The document discusses the role of anti-DNA IgG in the pathogenesis of cutaneous lupus erythematosus (CLE). It describes experiments examining the interaction of anti-DNA IgG with keratinocytes and its effects. Skin tissue samples from CLE patients and healthy controls were obtained and anti-DNA IgG clones were cultured. Experiments found that anti-DNA IgG binds to keratinocytes and induces apoptosis. It also binds to novel antigens like collagen III and SOCS1. This binding may amplify TWEAK/Fn14 signals and modulate SOCS1, contributing to macrophage chemoattraction and skin lesions in CLE. The document aims to better understand the pathogenic effects of anti-DNA IgG in C

1. Andrea Mazo Cañola
III semester
2018

2. ■ LUPUS : lupus erythematosus (LE) Its an autoimmune
inflammatory disease that includes a large spectrum of
manifestations, cutaneous manifestations include:
• Malar eminences in a “butterfly” distribution
INTRODUCTION
Interactions between susceptibility
genes and environmental factors result
in abnormal immune responses.
1. Activation of innate
immunity
2. reduced of immune
complexes and
apoptotic cells.

3. INTRODUCTION

4. OBJECTIVE
Elucidate the role of anti-DNA IgG
in the pathogenesis of CLE (
cutaneous lupus erythematosus )

5. MATERIALS AND METHODS
 Tissue sample of patients
Fresh skin tissues
• Patients with CLE, who received neither.
pharmaceutical nor physical therapies
n=11.
• Healthy controls n=4.
• Sera : tissue donors and other patients
n=34.
 Murine Anti-DNA IgG
• Trypanosoma equiperdum derived
kinetoplast DNA = natural dsDNA
antigen that induces the generation of
Anti DNA IgG
• Anti DNA IgG clones = BALB c/mice
were immunized with a mixture of
kinetoplast DNA and incomplete
Freund’s adjuvant
Anti DNA IgG were labelled with
• Fluorescein isothiocynate (FITC)
• rhodamine

6. MATERIALS AND METHODS
 Cell culturing: Murine PAM212 keratinocytes were grown in
Dulbecco's modified Eagle’s medium.
stimulation assays: cells starved in 2% fetal calf serum before the
addition :
• murine anti DNA , isotope control or recombinant mouseTWEAK (
inductor of apoptosis)
 Apoptosis :The cells apoptosis was quantitated
using a caspasa tag caspase 3,7 Kit

7. MATERIALSAND METHODS
 FLOW CYTOMETRY AND INMUNOFLUORESENCE
1.Technique that allows detecting a cell population in a sample in which other cell
samples predominate Information of each one of the cells analyzed and the
relationship that exits between the parameters of one cell and another.
2. Antibody-antigen reaction + fluorescent dye to the antibody, exposed to an
ultraviolet light microscope to be evidenced.
• PAM212 Cells + murine anti DNA IgG or control and FITC conjugated goat anti-
mouse IgG
• Cells + FITC or rhodamine anti DNA-IgG
 Pretreated with DNase I

8. MATERIALSAND METHODS
 Enzyme-linked immunosorbent assay
( ELISA )
It’s a serological laboratory test , in witch we can
identify an antibody or an antigen using (antibody or
antigen) absorber in a solid surface .
■ Identify the reaction between antigen- antibody
= conjugated antigamma globulin+ enzyme =
color
■ Color = amount of antibodies or antigent
• Affinities of anti-DNA IgG to ssDNA and ds DNA
• Blinding of collagen III and Ro52
 WESTERN BLOT
It’s a laboratory method that uses antibodies to identify
proteins after the proteins have been separated by
electrophoresis in a gel according to the size.
• western blot to the proteins lysates ( cell culture)
• Cell lysates + control IgG (-)
• Cell lysates + anti SOCS1 IgG (+)
• Cell lysates + anti DNA IgG
• B-actin bands as a control

9. RESULTS
Fig 2.B Fig 2.C

10. RESULTS
Fig 3.A
Fig 3.C

11. RESULTS
Fig 5.A

12. RESULTS
Fig 6.B Fig 6.D

13. DISCUSSION
AUTHOR WHAT DIDTHEY SAID? YES/NO
• Y. Liang et al TWEAK/Fn14 signals mediate the
pathogenicity of anti-DNA IgG in CLE.
As a key regulator of cytokine
production, SOCS1 is dysregulated in
the damaged tissues of lupus er-
ythematosus.
YES
• Y. Xia et al They also found a specific
interaction between anti-DNA IgG
and other collagens, including
collagen IV and matrigel.
NO
• A. Reich et al
• T.D.Golan et al
UVB irradiation triggers
photosensitization and
exaggerates skin lesions in lupus
erythematosus through inducing
Ro52 expression.
YES
• O.J. Park et al Murine anti-DNA IgG increases
both Fn14 ex- pression and the
effect ofTWEAK on keratinocytes
by increasing apoptosis and
cytokine production.
YES

14. CONCLUSSIONS
1 Try to develop a way to decrease the effects of anti DNA IgG in patients with lupus, to
attenuate the symptoms.
2 Anti DNA IgG, could be a determinant factor at the moment of evaluate the clinic of the patient
with lupus, because a lot of manifestations are caused by it .
 Anti-DNA IgG exists in skin lesions of lupus erythematosus and recognizes
novel antigens of collagen III and SOCS1, blinding to keratinocytes and induces
macrophage chemoattraction which may be related to the amplification of
TWEAK/Fn14 signals and the modulation of the SOCS1.

15. MAP