation Assay
3. Methods
3.1. Immunophenotyping
3.1.1. Preparation
of Antibody-Mixture
3.1.2. Preparation of Samples
3.1.3. Preparation of Cells from Lymphatic Tissue
3.2. Determination of Cytokines
3.3. Functional NK Cell Testing
3.4. T-Cell Dependent Antibody Reaction (TDAR)
3.4.1. Immunization of Cynomolgus Monkeys/Marmoset Monkeys
3.4.2. Determination of Anti-KLH-IgG/IgM
in Serum of Cynomolgus/Marmoset Monkeys
3.5. Bone Marrow Examination
3.5.1. Preparation of Smears
3.5.2. Staining of Smears
3.5.3. Microscopical Examination
3.6. Immunohistochemical Staining
3.7. Lymphocyte Proliferation Assay
3.7.1. Preparation of PBMCs from Blood Samples
3.7.2. Stimulation of PBMCs with Mitogens
3.7.3. Measurement of Proliferative Response
4. Notes
References
Dietert_Part-V.pdf
Dietert_Ch24.pdf
Chapter 24
Fundamentals of Clinical Immunotoxicology
1. Introduction: Basic Terminology and Concepts
2. Why Not Depend on Animal Studies?
2.1. When Immunomodulation Is a Good Thing: The Example of Vaccines
2.1.1. Do Vaccines Overload the Immune System?
2.1.2. Does Vaccination Lead to Autoimmunity?
2.1.3. Does Vaccination Lead to Increases in Asthma or Allergy?
2.1.4. Does Vaccination Lead to Autism?
2.2. When Immunomodulation Is Not a Good Thing: The Example of Immunomodulatory Antibodies
2.3. Clinical Considerations for Developmental Immunotoxicology
3. General Testing Considerations and Approach
3.1. Conducting Research in Humans
3.2. Specific Tests Available to Assess Immunotoxicity in Humans
3.2.1.Complete Blood Count and Differential
3.2.2. Immunoglobulin Concentrations
3.2.3. Delayed-Type Skin Testing
3.2.4. Phenotypic Analysis by Flow Cytometry
3.2.5. Specific Antibody Assessment
3.2.6. Cytokine Measurement
3.2.7. Natural Killer (NK) Cells
3.2.8. Neutrophil Function
3.2.9. Autoantibodies
4. The Future of Clinical Immunology?
4.1. The Promise of Synthetic Immune Systems
4.2. Not-Quite-Human Human Models
4.3. Replacement Strategy
4.4. Modification Strategy
5. Putting All the Pieces Together
6. Summary and Conclusions
References
Dietert_Part-VI.pdf
Dietert_Ch25.pdf
Chapter 25
In Vivo Functional Tests for Assessing Immunotoxicity in Birds
1. Introduction
1.1. Phytohemag-glutinin Skin Test for T Cell-Mediated Immunity
1.2. Hemagglutination Assay for Antibody Response to Immunizations with Sheep Red Blood Cells
2. Materials
2.1. Phytohemag-glutinin Skin Test for T Cell-Mediated Immunity
2.2. Hemagglutination Assay for Antibody Response to Immunizations with Sheep Red Blood Cells
2.2.1. Preparation of SRBC Suspensions
2.2.2. Immunization Injections
2.2.3. Blood Collection and Processing
2.2.4. Hemagglutination Microtiter Assay
3.Methods
3.1. Phytohemag-glutinin Skin Test for T Cell-Mediated Immunity
3.1.2. Injections
3.1.3. Measurement and Calculation of Response
3.2. Hemagglutination Assay for Antibody Response to Immunizations with Sheep Red Cells
3.2.1. Preparation of SRBC Suspensions
3.2.2. Immunization Injections
3.2.3. Blood Collection and Processing
3.2.4. Hemagglutination Microtiter Assay
4. Notes
References
Dietert_Part-VII.pdf
Dietert_Ch26.pdf
Chapter 26
In Vitro Testing for Direct Immunotoxicity: State of the Art
1. Introduction
2. In Vitro Testing for Direct Immunotoxicity
2.1. Myelotoxicity
2.2. Lymphotoxicity
2.2.1. Human Whole-Blood Cytokine Release Assay
2.2.2. Lymphocyte Proliferation Assay
2.2.3. Mixed Lymphocyte Reaction
2.2.4. Cytotoxic T-Lymphocyte Assay
2.2.5. Natural Killer Cell Assay
2.2.6. T Cell-Dependent Antibody Response
2.2.7. Dendritic Cell Maturation
2.2.8.Fluorescent Cell Chip
2.3. Additional Assay Considerations
2.3.1. Toxicogenomics
3. Discussion of In vitro Assays
3.1. The Roles of In Vitro Assays
3.1.1. Obtain Mechanistic Understanding of Immunotoxic Effects
3.1.2. Parallelogram Approach
3.1.3. Replace, Reduce and Refine the Use of Laboratory Animals
3.2. General Limitations of In Vitro Methods
3.3. Prevalidation Studies
3.4. Advantages of In Vitro Assays
3.5. Comparison of Whole Blood Cytokine Release Assay and Fluorescent Cell Chip
3.6. Using In Vitro Assay for Inter-Species Extrapolation
3.7. “Omics” Approaches to In Vitro Testing
3.8. The Future of In Vitro Testing
4. Conclusion
References
Dietert_Backmatter.pdf