inflammatory markers in tears & cxl

TEAR INFLAMMATORY MARKERS IN TEAR INFLAMMATORY MARKERS IN KERATOCONUS; POSSIBLE MODIFICATION KERATOCONUS; POSSIBLE MODIFICATION

AFTER CORNEAL CROSS-LINKINGAFTER CORNEAL CROSS-LINKING1st Symposium1st Symposium

RESEARCH IN OCULAR INFLAMMATIONRESEARCH IN OCULAR INFLAMMATIONBilbao, 22 January 2016Bilbao, 22 January 2016

Jose I. Recalde Jose I. Recalde Arantxa Acera Arantxa Acera Juan A. DuranJuan A. Duran

HYPOTHESISHYPOTHESIS

• Keratoconus has been classically defined as a Keratoconus has been classically defined as a progressive, non-inflammatory progressive, non-inflammatory

condition, condition, which produces a thinning and steepening of which produces a thinning and steepening of the cornea.the cornea.

• Recent studies Recent studies have shown a significant role of proteolytic enzymes, cytokines, have shown a significant role of proteolytic enzymes, cytokines,

and free radicals; therefore, although keratoconus does not meet all the classic and free radicals; therefore, although keratoconus does not meet all the classic

criteria for an inflammatory disease, the lack of inflammation has been criteria for an inflammatory disease, the lack of inflammation has been

questioned.questioned.

RESEARCH IN OCULAR INFLAMMATION Bilbao, 22 January 2016

HYPOTHESISHYPOTHESIS


The inadequate balance between pro-inflammatory cytokinespro-inflammatory cytokines, proteolitic enzymesproteolitic enzymes, and protease protease inhibitors, inflammatory modulators, and antioxidants inhibitors, inflammatory modulators, and antioxidants may lead to an altered corneal structure and function in keratoconus, triggering a vicious circle between oxidative stress, keratocyte apoptosis, and increased activity of metalloproteinases.

OBJECTIVESOBJECTIVES

1.1. To determine the expression of three inflammatory markers in tears of To determine the expression of three inflammatory markers in tears of patients with keratoconus.patients with keratoconus.

– METALLOPROTEINASE 9 (MMP 9)METALLOPROTEINASE 9 (MMP 9)– CST 4 (CYSTATIN-S) CST 4 (CYSTATIN-S) – S 100 A6 (CALCYCLIN) S 100 A6 (CALCYCLIN)

2.2. To ascertain wether treatment of corneal cross-linking with UVA radiation and To ascertain wether treatment of corneal cross-linking with UVA radiation and riboflavin (CXL) could have any influence over the expression of these riboflavin (CXL) could have any influence over the expression of these mediators over the medium and long term. mediators over the medium and long term.


STUDY DESIGNSTUDY DESIGN

• Prospective observational study.Prospective observational study.– Analysis of the expression of three inflamatory markers in tears of Analysis of the expression of three inflamatory markers in tears of

keratoconus patients after CXL ( up to 1 year follow up ) .keratoconus patients after CXL ( up to 1 year follow up ) .– Correlation with several clinical parameters of Ocular Surface Disease Correlation with several clinical parameters of Ocular Surface Disease

( OSD).( OSD).

• Demographics:Demographics:– 30 keratoconus eyes ( patients pending CXL ).30 keratoconus eyes ( patients pending CXL ).– 10 normal eyes ( control group ).10 normal eyes ( control group ).


MMP 9 MMP 9 (Metalloproteinase 9)(Metalloproteinase 9)

Description and functionDescription and function• Cleaved by MMP3 into its active Cleaved by MMP3 into its active

form of 83 kDa.form of 83 kDa.• Cleaves the precursor of IL-1B in its Cleaves the precursor of IL-1B in its

active form.active form.• Involved in extracelluar matrix Involved in extracelluar matrix

degradation.degradation.• Plays a central role in inflammation.Plays a central role in inflammation.• Inhibited by alpha2-macroglobulin Inhibited by alpha2-macroglobulin

and TIMPsand TIMPs

Biological ProcessesBiological Processes• Extracellular matrix disassembly.Extracellular matrix disassembly.• Regulation of apoptotic process.Regulation of apoptotic process.• Wound healing.Wound healing.• Inflammatory process.Inflammatory process.• Produces tissue damage and is a Produces tissue damage and is a

diagnostic indicator of OSD.diagnostic indicator of OSD.• Overexpressed in tears in Overexpressed in tears in

keratoconuskeratoconus


CST4 ( Cystatin-S )CST4 ( Cystatin-S )

Description and functionDescription and function

• Belongs to the cystatin family.Belongs to the cystatin family.• Protein of 14 kDa.Protein of 14 kDa.• Highly expressed in saliva, tears and Highly expressed in saliva, tears and

urine.urine.• Antibacterial and antiviral activity.Antibacterial and antiviral activity.• Not directly related with Not directly related with

inflammationinflammation• Acts as protease inhibitor.Acts as protease inhibitor.

Biological ProcessesBiological Processes

• Protection against microbial Protection against microbial infections.infections.

• Tear marker for human dry eye and Tear marker for human dry eye and blefaritis.blefaritis.

• Decreased concentration in tears in Decreased concentration in tears in keratoconus.keratoconus.


S 100 A6 ( Calcyclin )S 100 A6 ( Calcyclin )

Description and functionDescription and function

• Member of S100 protein family Member of S100 protein family ( calcium-binding ).( calcium-binding ).

• Small acidic proteins ( 10-12 kDa ).Small acidic proteins ( 10-12 kDa ).• Involved in regulation of cell cycle Involved in regulation of cell cycle

progression and differentiation.progression and differentiation.• Uniformly expressed in normal Uniformly expressed in normal

ocular surface cells.ocular surface cells.

Biological ProcessesBiological Processes

• Regulation of fibroblast Regulation of fibroblast proliferation proliferation

• Apoptosis.Apoptosis.• Oxidative Stress.Oxidative Stress.• Elevation of S 100A 6 level in Elevation of S 100A 6 level in

tears suggests damage of the tears suggests damage of the ocular surface.ocular surface.

• Tear marker for human dry eye.Tear marker for human dry eye.


INTERVENTIONINTERVENTION

• Epithelium-off cross-linking technique.Epithelium-off cross-linking technique.– Accelerated Cross-Linking.Accelerated Cross-Linking.– Pulsed light.Pulsed light.– 8 minutes´irradiation time.8 minutes´irradiation time.– Delivered energy , 8 j/cmDelivered energy , 8 j/cm22


AVEDRO KXLAVEDRO KXL®® SYSTEM SYSTEM

METHODSMETHODS


-80ºC

Visit 1 Visit 2 Visit 3 Visit 4

pre-CXL 3 months 6 months 12 months

BLAUBRAND intraMARK micropipetes 10 µl(Brand Gmbh, Wertheim, Germany)

METHODSMETHODS


It is not possible to analyze the three proteins at the same time using conventional ELISA, because the volume of the tear samples is not sufficient.

There are commercially available multiplexed arrays to detect interleukines, metalloproteinases and other proteins ( Q-Plex™, Quansis Biosciences, Utah, USA ).

There is no standard multiplexed array that includes our three proteins.

A specific custom made microarray has been developed for this study by Bioftalmik SL, Derio, Bizkaia, Spain .

METHODSMETHODS


Microarray scheme for the multiplexed detection and quantification of

proteins S100A6, CST4 and MMP9.

NaCl A C A+C A+B+CStd

t = 0h

t = 4h

t = 8h

t = 24h

RESULTSRESULTS


We are still collecting and analyzing data so we cannot show the results until we have completed the whole analysis.

Preliminary tests have been satisfactory for MMP 9 and CST4, but unfortunately there is still lack of sensitivity for S 100 A6 ( Calcyclin ).

At BIOFTALMIK they are working hard on this subject and we hope that in a few months we will be able to perform accurate and reliable analysis of all three inflammatory markers in the tear samples.

Evaluation of Surgical OutcomesEvaluation of Surgical Outcomes

• Function:Function:– Best Corrected visual acuity (BCVA).Best Corrected visual acuity (BCVA).– Ocular Surface disease Index (OSDI).Ocular Surface disease Index (OSDI).

• Ocular Surface:Ocular Surface:– Osmolarity (Tear Lab®).Osmolarity (Tear Lab®).– Ocular Surface Disease Index (OSDI).Ocular Surface Disease Index (OSDI).– Tear Break-Up Time (BUT).Tear Break-Up Time (BUT).– Schirmer Test.Schirmer Test.– Lacrimal Function IndexLacrimal Function Index

• Topographic Features:Topographic Features:– Maximum keratometry (K max). Maximum keratometry (K max). – Simulated keratometry in the central 3 mm (Sim K).Simulated keratometry in the central 3 mm (Sim K).– Pachimetry.Pachimetry.


Functional OutcomesFunctional Outcomes


Functional OutcomesFunctional Outcomes


8,65 pt

Control group Keratoconus

Ocular Surface EvaluationOcular Surface Evaluation



Control 299,5




Control 13,5




Control 18,3



Depending on Schirmer test and tear clearance> 74 = Good Ocular Surface< 74 = Less good OS

Topographic FeaturesTopographic Features



Thank you for your kind attention

inflammatory markers in tears & cxl

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