Rheumatoid Arthritis: Methods and Protocols

Preface
Contents
Contributors
Part I: Animal Models
	Chapter 1: Collagen-Induced Arthritis Models
		1 Introduction
		2 Materials
			2.1 Emulsion Preparation
			2.2 Animal Immunization
		3 Methods
			3.1 Emulsion Preparation (See Notes 3 and 4)
			3.2 Animal Immunization (See Note 7) (Fig. 1)
		4 Notes
		References
	Chapter 2: Human Xenograft Model
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation of PBMC from Peripheral Blood from RA Patients
			3.2 Trimming Explanted Joint Tissue
			3.3 Implantation (See Notes 4 and 5)
			3.4 Evaluation of Invasion of Synovium
		4 Notes
		References
	Chapter 3: Scaffolded Chondrogenic Spheroid-Engrafted Model
		1 Introduction
		2 Materials
			2.1 3D Culture of Chondrogenic Spheroids
			2.2 Implantation In Vivo
		3 Methods (See Note 4)
			3.1 3D Culture of Chondrogenic Spheroids
			3.2 Implantation In Vivo (See Note 7)
		4 Notes
		References
	Chapter 4: Denervation-Induced Sarcopenia Model
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 5: Long-Term Constant Subcutaneous Drug Administration
		1 Introduction
		2 Materials (See Note 1)
		3 Methods (See Note 3)
			3.1 Filling of ALZET Pump (See Note 4)
			3.2 Implantation
			3.3 Explanting Pumps
		4 Notes
		References
	Chapter 6: Clinical Scoring of Disease Activity in Animal Models
		1 Introduction
		2 Materials
			2.1 CIA Scoring System
			2.2 Evaluation of Paw Volume
		3 Methods
			3.1 CIA Scoring System
			3.2 Evaluation of Paw Volume by In Vivo MRI Scanning (See Note 3)
		4 Notes
		References
	Chapter 7: Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice
		1 Introduction
		2 Materials
			2.1 Sampling (Fixation and Decalcification)
			2.2 Embedding
			2.3 Sectioning
			2.4 Staining
			2.5 HE Staining
			2.6 TRAP Staining
			2.7 IHC for CRACM3 Antigen
		3 Methods
			3.1 Fixation and Decalcification
			3.2 Embedding
			3.3 Sectioning
			3.4 HE Staining
			3.5 TRAP Staining
			3.6 Immunohistochemistry (IHC)
			3.7 Observation
		4 Notes
		References
	Chapter 8: Preparation of Joint Extracts
		1 Introduction
		2 Materials (See Note 1)
		3 Methods
			3.1 Harvesting Synovium from Mouse Knee Joint
			3.2 Protein Extraction (See Note 6)
		4 Notes
		References
Part II: Therapeutic Approach
	Chapter 9: Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System
		1 Introduction
		2 Materials
			2.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
			2.2 Cell-Free Proteoliposome Synthesis Using Bilayer-Dialysis Method
			2.3 Preparation of Adjuvant-Containing Liposome
			2.4 Large-Scale Proteoliposome Antigen Production
		3 Methods
			3.1 Construction of Expression Plasmid for Cell-Free Protein Synthesis
			3.2 Small-Scale Test Proteoliposome Synthesis Using Bilayer-Dialysis Method
			3.3 Preparation of Adjuvant-Containing Liposome
			3.4 Large-Scale Proteoliposome Antigen Production
		4 Notes
		References
	Chapter 10: Reconstruction of Protein/Liposome Complex
		1 Introduction
		2 Materials
			2.1 Cell-Free Protein Synthesis
			2.2 Membrane Protein Solubilization
			2.3 Reconstituting the Membrane Protein
			2.4 Sucrose Density Gradient Centrifugation and SDS-PAGE
		3 Methods
			3.1 Cell-Free Protein Synthesis
			3.2 Solubilizing HRH1
			3.3 Preparation of Bio-beads SM-2
			3.4 Reconstituting HRH1
			3.5 Sucrose Density Gradient Centrifugation
			3.6 SDS-PAGE
		4 Notes
		References
	Chapter 11: Production of Neutralizing Antibody
		1 Introduction
		2 Materials
			2.1 Antigen Preparation (See Note 1)
			2.2 Mouse Immunization
			2.3 Preparation for Fusion
			2.4 ELISA for Screening
			2.5 Clone Selection and Expansion of Positive Clones
			2.6 Purification and Storage of mAbs
		3 Methods
			3.1 Antigen Preparation
				3.1.1 Homogenization Methods
				3.1.2 Syringe-to-Syringe Method (Fig. 2)
			3.2 Mouse Immunization
			3.3 Cell Fusion
			3.4 Hybridoma Screening (ELISA)
			3.5 Expanding the Hybridomas and Freezing Positive Clones
			3.6 Purification and Storage of mAbs
		4 Notes
		References
	Chapter 12: Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen
		1 Introduction
		2 Materials
			2.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
			2.2 Cell-Free Synthesis of Autoantigen Protein Array
			2.3 AlphaScreen
		3 Methods
			3.1 Construction of Transcription Templates for Cell-Free Protein Synthesis
			3.2 Cell-Free Synthesis of Autoantigen Protein Array
			3.3 AlphaScreen
		4 Notes
		References
	Chapter 13: Generation of Specific Aptamers
		1 Introduction
		2 Materials
			2.1 Oligonucleotide Library and Primers
			2.2 Selection
			2.3 Amplification
			2.4 Purification of Single-Strand DNA (ssDNA)
		3 Methods
			3.1 DNA Selection from Oligonucleotide Library
			3.2 Amplification
			3.3 Preparation of ssDNA
		4 Notes
		References
	Chapter 14: Detailed Protocol for Predicting 3D Structure of DNA Aptamers and Performing In Silico Docking Calculations
		1 Introduction
		2 Methods
			2.1 Software Installation and Environment Setup
			2.2 Sequence Input and Structure Prediction
				2.2.1 Sequence Modification
				2.2.2 3D Structure Prediction
				2.2.3 Structure Minimization
				2.2.4 Preparing for Docking with Chimera
			2.3 Docking with Hdock
		3 Limitations
		References
	Chapter 15: RNA Interference Ex Vivo
		1 Introduction
		2 Materials
			2.1 Isolation of T Cells from Peripheral Blood
			2.2 Transfection of siRNA into Primary T Cells Using Oligofectamine
			2.3 Lentiviral-Mediated shRNA Transfection
		3 Methods (See Note 2)
			3.1 Isolation of T Cells from Peripheral Blood
			3.2 Transfection of siRNA into Primary T Cells Using Oligofectamine
			3.3 Lentiviral-Mediated shRNA Transfection (See Note 6)
		4 Notes
		References
	Chapter 16: Lentiviral-Mediated Systemic RNA Interference In Vivo
		1 Introduction
		2 Materials
			2.1 Lentiviral Titration Using Quantitative Real-Time PCR (qRT-PCR)-Based Methods
			2.2 Intraperitoneal Injection of shRNA-Encoding Lentiviral Particles
			2.3 Determination of Integrated Lentiviral Copies in Tissues
		3 Methods (See Note 1)
			3.1 Lentiviral Titration
			3.2 Intraperitoneal Injection of shRNA-Encoding Lentiviral Particles (See Note 3)
			3.3 Determination of Number of Integrated Provirus Copies in Tissue
		4 Notes
		References
	Chapter 17: Lentiviral Production Platform
		1 Introduction
		2 Materials (See Note 1)
			2.1 Production of Lentiviral Particles
			2.2 Preparation of Concentrated Viral Stock
		3 Methods (See Notes 5 and 6)
			3.1 Production of Lentiviral Particles
			3.2 Preparation of Concentrated Viral Stock
				3.2.1 Ultracentrifugation
				3.2.2 PEG Precipitation
		4 Notes
		References
	Chapter 18: Mesenchymal Stem Cell Engineering
		1 Introduction
		2 Materials (See Note 1)
			2.1 Isolation of Synovium-Derived MSC
			2.2 Knockout of Target Gene Using Via CRISPR/Cas9 (See Note 3)
		3 Methods
			3.1 Isolation of MSC from Human-Derived Synovium
			3.2 Knockout Genes at Chromosomal Level in MSC
		4 Notes
		References
Part III: Evaluation of Immunological Status
	Chapter 19: Screening of Ca2+ Influx in Lymphocytes
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 20: Single-Cell Ca2+ Imaging
		1 Introduction
		2 Materials
		3 Methods
			3.1 Single-Cell Ca2+ Imaging
			3.2 Image Analysis (See Note 12)
		4 Notes
		References
	Chapter 21: Electrophysiological Methods to Measure Ca2+ Current
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 22: Evaluation of Mitochondrial Respiratory Function in Murine Splenocytes
		1 Introduction
		2 Materials
			2.1 Hydrate Cartridge
			2.2 Cell Preparation for Seahorse XF Cell Mito Stress Test
			2.3 Running Mito Stress Test
		3 Methods
			3.1 Hydrate Cartridge
			3.2 Cell Preparation for XFp Mito Stress Assay
			3.3 Run XFp Mito Stress Assay
		4 Notes
		References
	Chapter 23: The Functional Assessment of T Cells
		1 Introduction
		2 Materials
			2.1 Analysis of Cell Surface Markers
				2.1.1 Lymphoid Cell Preparation
				2.1.2 Enrichment of T Cells
				2.1.3 Detection of Cell Surface Markers by Flow Cytometer (FCM)
				2.1.4 Detection of the Cytoplasmic Molecules by FCM
			2.2 Analysis of the mRNA
				2.2.1 RNA Preparation
				2.2.2 cDNA Synthesis
				2.2.3 PCR
			2.3 Measurement of the Cytokines
				2.3.1 In Vitro Stimulation of T Cells
				2.3.2 Intracellular Cytokine Staining (See Also Subheading 2.1.4)
				2.3.3 ELISA (Enzyme-Linked Immunosorbent Assay)
			2.4 Measurement of the Cytotoxic Activity
				2.4.1 Measurement of NK Activity (51Cr Release Assay)
				2.4.2 Measurement of CTL Activity (Non-RI CTL Assay)
		3 Methods
			3.1 Analysis of the Cell Surface Markers
				3.1.1 Lymphoid Cells
				3.1.2 Preparation of Single-Cell Suspension of the Spleen and Lymph Nodes
				3.1.3 Enrichment of T Cells by Panning Method
				3.1.4 Enrichment of T Cells by Magnetic Beads Method and by the Cell Sorting with FCM
				3.1.5 Detection of Cell Surface Markers by FCM with Direct Staining Method
				3.1.6 Detection of Cell Surface Markers by FCM with Indirect Staining Method
				3.1.7 Detection of the Cytoplasmic Molecules by FCM
			3.2 Analysis of the mRNA
				3.2.1 Preparation of RNA from Cells
				3.2.2 DNaseI Treatment of Cellular RNA
				3.2.3 cDNA Synthesis
				3.2.4 PCR
			3.3 Measurement of the Cytokines
				3.3.1 In Vitro Stimulation of T Cells
				3.3.2 Measure Cytokines by ELISA
				3.3.3 Intracellular Cytokine Staining
				3.3.4 PCR Analysis of the Transcript Prepared from the In Vitro Stimulated Cells
			3.4 Measurement of the Cytotoxic Activity
				3.4.1 51Cr Release Assay (Fig. 7)
					51Cr Labeling of Target Cells
					Cytotoxicity Assay
				3.4.2 Non-RI CTL Assay (Fig. 8)
					CFSE Labeling of Target Cells
					Measurement of Cytotoxicity of Effector Cells
		4 Notes
		References
	Chapter 24: Release of Antibodies and Cytokines from B Cells
		1 Introduction
		2 Materials
			2.1 Measurement of Total IgG Level Using Enzyme-Linked Immunosorbent Assay (ELISA) (See Note 1)
			2.2 Rapid Latex Test for RF
			2.3 Preparation of Pan B Cells for Cytokine Release Assay
		3 Methods
			3.1 Measurement of Total IgG Level Using ELISA
			3.2 Rapid Latex Test for RF (See Note 10)
			3.3 Preparation of Pan B Cells for Cytokine Release Assay
		4 Notes
		References
	Chapter 25: Evaluation of Autoreactive Responses
		1 Introduction
		2 Materials
			2.1 Preparation of Single-Cell Suspension of CIA Mice-Derived Splenocytes
			2.2 Assessment of T-Cell Responses to CII
		3 Methods
			3.1 Preparation of Single-Cell Suspension of CIA Mouse-Derived Splenocytes (See Note 3)
			3.2 Assessment of T-Cell Responses to CII
		4 Notes
		References
	Chapter 26: Macrophage Polarization and Osteoclast Differentiation
		1 Introduction
		2 Materials
			2.1 Isolation of Bone Marrow-Derived Macrophages
			2.2 M1/M2 Polarization
			2.3 Cellular Metabolism Assay
			2.4 Osteoclast Differentiation
			2.5 TRAP Activity Assay
			2.6 TRAP Staining
		3 Methods
			3.1 Isolation of Bone Marrow-Derived Macrophages
			3.2 M1/M2 Polarization
				3.2.1 Cellular Metabolism Assay (Preparation from the Day before to the Day of Assay)
				3.2.2 Cellular Metabolism Assay (Glycolysis Stress Test)
				3.2.3 Cellular Metabolism Assay (Mito Stress Test)
			3.3 Osteoclast Differentiation
			3.4 TRAP Activity Assay
			3.5 TRAP Staining
		4 Notes
		References
	Chapter 27: Scanning Electron Microscopic Analysis of the Bone-Resorption Activity in Mature Osteoclasts
		1 Introduction
		2 Materials
			2.1 Isolation of Mature Osteoclasts Using Collagen Films (See Note 1)
			2.2 Hematoxylin Staining
			2.3 FE-SEM Observation
		3 Methods
			3.1 Isolation of Mature Osteoclasts Using Collagen Films
			3.2 Hematoxylin Staining
			3.3 FE-SEM Observation
		4 Notes
		References
	Chapter 28: Animal Models of Vasculitis
		1 Introduction
		2 Materials
			2.1 Candida albicans Water-Soluble Glycoprotein (CAWS)
			2.2 Induction of CAWS-Induced Vasculitis
			2.3 Immunohistochemical Staining for Cell Proliferation, Hypoxia, and Angiogenesis
			2.4 Dihydroethidium Staining
			2.5 Real-Time RT-PCR
			2.6 Immunoblot Analysis
		3 Methods
			3.1 Preparation of CAWS
			3.2 Induction of CAWS-Induced Vasculitis
			3.3 Immunohistochemical Staining for Cell Proliferation, Hypoxia, and Angiogenesis
			3.4 Dihydroethidium Staining
			3.5 Real-Time RT-PCR
			3.6 Immunoblot Analysis
		4 Note
		References
	Chapter 29: Evaluation of Skin Damage Under UV Exposure
		1 Introduction
		2 Materials
			2.1 UVA-Photoaging Model
			2.2 Silicone Replica Method for Detection of Reticular Pattern
			2.3 Preparation for Histological Analyses Using Paraffin Section
			2.4 HE Staining
			2.5 Fontana-Masson Staining
			2.6 Immunostaining for Ki67
			2.7 Preparation for Histological Analyses by Frozen Section
			2.8 Detection for Collagen Degradation on Frozen Section
			2.9 Detection of DNA Damage
		3 Methods
			3.1 UVA-Photoaging Model
			3.2 To Detect Visible Changes in the Reticular Pattern on the Dorsal Skin Surface
			3.3 To Detect Histological Changing of Dorsal Skin Tissue (Embedding and Sectioning Process)
			3.4 HE Staining for Epidermal Hyperplasia Analysis Using Paraffin Section
			3.5 Fontana-Masson Silver Staining for Melanin Pigmentation Analysis Using Paraffin Section
			3.6 Ki67 Immunostaining for Detection of Proliferating Cells in the Epidermal Basal Layer Using Paraffin Section
			3.7 To Detect Collagen Degradation in the Dermis Using a Frozen Section
			3.8 To Detect DNA Damage by Oxidative Stress in Dorsal Skin Tissue
			3.9 To Detect DNA Damage by Oxidative Stress Using Urine Samples
		4 Notes
		References
	Chapter 30: Bulk RNA-seq Assessment of Murine Spleen Using a Portable MinION Sequencing Device
		1 Introduction
		2 Materials (See Note 1)
			2.1 Total RNA Extraction (See Note 1)
			2.2 Library Preparation for RNA-seq
			2.3 Computer Environment
			2.4 Software Installation
			2.5 Reference Database
		3 Methods
			3.1 Total RNA Extraction from Spleen
			3.2 Library Preparation for RNA-seq (See Note 6)
			3.3 Establishment of Bioinformatic Pipeline
				3.3.1 Basecalling with Guppy Basecaller
				3.3.2 Concatenating FASTQ Files
				3.3.3 Generating Reference Data
				3.3.4 Mapping to Reference Genome with Minimap2
				3.3.5 SAM to BAM Conversion, Sorting, and Indexing
				3.3.6 Counting Features with FeatureCounts
				3.3.7 Data Frame Creation for Downstream Analysis
		4 Notes
		References
Part IV: Clinical Approach
	Chapter 31: Institutional Review Board Considerations for Clinical Trials
		1 Introduction of IRBs
		2 How to Apply to IRB
			2.1 Determine If Project Requires IRB Approval
			2.2 Complete IRB Research Project Application
			2.3 Prepare Informed Consent Document(s)
			2.4 Submit Proposal  Form
			2.5 Make Adjustments as Necessitated by IRB Review Until Approved
			2.6 Report Changes and Annual Renewal Authorization (If Needed)
		3 Notes
		Reference
	Chapter 32: Design an Intervention Study
		1 Introduction
		2 Methods
			2.1 Choice of Intervention and Control
			2.2 Selection of Subjects
			2.3 Informed Consent
			2.4 Baseline Measurement
			2.5 Bank Specimens
			2.6 Randomized Allocation
			2.7 Blinding
			2.8 Outcome Measurements
				2.8.1 Efficacy Outcomes
				2.8.2 Safety Outcomes
		3 Notes
		References
	Chapter 33: Assessment of Disease Activity, Structural Damage, and Function in Rheumatoid Arthritis
		1 Introduction
		2 Methods
			2.1 Assessment of Disease Activity
				2.1.1 Disease Activity Score in 28 Joints (DAS28) (See Note 1)
				2.1.2 Simplified Disease Activity Index (SDAI) (See Note 2)
				2.1.3 Clinical Disease Activity Index (CDAI) (See Note 3)
				2.1.4 American College of Rheumatology (ACR) Core Set
				2.1.5 2022 ACR/European Alliance of Associations for Rheumatology (EULAR) Remission Criteria
			2.2 Functional Assessment
				2.2.1 Health Assessment Questionnaire-Disability Index (HAQ-DI) (See Note 4)
				2.2.2 Short Form-36 (SF-36) (See Note 5)
				2.2.3 European Quality of Life-5 Dimensions (EQ-5D-5L) (See Note 6)
			2.3 Assessment of Structural Damage of Joints
				2.3.1 The van der Heijde Modification of the Sharp Method (the Sharp/van der Heijde Method)
				2.3.2 The Larsen Method
		3 Notes
		References
	Chapter 34: Assessment of Musculoskeletal Ultrasound of Rheumatoid Arthritis
		1 Introduction
		2 Methods
			2.1 MSUS Findings
				2.1.1 Synovitis
				2.1.2 Bone Erosion
				2.1.3 Tenosynovitis
			2.2 Positioning of MSUS in the Management of  RA
				2.2.1 Early Diagnosis
				2.2.2 Assessment of Disease Activity
				2.2.3 Definition of Remission
				2.2.4 Treatment Strategy Utilizing  MSUS
		3 Notes
		References
	Chapter 35: 16S rRNA Gene Amplicon Analysis of Human Gut Microbiota
		1 Introduction
		2 Materials
			2.1 Stool Sampling (See Note 1)
			2.2 DNA Isolation
			2.3 Library Preparation
			2.4 Sequencing
		3 Methods
			3.1 Stool Sampling
			3.2 DNA Isolation
			3.3 Library Preparation
			3.4 Sequencing
		4 Notes
		References
Index