keratoconus ( kc ) is usually a bilateral , progressive ectatic corneal disorder , usually appearing in puberty [ 13 ] .
the corneal stroma becomes thinner and protrudes , causing the typical conical shape that leads to irregular astigmatism , myopia , and the decrease of visual acuity [ 13 ] .
the apex of the protrusion can most commonly be found in inferotemporal orientation from the center of the cornea [ 1 , 2 ] .
the incidence of kc is around 1  :  2,000 in the general population .
however , recent studies have suggested that inflammatory factors play a key role in the pathomechanism of the disorder .
elevated levels of interleukin- ( il- ) 1b , il-4 , il-5 , il-6 , il-8 , and il-17 ; tumor necrosis factor ( tnf)- , - ; interferon- ( ifn- )  ; matrix metalloproteinase- ( mmp- ) 1 , mmp-3 , mmp-7 , mmp-9 , and mmp-13 ; cathepsin b ; and lipocalin-1 have been found in the tears of patients with keratoconus [ 411 ] . decreased levels of il-4 , il-10 , il-12 , and il-13 ; tnf- ; ifn- ; chemokine ( c - c motif ) ligand 5 ( ccl5/rantes , regulated on activation , normal t cell expressed and secreted ) ; lipophilin c ; lipophilin a ; lactoferrin ; -fibrinogen ; zinc-2-glycoprotein ( zag ) ; immunoglobulin a ( iga ) ; immunoglobulin -chain ( igkc ) ; polymeric immunoglobuline receptor ( pigr ) ; phospholipase a2 ; cystatin s ; cystatin sn and cystatin sa have also been found in the tear fluid [ 4 , 8 , 1014 ] .
kenney et al . have found a decreased level of tissue inhibitor of metalloproteinase-1 ( timp-1 ) in kc corneas , compared to normal corneas .
in addition , an association between kc and bronchial asthma has been identified almost 50 years earlier .
several studies have demonstrated a strong association between kc , asthma , and other immune disorders , pointing to the crucial role of the immune system in the pathogenesis of keratoconus [ 17 , 18 ] . in the past few decades , slit - imaging technologies provided further improvement in corneal imaging .
nowadays , we can measure not only the front but also the back surface of the cornea with pachymetric mapping and can typify corneal architecture in three dimensions . furthermore , ambrsio et al .
established numerous indices to improve the screening of the ectasia [ 19 , 20 ] .
there are only a few preliminary studies examining the association between mediators ( mainly cytokines ) in the tear fluid and the severity of keratoconus .
in a recent study , shetty et al .   examined the association between mmp-9 , il-6 , tnf- , and different stages of kc .
the crucial limitations of these studies [ 5 , 13 , 21 , 22 ] are the small number of patients , or the few examined mediators , or the lack of subclinical cases . in the present study ,
our goals were to determine associations between the different types of mediators in tear fluid 
il-6 , il-10 , chemokine ( c - x - c motif ) ligand 8 ( cxcl8)/il-8 , ccl5/rantes , mmp-9 , mmp-13 , timp-1 , tissue plasminogen activator ( t - pa ) , and plasminogen activator inhibitor ( pai-1)in the whole spectrum of keratoconic eyes ( suspect , subclinical , and manifest cases of keratoconus ) and normal eyes .
an additional goal was to explore associations between these mediators and the scheimpflug parameters which characterize the severity of keratoconus .
our aim was also to examine the relationship between the scheimpflug imaging parameters and bronchial asthma in keratoconus .
in this prospective study , patients with keratoconus and normal subjects were recruited from the outpatient unit , department of ophthalmology , faculty of medicine , university of debrecen , hungary .
we examined patients with keratoconus at all stages ( severe , moderate , and mild kc , subclinical kc or forme fruste kc ) and normal , control patients .
we have categorized the participants based on the clinical stage of keratoconus , but group allocation was not a factor in the analysis .
an eye was diagnosed as having keratoconus where it had one or a combination of the following clinical signs : central or paracentral stromal thinning of the cornea , conical protrusion , fleischer 's ring , vogt 's striae by slit - lamp examination , and topographic changes .
the stages of kc were divided between mild , if the steepest keratometric reading ( k2 ) was < 45 diopters ( d ) ; moderate , when k2 was between 45 and 52  d ; and severe if k2 was > 52  d [ 13 , 24 ] . at present , there are no specific or universally accepted criteria categorizing an eye as having subclinical kc or forme fruste kc ( ffkc ) .
the criteria for diagnosing subclinical kc or ffkc were defined as one or a combination of the following clinical signs : if it was the fellow eye of a patient with keratoconus and had a normal cornea on slit - lamp examination ( in several cases , keratoplasty or corneal cross - linking was performed on the keratoconic eye ) , or using a pentacam , the bad - d ( belin - ambrsio deviation index ) , art max ( maximum ambrsio 's relational thickness ) , and ppi ave ( average pachymetric progression index ) ; or where the back elevation values at the thinnest location ( b.ele.th . ) were not in the normal range of the pentacam ; or where the criteria of kc did not apply but the maximum keratometric reading of the front surface ( kmax front ) was more than 47  d. we included a randomly selected eye from each participant , although both eyes were examined .
eye selection was based on generating random values using microsoft excel set to produce numeric indicators with equal probabilities for either eye .
altogether , 69 patients ( mean ( sd ) age 30.7 ( 10.3 ) , range 1368 years ) with the following diagnoses  severe kc : 25 , moderate kc : 21 , mild kc : 5 and subclinical kc : 18and 19 normal controls ( mean ( sd ) age 31.7 ( 11.5 ) , range 1867 years ) were enrolled in the study .
the 69 eyes of the patients represented the whole spectrum of the abnormal , ectatic , and keratoconic corneas , although no such group allocation was made .
the exclusion criteria included the existence of active inflammatory or infective systemic or ocular disease , eye rubbing , and current treatment with systemic or local anti - inflammatory drugs .
those eyes that were affected by previous ocular surgery or trauma , as well as those patients who were pregnant or lactating during the course of the study , were also excluded . written informed consent from each participant , as well as permission from the university of debrecen institutional ethics committee , was obtained prior to enrollment .
the tenets of the declaration of helsinki were followed in all procedures during the study .
both eyes of each participant underwent ophthalmological evaluation , including clinical history ( especially bronchial asthma and contact lens usage ) , automated kerato - refractometry ( kr-8900 ; topcon co , tokyo , japan ) , uncorrected and corrected distance visual acuity determinations , slit - lamp biomicroscopy ( under low illumination to avoid reflex tearing ) , rotating scheimpflug tomography ( pentacam hr , oculus optikgerte gmbh , wetzlar , germany ) , and nonstimulated tear sample collection with glass capillaries .
all eyes were examined with a pentacam hr ( software version 1.16r26 and 1.17r139 ) without the application of any eye drops .
three sequential scans were taken in each eye by the same trained examiner . in short
, the patients were asked to keep both eyes open and fixate on the target , in the center of the blue fixation beam .
the examiner with the joystick then focuses and obtains a correct alignment with the corneal apex and a perfect focus , and the instrument automatically takes 25 scheimpflug images within two seconds .
the quality of images was checked under the quality specification ( qs ) window and only the correct measurements (  qs  reads  ok  ) were accepted ; if the comments were marked yellow or red , the examination was repeated [ 25 , 26 ] .
the following parameters were exported to microsoft excel ( microsoft corp , redmond , washington ) : holladay equivalent keratometry values in the flat ( k1 ) and steep ( k2 ) meridian ; maximal keratometry of the front surface ( kmax front ) ; corneal astigmatism ( astig ) ; corneal thickness at the pupil 's center ( pachy pupil ) , at the apex ( pachy apex ) and at the thinnest point of the cornea ( pachy min ) ; index of surface variation ( isv ) ; index of vertical asymmetry ( iva ) ; keratoconus index ( ki ) ; central keratoconus index ( cki ) ; index of height asymmetry ( iha ) ; index of height decentration ( ihd ) ; front and back elevation at the thinnest location ( f.ele.th . and b.ele.th . ) ; minimal , maximal , and average pachymetric progression indices ( ppi min , ppi max , and ppi ave ) ; ambrsio 's relational thickness ( art ) ; and belin - ambrsio deviation index ( bad - d ) .
nontraumatic tear collection was carried out using sterile thin glass microcapillary tubes from the inferior meniscus , without anesthetic drops or stimulation .
tears were collected for two minutes and then promptly transferred to eppendorf tubes and frozen at 80c without centrifugation , within 15 minutes of collection .
in tear samples , the concentrations of il-6 , il-10 , cxcl8/il-8 , ccl5/rantes , mmp-9 , mmp-13 , timp-1 , t - pa , and pai-1 were measured using the cytometric bead array method .
combined flowcytomix simplex kits were used with the suitable flowcytomix basic kit , with minor modifications to the manufacturer 's orders ( ebioscience , bender med systems gmbh , vienna , austria ) . briefly stated , 12.5  l of tear samples ( diluted samples , if necessary ) or serial dilutions of mixed standard cytokines were added to a 12.5  l suspension of fluorescent cytokine capture beads in multiwell filter microplates . added to the wells were 12.5 microliters of biotin - conjugated anti - cytokine antibodies , after which the plates were incubated for two hours on a microplate shaker .
phycoerythrin - conjugated streptavidin was added to the plate wells , followed by an additional incubation period of one hour and washed as described before .
a 150  l assay buffer was applied to the wells , after which multiparametric data acquisition was executed using a facs array cytometer ( bd biosciences immunocytometry systems , san jose , ca ) .
additional serial dilutions of the standard were applied to achieve better sensitivity and modified standard curves were thus generated in the analysis .
the subsequent detection limits were as follows : il-6 : 1.2  pg / ml ; il-10 : 1.9  pg / ml ; cxcl8 ( il-8 ) : 0.5  pg / ml ; ccl5 ( rantes ) : 25  pg / ml ; mmp-9 : 95  pg / ml ; mmp-13 : 50  pg / ml ; timp-1 : 28  pg / ml ; t - pa : 4.8  pg / ml ; and pai-1 : 13.5  pg / ml . we categorized the participants for descriptive purposes based on clinical severity ( group allocation was not used as a variable in the analysis ) .
mediator concentration variables were inspected for the distribution shape and natural log transformed ( for t - pa and pai-1 , square root transformed ) to improve normality .
similarly , pentacam parameters were subjected to one of these transformations if that improves distributional symmetry .
pentacam parameters were unified in a composite index referred to as the standardized pentacam score .
this was calculated by centering , standardizing , and direction correcting the source variables ( so that the higher values invariably represent more severe pathologies ) , running a principal component analysis and deriving the first principal component .
associations between all possible pairs of mediator levels , as well as those between mediators in the tear fluid and scheimpflug parameters , were evaluated using simple linear regression , including a quadratic term by a curvature in the relationship , if required .
associations between pairs of mediators and the standardized pentacam score were assessed using multiple linear regressions adjusted for age , the presence of asthma , and contact lens usage .
mediator variables were used in linear and quadratic forms , and interactions between those terms were also included in order to accommodate the model to curvatures in the outcome space .
relationships were expressed as the overall significance of the mediator pair effect and also as additive differences in the standardized pentacam score at sample - covered locations , defined by mediator pair concentrations versus an arbitrary reference point .
a number of significant positive associations were observed between pairs of mediator concentrations in tear fluid collected from kc patients and subjects with normal eyes , as shown in table 1 .
significant positive associations were found between bad - d and cxcl8 , bad - d and mmp-9 , and k2 and mmp-9 .
significant negative associations were found between pachy min and cxcl8 and pachy min and t - pa ( table 2 ) .
a significant association was found between the timp-1 concentration , the mmp-9 concentration , and the standardized pentacam score : the combination of high timp-1 and low mmp-13 levels was characterized by a low score , while high levels of both mediators  as well as low timp-1 concentrations coupled with moderate mmp-9 levels  were associated with an elevated score ( table 3 ) .
significant associations were also found between the concentrations of a number of other pairs of mediators and the standardized pentacam score as shown in table 4 . there was a history of asthma in five patients ( 7.25% ) and one of contact lens usage in 15 patients ( 21.74% ) .
a strong , significant positive association between asthma and the standardized pentacam score was found by linear regression adjusted for age and contact lens usage .
asthmatic patients ' scores were , on average , an estimated 5.7 units ( 95% ci : 2.0 to 9.4 , p = 0.003)or 1.32 standard deviations  higher than those of subjects without the condition .
to the best of our knowledge , this is the first study that aimed to reveal associations between pairs of mediators and the severity of keratoconus , evaluated using a pentacam . despite the intensive clinical and biochemical investigations , the pathogenesis of keratoconus is not yet known in detail .
classically , keratoconus was considered a noninflammatory disease ; however , recently published articles have suggested that inflammation is involved in the pathogenesis of kc [ 4 , 714 ] . in the tear fluid of keratoconic patients , elevated levels of il-6 , tnf- , and
mmp-9 were detected , and il-6 and tnf- levels were also elevated in subclinical cases [ 5 , 6 ] .
jun et al . found high level of il-6 and low levels of il-12 , tnf- , ifn- , il-4
, il-13 , and ccl5 in the tear fluid of kc patients . partly in line with these results , increased tear levels of mmp-1 , mmp-3 , mmp-7 , mmp-9 , and mmp-13 ; il-4 , il-5 , il-6 , and il-8 , and tnf- , - were found in keratoconus . in keratoconic corneas , a decrease in timp-1 mrna
only few studies investigated the associations between a range of cytokines in the tear fluid and the severity of keratoconus .
the limitations of these reports are the small number of patients , or the few examined mediators , or the lack of subclinical cases [ 5 , 13 , 21 , 22 ] . in the current study
, we have determined the associations between nine mediators ( il-6 , il-10 , cxcl8/il-8 , ccl5/rantes , mmp-9 , mmp-13 , timp-1 , t - pa , and pai-1 ) in the tear fluid of keratoconic patients covering the whole spectrum of the disease ( from subclinical to manifest keratoconus ) . in accordance with earlier studies
, we proved that different mediators  including cytokines , chemokines , enzymes , and inhibitors  in the tear fluid cooperate and take part in a complex immunological network [ 4 , 5 , 714 , 21 ] .
significant associations were explored between the concentrations of different mediators ( between il-6 and cxcl8 ; between ccl5 , cxcl8 , and mmp-9 ; between timp-1 , mmp-9 , mmp-13 , and t - pa ; and between t - pa , cxcl8 , ccl5 , and pai-1 ) and the standardized pentacam score , which is a composite index statistically unifying all pentacam parameters . as far as we know , there are no studies evaluating the association between different tear mediators and pachy min and also bad - d , which was designed to present comprehensive data based on anterior and posterior corneal elevation and a pachymetric evaluation .
our results support the linkage between the complex network of the various mediators and the comprehensive pentacam indices . based on our results , inflammation not only seems to be involved in the pathogenesis of keratoconus , but also plays a crucial role in the pathological corneal processes from the initial stage to the final one .
we have found only a few significant associations between the single mediators and pentacam indices , highlighting the fact that mediators , including cytokines , take part in a complex cascade .
it is in line with evidence showing that various multitargeted mediators in the serum collaborate in different diseases , suggesting that examination of only one or a few mediators is not enough to explore complex immunopathological processes [ 13 , 28 , 29 ] .
mmps are zinc - dependent endopeptidases , which participate in degrading and remodeling the extracellular matrix , thereby maintaining its integrity during normal conditions . nevertheless , under pathological conditions
timp-1 is the inhibitor of mmps , which prevents pro - mmp activation and , furthermore , presents antiapoptotic properties [ 15 , 30 , 31 ] .
mmp-13 was categorically reported in keratoconus , suggesting that it plays a role in intra- and extracellular pathological collagen destruction .
, we have found significant associations between timp-1 concentration , mmp-13 concentration , and the standardized pentacam score .
the combination of high timp-1 and low mmp-13 levels was characterized by a low score , while high levels of both mediators  as well as low timp-1 concentrations coupled with moderate mmp-9 levels  were associated with elevated scores .
additionally , significant positive associations were found between mmp-9 and bad - d , and also with k2 . based on our study and others ,
an active form of t - pa converts plasminogen to plasmin and can also degrade several components of the extracellular matrix , triggering the activation of the mmp pathway .
numerous interactions have been observed between the fibrinolytic and mmp systems taking part in proteolytic activation .
plasminogen activators are partially regulated by pais , inhibiting this cascade system and , therefore , influencing kc progression .
different growth factors and cytokines induce the pai-1 gene and inhibit the activity of the t - pa enzyme .
significant associations between the severity of keratoconus and pairs of t - pa / timp-1 and t - pa / pai were detected in our study .
in addition to this observation , significant negative associations were found between the corneal thickness at the thinnest point of the cornea and t - pa .
apart from the elements of the proteolytic and fibrinolytic systems , we have examined different cytokines , such as the proinflammatory il-6 and chemokine cxcl8/il-8 and the anti - inflammatory th2 cytokine il-10 ; we found that all of these cytokines cooperate with each other and play a crucial role in the pathogenesis of kc .
the significant positive association between bad - d and cxcl8 and the significant negative correlation between pachy min and cxcl8 highlight the roles of the chemokines .
the infiltration of eosinophils , mastocytes , and t lymphocytes and the release of several inflammatory mediators play an important role in asthma 's pathogenesis [ 36 , 37 ] .
different proteins are altered in the serum of these patients , such as ige ; il-1 , il-4 , il-6 , il-8 , and il-13 ; ccl5 ; tnf- ; mmp-9 ; timp-1 ; t - pa ; and serum angiopoietin-1 [ 36 , 3843 ] .
due to an increase in the number of known mediators , additional anti - inflammatory options are becoming available in the therapy of asthma . the connection between asthma and other atopic diseases with keratoconus was first published almost 50 years ago and has been confirmed several times since then [ 1618 ] .
we found a strong significant positive association between asthma and the severity of keratoconus , meaning that asthmatic patients have 5.7 higher score than nonasthmatic subjects .
based on our study , bronchial asthma has an impact on the severity of keratoconus .
this result albeit was a secondary outcome of our study and confirms the previous hypothesis related to the connection between kc and asthma .
the strengths of our study are the large number of participants ( 88 subjects ) and the consideration of a wide range of tear mediators that could be associated with pentacam parameters .
our study has limitations , such as the lack of examination of enzyme activities , as well as the fact that we did not examine the progression of keratoconus and the effect of asthma medication .
in addition , this study does not exclude the role of other inflammatory molecules in the pathophysiology of kc .
it would be interesting to measure more and different types of mediators , but this remains to be determined in subsequent studies .
however , several new correlations can be revealed from our results that can be the basis for further study of this topic .
keratoconus has a complex pathomechanism , in which many different cytokines , chemokines , enzymes , and inhibitors are involved .
this study reveals the cooperation of the different mediators in tear fluid all taking part in this complex immunological network .
as far as we know , this is the first study to reveal associations between tear mediators and bad - d or pachy min .
our study confirms that inflammation is involved in the pathogenesis of keratoconus . as a next step ,
the precise role of these mediators needs to be defined , as well as examination of the progression of kc and exploration of other mediators ' functions .
these studies might then serve as a platform for finding targets for local inhibition of pathological corneal thinning , or eventual treatment .
further prospective studies are required to examine the effect of the systemic treatment of asthma on the pathomechanism of keratoconus .
 
 purpose . to determine associations between mediators in tears in the whole spectrum of keratoconus ( kc ) ; to explore connections between mediators and scheimpflug parameters ; to examine correlations between scheimpflug parameters and bronchial asthma . methods . 
 tear samples were collected from 69 patients and 19 controls . 
 concentrations of mediators  il-6 , -10 ; cxcl8 , ccl5 ; mmp-9 , -13 ; timp-1 ; t - pa , pai-1were measured by cytometric bead array . measured pentacam parameters include keratometry values ( k1 , k2 , kmax ) , corneal thickness ( pachy pupil , apex , min ) , and elevations and indices ( including belin - ambrsio deviation ( bad - d ) ) . results . 
 a number of significant positive associations were observed between pairs of mediator concentrations . 
 significant positive correlations were found between bad - d and cxcl8/mmp-9 and k2 and mmp-9 . 
 significant negative associations were explored between pachy min and cxcl8/t - pa . 
 significant associations were found between pairs of mediators ( il-6 and cxcl8 ; ccl5 and cxcl8/mmp-9 ; timp-1 and mmp-9/-13/t - pa ; t - pa and cxcl8/ccl5/pai-1 ) and the severity of kc . 
 significant positive correlation between asthma and the severity of kc was explored . 
 conclusion . 
 cooperation of different mediators in tears all taking part in the complex pathomechanism of keratoconus was revealed . 
 our research verifies that inflammation plays a crucial role in the pathogenesis of kc . 
 additionally this study confirms the effect of bronchial asthma on keratoconus . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion
5. Conclusion