Chapter
The Basal Cell Shrinkage Hypothesis
Novel Concepts and Future Directions
in Pemphigus Research
Chapter 2:
Patients, Sera,
and Experimental Models
Purification of Total IgG Fractions and Anti-Dsg3-L IgG
In Vitro Model of PV Using Keratinocyte Monolayers
In Vitro Models with Mouse Skin Organ Cultures
In Vivo Passive Transfer of Sera Using Neonatal Mice
Standard Immunofluorescence Microscopy and Histology
Evaluatiotion of Acantholysis in Cell Monolayers
Selection of Patients and Characterization of Sera
PV Serum Induces Specific Changes in KIF Arrangement
Detection of Acantholysis Induced by Concentrated PV Sera
through Dsg3 Staining
In Vitro Model of PV with Mouse Skin Cultures
In Vivo Model of PV with Passive Transfer of Sera in Neonatal
Mice
Chapter 3:
Optimization of the Experimental
Procedures: Semi-Quantitative
LCIF Microscopy, Cirillo’s HSU
Buffer, KAD Medium
Culture Conditions and In Vitro Models
Western Blot Analysis and Immunoprecipitation
In Vitro Model of PV Using Cultured Keratinocytes
Semiquantitative Living Cell Immunofluorescence (LCIF)
Microscopy
Optimization of Extraction Rate and Expression Levels of Dsgs
Cell-Cell Detachment and KIF Collapse Can Be Revealed by PV
Igg Staining
Time-Course of PV-Induced Acantholytic Dysmorphisms on Keratinocytes
Chapter 4:
Study of Anti-Desmoglein
Autoimmunity: Pemphigus
Vulgaris as a Desmoglein-
Remodeling Disease
Sera, Total IgG Fractions, and Purified Anti-Dsg3-L IgG
Cell Cultures and Treatments
Protein Extraction and Western Blot Analysis
Standard and Semiquantitative Living-CellImmunofluorescence (LCIF) Microscopy
Pemphigus Serum Induces Reduction of Dsg3 Half-Life
PV IgG do Not Induce an Immediate Disruption of AssembledDesmosomes
PV IgG-Bound Dsg3 is Not Early Internalized but Does NotForm Desmosomes
Anti-Dsg1 IgG-Containing Sera Induce Internalization of Dsg1but Not Its Early Depletion from Desmosomes
PF and PF-Like Autoantibodies Mobilize PrevalentlyNon-Clustered Dsg1
Changes in Dsg1 Synthesis in Response toAnti-Dsg1 Antibodies
Cleavage of Dsg3 in Keratinocytes Exposed to PV Serum
Depletion of Dsg3 and Its Localization during Acantholysisby Semiquantitative LCIF
Depletion of Dsg3 by PV Serum and IgG Fractions
High-Dose Anti-Dsg3-L Can Induce Acantholysis andReduction of Dsg3 Levels
Chapter 5: Role of Non-Desmoglein andNon-IgG Autoimmunity in PV
Preparation of IgG-Free Sera and Igg Purification
Cell Cultures, Treatments and siRNA transfections
Protein Extraction, Western Blotting, and Immunoprecipitation
Gel Purification of 130 kDa Bands
Immunofluorescence Microscopy
Metabolic activity Assay Using MTT
PV IgG Immunoprecipitate a PBMC 130 kDa Protein Other than Dsg3
The Novel 130 kDa Antigen Is Expressed on PBMC Surface
PV IgG-depleted Sera Induce Dramatic Morphological Changeson Keratinocytes
Effects of IgG-free PV Sera on Keratinocyte Viability and Cell-Cell Adhesion
PERK expression is altered in pemphigus vulgaris and isdependent on non-IgG factors
Chapter 6:
Downstream Signaling Involved
in Acantholysis: Part I: Changes
in Cell Cycle and Protein
Phosphorylation
Keratinocyte Culture Experiments
Protein Phosphorylation Assay
Immunofluorescence and Immunohistochemistry
Morphometric Analysis of Cell-Cell Detachment
PV Sera Affect Cell Phosphorylation Status
PV serum promotes activation of a number protein kinases in kerotinocytes
PV Sera Modulate Dose-Dependently cdk2 Expression butModestly Other Kinases
Silencing of cdk2 Prevents Cell-Cell Detachment Inducedby PV Sera
Chapter 7:
Downstream Signaling Involved
in Acantholysis: Part II:
Secretion of Proteinases
and the “Specific Proteolysis
Hypothesis” of Pemphigus
Keratinocyte Culture Experiments
Quantitation of Apoptosis
Western Blot Analysis and Immunofluorescence
MMP-9 Is over-Expressed in Keratinocyte Mololayers Exposedto PV Serum
MMP-9 Overexpression in the Organ-Culture Model of PV
Secretion of MMP9 in Keratinocyte Intercellular SpacesPrecedes Cell-Cell Detachment
Secretion of MMP-9 in Culture Supernatants
Processing of Dsg3 by Gelatinases
The Specific Proteolysis Hypothesis
Beyond Anti-Desmoglein Autoimmunity
Chapter 8:
Microarray Analysis of Diseased
Tissues: Cell Adhesion Is Down-
Regulated on the Gene
Level in Pemphigus
Passive Transfer of Sera in Neonatal Mice
Cell Cultures and Treatments
RNA Extraction, Microarrays Analysis, and RT-PCR
Modulation of Gene Expression by PV Sera in KeratinocytesDetected by DNA Microarray
Evidence that cdk2 Inhibition through Roscovitine Prevents
Acantholysis in the Neonatal Mouse Model of PV
Modulation of Gene Expression by PV Sera in Mouse Skin
Lesions Detected by DNA Microarray
Shared Microarray Results
Chapter 9:
Pharmacological Block
of Acantholysis: Towards Novel
Therapies for the Treatment
of Pemphigus
Passive Transfer of Sera in Neonatal Mice
Cell Cultures and Treatments
RNA Extraction, Microarrays Analysis, RT-PCR,and Ontology Assessment
Standard Immunofluorescence Microscopyand Histology
Inhibition of Phosphorylation Events PreventsPemphigus Acantholysis
Evidence that cdk2 Inhibition through Roscovitine PreventsAcantholysis in the Neonatal Mouse Model of PV
Cdk2 Partially Modulates Gene Expression by PV Sera inMouse Skin by DNA Microarray