Transcript Style A 36 by 48 tall - Loyola University Chicago

Proteomic Analysis of Ocular Discharges and Plasma From Patients with
Stevens Johnson Syndrome/Toxic Epidermal Necrolysis
Julia Malalis, Christine Mata, Daniel Kahn, Amy Lin, Michael Mosier, Charles Bouchard, Josephine Cunanan, Omer Iqbal, Debra Hoppensteadt and Jawed Fareed
Departments of Ophthalmology, Pathology and Surgery, Loyola University Health System, Maywood, IL 60153
Abstract
Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are life-threatening,
immune-complex hypersensitivity reactions that affect the skin and mucous membranes,
often resulting in significant ocular inflammation. The purpose of this study was to determine
and compare the proteomic and coagulation profile of plasma and mucosal discharge
samples in affected and unaffected patients.
Following institutional review board approval, plasma and swabs from ocular, oral, and skin
lesions were obtained from patients with clinically suspected SJS/TEN (n=9). Four patients
had biopsy-proven SJS/TEN. Five patients had alternative skin diagnoses not consistent with
SJS/TEN and were considered abnormal controls. Samples from normal healthy controls
were also obtained. Following centrifugation, the plasma was frozen at -70°C and later
thawed and analyzed to determine thrombin-antithrombin complex (TAT, Dade®, Marburg,
Germany), Prothrombin fragment (F1.2, Dade®), plasminogen activator inhibitor-1 (PAI-1,
Diagnostica Stago®, Asnieres Sur Seine, France), ZYMUPHEN platelet microparticle activity
(Hyphen® BioMed, Neuville-Sur Oise, France), HEMOCLOT protein C (Stago®), and
STACHROME antithrombin (ATIII, Stago®), using ELISA kits as per manufacturer’s
instructions. The swabs were immediately frozen at -70°C and later thawed. The discharges
were isolated following addition of 0.25 ml of saline to each swab and double centrifugation.
The discharges and plasma samples were analyzed using SELDI-TOF technique.
Methods
Coagulation Cascade
Hemostatic Alterations
SELDI TOF
Activation markers of thrombogenesis,
such as TAT and F1.2, can be measured
This instrument was used to profile the protein components
in ocular and other discharge fluids
PAI-1
(ng/ml)
Microparticles
(nM)
Protein C
ATIII %
Patient #1
5/25/2012
1.64
353.21
47.19
4.1
132.7
115.2
Patient #1
6/1/2012
2.53
625.1
55.64
26.1
142.4
110.3
Patient #1
6/8/2012
14.65
561.6
32.6
12.3
3.7
23.5
Patient #4
9/2/2012
6.2
181.71
77.65
9.7
83.2
73.9
MEAN
6.3
430.4
53.3
13.1
90.5
80.7
ST DEV
5.9
202.4
18.8
9.3
63.4
42.4
TAT (ng/ml)
F1.2
(pmol/L)
PAI-1
(ng/ml)
Microparticles
(nM)
Protein C
ATIII %
Patient #2
8/31/2012
7.74
486.67
125.52
15.5
18.4
108.5
Patient #3
9/14/2012
5.9
296.06
25.25
9.7
91
86.5
Patient #5
11/12/2012
3.39
146.77
69.02
69.4
8.8
12.4
MEAN
5.7
309.8
73.3
31.5
39.4
69.1
ST DEV
2.2
170.4
50.3
32.9
44.9
50.3
TAT (ng/ml)
F1.2
(pmol/L)
PAI-1
(ng/ml)
Microparticles
(nM)
Protein C
ATIII %
1.02
190.08
76.16
24.25
146.2
128.6
Results
Normal*
*represents replicate analysis of a normal pool plasma from healthy volunteers (n=8-10).
Procoagulant factors and unique peaks suggestive of protein upregulation of various
molecular weights may lead to the development of targeted therapy aimed to attenuate local
and systemic inflammatory processes in patients with SJS/TEN.
Date
STACLOT
Protein C
Time (sec)
STACLOT
% Protein C
Platelet Microparticle
5/25/2012
100.6
41.58%
86.64
6/1/2012
155.5
143.58%
34.15
6/8/2012
190.7
208.97%
13.80
Patient #3
129.2
94.72%
14.92
Patient #4
133.5
102.70%
87.20
Patient #5
88.7
19.48%
18.23
Patient #6
73.2
0.00%
28.21
Patient #7
139.8
114.41%
9.01
12/7/2012
143.0
120.35%
12.03
12/13/2012
126.2
89.14%
24.02
12/20/2012
143.2
120.73%
17.79
127.5
91.56%
6.55
Patient #1
SELDI-TOF spectra depicting the MW range of 3kDa-20kDa of a normal mucosal swab. This particular
sample exhibits peaks at 3.4, 5.3, 10.1, 11.3 and 14.7 kDa.
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are a spectrum of
rare and potentially fatal immune-complex hypersensitivity disorders triggered by infections or
medications. SJS and TEN are characterized by diffuse epidermal necrosis and are also two
of the most devastating ocular surface diseases that may lead to extensive corneal damage
and loss of vision.1,2,3
Although it is well established that epidermal and mucosal involvement in SJS and TEN
occurs through immune mediated, drug hypersensitivity reactions initiated by cytotoxic T
lymphocytes, the immunopathology of these diseases is not completely understood.3 Current
literature suggests separation of epidermis from dermis as a result of keratinocyte apoptosis.
Analysis of blister fluid of affected patients supports the idea that keratinocyte apoptosis
occurs through the Fas-FasL interaction and the release of perforin, granzyme B, and
granulysin.1,3,4
F1.2
(pmol/L)
BioRad Richmond, CA
Analyses of the SJS/TEN plasma samples revealed a marked increase in the TAT complexes
(6.3±5.9µg/ml), F1.2 (430.4±202.4 pmol/L), platelet microparticles (13.1±9.3nM) and
protein C levels (90.5±63.4 %), with a corresponding decrease in PAI-1 (53.3±18.8ng/ml)
and antithrombin levels (80.7±42.4 %) compared to normal control human plasma,
suggesting a procoagulant state. Protein chip array of the SJS/TEN skin and oral mucosal
samples exhibited multiple peaks suggestive of protein upregulation in SJS-positive patients.
These peaks were not present in the control group.
Purpose
TAT (ng/ml)
Patient #8
Patient #9
Conclusions
SELDI-TOF spectra depicting the MW range of 20kDa-150kDa of a normal mucosal swab. This particular
sample exhibits peaks at 22.8 kDa.
The molecular mediators in SJS/TEN, specifically TNF-a, can activate the coagulation
process and lead to a hypercoagulable state with microvascular thrombosis.5,6 Restoration of
hemostatic processes may help alleviate the ocular and dermatologic inflammatory
manifestations. Furthermore, we believe there might be a correlation between these
mediators and the clinical ocular surface disease. Finally, our study will provide valuable
information on the pathogenesis of these diseases which may lead to the development of
novel approaches to improve ocular surface morbidity.
SELDI-TOF spectra depicting the MW range of 3kDa-20kDa of a SJS positive mucosal swab. This
particular patient has peaks at 3.4, 4.1, 4.8, 7.5, 9.4, 10.2, 11.7, 15.1 and 15.8 kDa. There is an increase in
the total number of proteins/fragments within the sample due to excessive proteolysis in SJS.
Our study suggests that multiple mediators in SJS/TEN may activate the coagulation
process which may lead to a hypercoagulable state with microvascular thrombosis. The
alterations of such markers as TAT, F1.2, PAI-1, Microparticles, Protein C, and ATIII are
consistent with the levels observed in hypercoagulation. Restoration of hemostatic
processes may help improve the ocular and dermatologic inflammatory manifestations.
The proteomic analysis of the discharge fluids suggest an upregulation of proteolysis
resulting in the generation of various molecular weight components in the SJS positive
group, in contrast to the normals. However, ocular swabs did not reveal any discernible
differences. Additional studies are in progress to characterize these peaks. Future
studies, such as the one reported in this communication, will provide valuable information
on the pathogenesis of these diseases and lead to the development of novel approaches
to improve ocular surface morbidity.
Acknowledgements
The authors are grateful to The Richard A. Perritt Charitable Foundation and The Illinois
Society for the Prevention of Blindness for the partial funding for this project. We are also
thankful to Dean Linda Brubaker and Dr. Richard Kennedy for their support.
Key References
Left: Severe keratoconjunctivitis with pseudomembranes in acute SJS/TENS
Right: Lid margin keratinization in chronic SJS/TENS
SELDI-TOF spectra depicting the MW range of 20kDa-150kDa of a SJS positive mucosal swab. This
particular patient has peaks at 23.4, 25.6, 45.1, 51.5, 67, 79.5 and 138 kDa. When compared to a normal
sample, there is an increase in the total number of proteins/fragments within the sample due to excessive
proteolysis. The generated protein fragments require further characterization.
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2. Ueta M, Sotozono C. Association of Fas Ligand gene polymorphism with Stevens-Johnson syndrome. Br J Ophthalmol.
2008 Jul;92(7):989-991.
3. Lin A, Patel N. Management of Ocular Conditions in the Burn Unit: Thermal and Chemical Burns and Stevens-Johnson
Syndrome/Toxic Epidermal Necrolysis. J Burn Care Res. 2011 Sept;32(5):1-14.
4. Chung WH, Hung SI. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and
toxic epidermal necrolysis. Nat Med. 2008 Dec;14(12):1343-1350.
5. Levi M, ten Carte Hu, van der Poll T, van Deventer SJH. Pathogenesis of disseminated intravascular coagulation in
sepsis. JAMA. 270:975-979, 1993
6. Morrison DC, Ryan JL: Endotoxin and disease mechanisms. Annu Rev Med. 38:417-432, 1987.