Cornea 194 492496 2000
`
`2000 Lippincott Williams Wilkins Inc Philadelphia
`
`Tnterleukin-6 Levels in the Conjunctival Epithelium
`of Patients with Dry Eye Disease Treated with
`Cyclosporine Ophthalmic Emulsion
`
`Pflugfelder M.D Zhonghua Ji M.D
`Kathleen Turner D.V.M Stephen
`William Feuer M.S Michael Stern PhD and Brenda
`Reis PhD
`
`Purpose To evaluate interleukin-6 IL-6 levels in the conjuncti
`val epithelium of patients with moderate to severe dry eye disease
`ophthalmic emul
`before and after
`treatment with cyclosporin
`sion CsA or its vehicle Methods Conjunctival cytology speci
`6-month
`mens were obtained from subset of patients enrolled in
`trial of the efficacy and safety
`randomized double-masked
`clinical
`months of BID
`of topical CsA at baseline and after
`and
`13 0.1% cy
`emulsion
`treatment with 0.05% cyclosporine
`10 RNA was
`emulsion
`or vehicle
`closporine
`extracted and
`competitive reverse transcriptase polymerase chain
`reaction RT-PCR was used to evaluate the levels of mRNA en
`IL-6 and
`coding the inflammatory cytokine
`housekeeping
`gene
`G3PDH Levels of IL-6 and G3PDH were measured
`and com
`pared Results There was no change from baseline in the level of
`G3PDH after
`months
`in any group IL-6 normalized for
`or
`G3PDH IL-6/G3PDH ratio was not different
`from baseline at
`months
`but showed
`significant decrease from baseline
`in the
`group treated with 0.05% CsA
`months No
`0.048 at
`differences were noted and no correla
`significant between-group
`tion was observed between the change in IL-6/G3PDH and corneal
`staining Conclusions This preliminary small-cohort
`fluorescein
`study showed
`decrease in IL-6 in the conjunctival
`epithelium of
`moderate to severe dry eye patients treated with 0.05% CsA for
`months The observed decrease suggests that dry eye disease in
`volves immune-mediated inflammatory processes that may be de
`creased by treatment with topical ophthalmic cyclosporine
`Key Words ConjunctivaCyclosporin ADry eye disease
`
`Interleukin-6-Keratoconjunctivitis
`
`sicca
`
`Specific evidence of chronic immune activation of the conjunc
`tival epithelium in patients with dry eye suggests
`that cell-
`inflammatory processes may play an important role in
`mediated
`the pathogenesis of dry eye disease These immunopathologic
`of immunologic adhesion
`increased expression
`findings include
`i.e HLA class II and ICAM-1 antigens and inflam
`molecules
`
`Submitted September 13 1999 Revision received January
`cepted January 15 2000
`From the Bascom Palmer Eye Institute K.T S.C.P Z.J W.J.F De
`partment of Ophthalmology University of Miami School of Medicine
`Miami Florida and Allergan Inc MS B.L.R Irvine California
`U.S.A
`Supported by Allergan Inc 2525 Dupont Drive Irvine CA 927 13-9534
`U.S.A
`
`2000 Ac
`
`Address
`
`conespondence
`
`requests to S.C Pflugfelder M.D
`Bascom Palmer Eye Institute 900 NW 17th Street Miami FL 33136
`U.S.A
`
`and reprint
`
`492
`
`in these pa
`IL-6 IL-8 and TNF-ci
`IL-i
`matory cytokines
`tients.57 Among the inflammatory cytokines
`evaluated
`levels of
`interleukin-6 IL-6 showed the greatest elevation in eyes with dry
`eye disease when compared to normal eyes.6
`Further evidence that
`immune-mediated inflammatory processes
`are involved in the pathogenesis of dry eye disease comes from
`topical use of the immunomodulatory agent cyclospor
`reports that
`inc can improve the signs and symptoms of dry eye.8 However
`it has not been determined whether
`the use of topical cyclosporine
`can cause measurable decreases in any of the inflammatory mark
`ers that are associated with dry eye disease
`The purpose of this study was to evaluate the level of the in
`of patients with kera
`flammatory cytokine IL-6 in the conjunctiva
`
`toconjunctivitis sicca enrolled in
`
`ent concentrations
`
`and
`
`randomized trial of two differ
`ophthalmic emulsion CsA
`of cyclosporin
`castor oil emulsion vehicle IL-6 was chosen because pre
`the level of IL-6 mRNA is elevated
`vious studies have shown that
`epithelium of patients with Sjogrens syndrome
`in the conjunctival
`keratoconjunctivitis sicca.56 Furthermore the level of IL-6 mRNA
`was found to decrease in bronchoalveolar
`lavage cells obtained
`rejection who were treated
`
`from patients with acute lung transplant
`
`with aerosolized cyclosporine
`
`PATIENTS AND METHODS
`
`Patients
`
`Evaluation
`
`of the level of IL-6 mRNA in the conjunctiva was
`performed on conjunctival cytology specimens from subset of
`double-masked
`vehicle-
`patients enrolled in
`prospective
`trial of topical cyclosporine emul
`controlled multi-center clinical
`sion CsA for the treatment of dry eye disease.9 Adult patients of
`either sex were eligible for participation if
`they presented with
`diagnosis of moderate to severe dry eye disease as defined by the
`following criteria
`
`Schirmer test without anesthesia of
`
`mmJS minutes in at
`mmJS
`
`staining of
`
`least one eye If Schirmer test without anesthesia
`minutes then Schirmer with nasal stimulation had to be
`mm/S minutes in the same eye
`sum of corneal and interpalpebral conjunctival
`in the same eye where corneal staining was
`baseline Ocular Surface Disease Index OSDI score of 0.1
`with no more than three responses of not applicable and
`Facial Expression Scale
`on the Subjective
`score of
`The OSDI
`
`is
`
`questionnaire
`
`consisting of 12 questions each
`
`FAMY CARE - EXHIBIT 1014-0001
`
`

`
`CONJUNCTIVAL IL-6 IN DRY EYE DISEASE
`
`493
`
`Baseline
`
`Month
`
`Month
`
`Baseline
`
`Month
`
`Month
`
`433 PDH
`1O-
`
`IL6
`1O-
`
`G3PDH
`IO-
`
`JUt
`
`1ff5
`
`G3PDH
`1O
`
`IL-6
`1O
`
`G3PDH
`
`10-s
`
`IL-6
`o-
`
`G3PDH
`1O
`
`IL-6
`IO
`
`G3PDH
`1O
`
`IL-6
`1O
`
`aniol/gi
`
`amol/pi amol4Ll amot4tl amol/pi
`
`amoI4tI
`
`amollpl
`
`amol/pi amoL4fl amol/pi
`
`amoI4tl
`
`amol/jil
`
`21
`
`71
`
`Ethidium bromide-stained
`
`FIG
`Initial PCIR was performed using high and low concen
`agarose gel
`trations of mimics for G3PDH i0- and 10 attomole/pL and IL-6 i0- and 10
`attomole/pL The
`percentage of product G3PDH or IL-6 per total amplified product gene mimic in the lane is given
`the bottom amol
`attomole
`
`at
`
`rated from
`
`none of the time to
`
`evaluation of the impact of
`
`all of the time for the
`patients dry eye disease on his or her
`The overall score was calculated
`by
`vision-related functioning
`dividing the sum of the responses for all questions answered by the
`score and then multiplying by 100 Thus overall
`total possible
`no disability to 100
`scores ranged from
`
`complete disabil
`
`puncta within
`
`if
`
`punctal plugs during the study permanent occlusion of lacrimal
`months of the study or
`they were pregnant
`pregnancy Patients were also excluded if
`lactating or planning
`they appeared to have end stage lacrimal gland disease Schirmer
`nmil5 minutes or if
`their dry
`reading with nasal stimulation of
`eye disease was secondary to the destruction of conjunctival goblet
`cells or scarring Any patient who no longer met the criteria for
`moderate to severe dry eye as defined above after completing the
`run-in phase was excluded from enrollment
`2-week
`in the treat
`ment phase of the study
`Conjunctival epithelial samples were obtained at baseline at
`months and at months from four participating sites in the United
`States Informed consent was obtained from all patients before the
`study The study complied with the Tenets of the Declaration
`of
`by im
`Helsinki Conjunctival epithelial samples were obtained
`nitrocellulose membrane
`pression debridement with
`from the
`of the worse eye The worse eye
`temporal bulbar conjunctiva
`as the eye with the worse Schirmer tear
`was defined
`and the worse sum of corneal and interpalpe
`without anesthesia
`bral conjunctival staining If both eyes were comparable then the
`right eye was used The membranes were placed in Trizol RNA
`lysis buffer Life Technologies Rockville MD U.S.A and im
`mediately frozen at 80
`Samples were shipped in dry ice by
`commercial overnight delivery service to the central
`laboratory at
`the University of Miami School
`of Medicine Upon arrival
`samples were placed in 80
`the RNA was ex
`freezer until
`
`test value
`
`tracted
`
`Cornea Vol 19 No
`
`2000
`
`artificial
`
`visits if
`
`ity
`To be eligible for enrollment signs and symptoms of dry eye
`despite conventional management
`disease must have presented
`which may have included
`tear drops gels and ointments
`sympathomimetic agents parasympathomimetic agents and punc
`they were deemed
`tal occlusion Eligible patients were enrolled if
`capable of following the study protocol and were considered likely
`to complete the treatment period and to return for all scheduled
`they had normal
`lid position and closure and if
`they had
`retinopathy study vi
`best-corrected
`early treatment of diabetic
`sual acuity score of 0.7 logmar or better
`in each eye
`Patients were excluded from the study if
`they had used systemic
`or topical ophthalmic cyclosporine within 90 days before
`the
`study Other exclusion criteria included
`the presence or history of
`any systemic or ocular disorder or condition including
`ocular
`surgery trauma and disease that could possibly interfere with the
`interpretation of the study results current or recent use of topical
`that could affect
`ophthalmic or systemic medications
`dry eye
`condition known hypersensitivity to any component of the study
`lens wear during the
`or procedural medications required contact
`month or anticipated
`
`study recent within
`
`use of
`
`temporary
`
`FAMY CARE - EXHIBIT 1014-0002
`
`

`
`494
`
`TURNER ET AL
`
`Baseline
`
`Month
`
`Month
`
`O3PDH
`
`1L-6
`
`G3PDH
`
`1L6
`
`G3PDH
`
`TL-6
`
`cDNA- Marker
`
`FIG
`IRNA
`final gels for each patient
`Example of
`sample G3PDH primers were reacted with 1O at
`tomole/pL mimic and IL-6 with 1O attomole/pL
`mimic The upper band in each lane is the amplified
`product and the lower band is the amplified mimic
`are G3PDH and the G3PDH mimic
`Lanes
`and
`and
`are IL-6 and the IL-6 mimic Lanes
`and
`are month
`are baseline lanes
`is cDNA-PCIR
`and
`are month
`lanes
`lane
`reaction mixture without cDNA and lane
`is the
`DNA marker
`IX Boehringer
`IN
`Indianapolis
`U.S.A.
`
`lanes
`and
`
`CTG GAT TGC AGA GTT
`downstream primer sequence is
`AAG TTT
`constitutively expressed gene in all
`Cyclophilin is
`human cells that serves as the cyclosporine-binding protein.3 The
`PCR products were separated by
`.6% agarose gel electrophoresis
`and the intensity of the band was visually graded from no band
`strong cDNA samples that yielded
`bands were
`or
`used for competitive reverse transcriptase polymerase chath reaction
`RT-PCR analysis of IL-6 and G3PDH housekeeping gene levels
`cDNA samples that yielded
`bands were resynthesized
`or
`
`to
`
`Semiquantitative Analysis of mRNA Levels by
`Competitive RT-PCR
`The relative levels of RNA transcripts encoding the inflamma
`protein G3PDH were
`IL-6 or the housekeeping
`competitive PCR technique This technique was
`pL of sample cDNA to the PCR mixture
`performed by adding
`pL of primers specific
`described above containing
`for IL-6 or
`
`tory cytokine
`evaluated by
`
`using
`
`RNA Extraction and cDNA Synthesis
`RNA was extracted
`from the conjunctival
`epithelial samples
`previously reported protocol.5 Complementary DNA
`cDNA was synthesized with
`first strand cDNA synthesis kit
`pg of total RNA
`Boehringer Indianapolis IN U.S.A using
`Reverse transcriptase
`negative controls were performed for each
`RNA sample The efficiency of cDNA synthesis was assessed by
`evaluating the level of cyclophilin mRNA in each sample by poly
`merase chain reaction PCR amplification using 35 cycles We
`pL of cDNA to the PCR reaction mixture containing
`added
`pL
`pL deoxy-nucleotide triphosphates 10
`of lOx reaction buffer
`mmolJL deoxyadenosine
`triphosphate 10 mmolJL deoxycytidine
`triphosphate 10 mmol/L deoxyguanosine
`triphosphate and
`10
`mmolJL deoxythymidine triphosphate
`39.7 pL sterile water 0.3
`pL Taq DNA polymerase
`units/pL and
`pL of primers spe
`The upstream cyclophilin primer sequence
`cific for cyclophilin.2
`ATG GTT AAC CCC ACC GTG TTC GAC
`and
`the
`
`is
`
`TABLE
`
`Average percent of product
`
`G3PDH in lane
`
`Mimic 10
`
`attomole
`
`Mimic 10
`
`attomole
`
`lL-6 in lane
`
`Mimic 10
`
`attomole
`
`Mimic 10
`
`attomole
`
`Cyclosporine
`dose
`
`Vehicle
`
`Mo
`
`Mean
`
`47
`
`Std
`dev
`
`20
`
`Mean
`
`74
`
`Std
`dev
`
`19
`
`value
`
`0.002
`
`Mean
`
`21
`
`Std
`dev
`
`13
`
`18
`
`Mean
`
`41
`
`29
`
`Std
`dev
`
`27
`
`24
`
`value
`
`0.008
`
`0.005
`
`0.05%
`
`0.10%
`
`31
`
`32
`46
`
`27
`
`53
`37
`
`29
`
`24
`
`33
`
`22
`22
`
`22
`
`18
`20
`
`25
`
`17
`
`48
`
`70
`70
`
`46
`
`77
`57
`
`35
`
`40
`
`43
`
`28
`32
`
`43
`
`16
`31
`
`38
`
`22
`
`0.051
`
`0.001
`0.005
`
`0.026
`
`0.003
`0.012
`
`0.388
`
`0.025
`
`17
`
`34
`
`26
`
`34
`12
`
`16
`
`12
`
`26
`
`22
`
`27
`15
`
`19
`
`16
`
`61
`
`41
`
`53
`37
`
`31
`
`32
`
`10
`30
`
`34
`
`28
`17
`
`25
`
`26
`
`0.091
`0.005
`
`0.081
`
`0.004
`0.002
`
`0.003
`
`0.009
`
`Cornea Vol 19 No
`
`2000
`
`FAMY CARE - EXHIBIT 1014-0003
`
`

`
`CONJUNCTIVAL IL-6 IN DRY EYE DISEASE
`
`TABLE
`
`Change Th percent G3PDH from base/The
`
`Group
`
`Vehicle
`0.05% GsA
`0.10% GsA
`
`3-mo
`mean
`
`12
`15
`4.4
`
`SD
`
`31
`
`33
`
`27
`
`value
`
`0.4
`
`0.2
`
`0.6
`
`Range
`
`61 to 17
`72 to 28
`45 to 47
`
`6-mo
`mean
`
`13
`
`3.7
`
`12
`
`SD
`
`25
`
`27
`
`33
`
`value
`
`0.14
`
`0.6
`
`0.3
`
`495
`
`Range
`1521
`3864
`5535
`
`G3PDH.6 Both the IL-6 and G3PDH PCR reactions were run for
`35 cycles The primer sequences for IL-6 and G3PDH were chosen
`the PCR product was due to
`an intron to confirm that
`to span
`the am
`amplification of cDNA not genomic DNA The size of
`plified IL-6 cDNA product was 627 base pairs and the size of the
`amplified G3PDH cDNA product was 983 base pairs Each reac
`tion tube used to amplify the IL-6 cDNA was spiked with
`spe
`cifically designed mimic template Clontech Palo Alto CA
`U.S.A that contained DNA sequences that were complimentary
`to the IL-6 PCR primers These primer-specific sequences were
`nonspecific DNA fragment in
`and
`ends of
`ligated to the
`the v-erbB gene The mimic templates
`this case
`portion of
`compete with the IL-6-specific cDNA sequences in the conjunc
`tival epithelial specimens for the reaction primers and they have
`length in base pairs after PCR amplification than the
`different
`cytokine amplification product The added mimics also served as
`tube Two concentrations
`in each
`internal controls
`reaction
`of
`
`for
`
`mimic 10 and 106 attomole/pL
`were used for each sample
`PCR reactions
`for G3PDH were also spiked with two concentra
`tions 10 and i04 attomole/pL
`of mimic with G3PDH
`sequences PCR reactions
`IL-6 and G3PDH
`primer-specific
`month
`and month
`were performed on baseline month
`samples simultaneously for each subject The levels of PCR prod
`by 1.6% agarose gel electrophoresis Gels
`ucts were evaluated
`were stained with ethidium bromide 10 mg/mL and were photo
`Polaroid camera Cambridge MA U.S.A. Base
`graphed with
`for IL-6 and G3PDH
`PCR products
`line month
`and month
`and their mimics from each subject were run together on one
`Two final gels one for the lower and the other
`agarose gel Fig
`for the higher mimic concentration were run for each subject Fig
`The mimic concentration
`bands for IL-6
`yielding measurable
`and G3PDH that allowed
`between at
`comparisons
`or baseline to month
`points baseline to month
`was used for the final analysis
`month
`Photographs of each gel were scanned with
`
`least
`
`two time
`
`or month
`
`to
`
`Hewlett Packard
`
`expressed as
`
`ScanJet Palo Alto CA U.S.A. The integrated optical density of
`the IL-6 G3PDH and their mimic bands in the scanned images
`was measured Levels of IL-6 and G3PDH in each
`lane were
`in each lane e.g
`fraction of total amplified product
`IL-6/
`IL-6 mimic Levels of
`the percentage of IL-6
`G3PDH in each treatment group were compared over
`time to de
`the amount of G3PDH changed significantly with
`termine whether
`CsA treatment Levels of IL-6 were evaluated at each
`time point
`in relation to the level of G3PDH
`baseline month
`and month
`in each sample by dividing the percentage of IL-6 by the G3PHD
`normalized IL-6
`
`Statistical Analysis
`test was used to confirm that IL-6
`two-tailed paired Student
`and G3PDH as
`the product plus mimic total
`percentage of
`decreased with sample dilution for each group at each
`of G3PDH and IL-6 changes
`period Within group comparisons
`were performed with the two-tailed paired Student
`test Changes
`in the normalized IL-6 percentage from baseline to 3- and 6-month
`for each
`subject and between-group
`follow-up were calculated
`in the extent of change were examined with one-way
`analysis of variance
`
`differences
`
`follow-up
`
`RESULTS
`
`tive internal control
`
`Mimic as
`Competitive Internal Control
`Mimics were added to the PCR reactions to act both as
`for the PCR reaction
`and as
`template used for calculating
`the percentage of amplified cytokine
`or housekeeping protein To confirm the competitive nature of the
`mimic the percentage of IL-6 and G3PDH in each lane was com
`low mimic concentrations
`For each
`pared using high and
`ment group and time point the percentage of IL-6 and G3PDH
`of added mimic decreased Table
`increased as the concentration
`
`posi
`
`competitive
`
`treat
`
`TABLE
`
`Change Th %IL-6/%G3PDH ratio from base/The
`
`GsA 0.05%
`
`GsA 0.1%
`
`Vehicle
`
`Pvaluea
`
`Dayob
`
`1.1441.005
`
`n14
`
`Change from baseline
`Month
`0.036
`0.643
`n10
`0.862
`
`Within
`
`Month
`
`Within
`
`0.626
`1.025
`n13
`0.048
`
`1.401 1.198
`n12
`
`0.8460.663
`
`n10
`
`0.095
`
`0.384
`
`1.430
`n10
`0.839
`
`1.248
`
`n8
`
`0.413
`
`2.269
`
`5.524
`
`n7
`
`0.319
`
`0.460
`0.749
`n10
`0084d
`
`0.438
`
`0.221
`
`0.853
`
`values from one-way analysis of variance
`Among-group
`IL-6 to G3PDH
`bValues are mimic corrected ratios of
`value from paired-t-test
`Within-group
`Note CsA
`cyclosporine ophthalmic emulsion Values shown are
`mean
`decrease
`standard deviation
`negative value indicates
`from baseline
`
`Changes from Baseline
`There were no significant differences between G3PDH levels at
`This demon
`months
`in any treatment group Table
`or
`strates that the levels of the housekeeping protein G3PDH are not
`changed by CsA treatment
`The levels of IL-6 were then normalized to the G3PDH levels to
`in IL-6 mRNA levels At
`identify treatment-related
`changes
`months posttreatment
`significant decrease in the ratio of the
`percentage of IL-6 to G3PDH was observed in the group treated
`with 0.05% CsA but not
`in the vehicle or 0.10% CsA treatment
`The ratio of the percentage of IL-6 to G3PDH was
`groups Table
`not significantly different from baselthe at month
`in any treatment
`group There were no significant
`differences in the between-group
`ratios of the percentage of IL-6 to the percentage of G3PDH
`
`Cornea Vol 19 No
`
`2000
`
`FAMY CARE - EXHIBIT 1014-0004
`
`

`
`496
`
`TURNER ET AL
`
`DISCUSSION
`
`REFERENCES
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`disease Ophthalmology 2000 1076319
`Silverman MH
`10 Laibovitz RA Solch
`OConnell
`Andriano
`Pilot trial of cyclosporine 1% ophthalmic ointment in the treatment of
`keratoconjunctivitis sicca Cornea 1993 1231523
`11 lacono AT Smaldone GC Keenan RJ et al Dose-related
`reversal of
`acute lung rejection by aerosolized cyclosporine Am Respir Crit
`
`Care Med 1997
`human T-cell cyclophilin EMBO 1987
`Mihatsch MJ Foxwell
`13 Ryffel
`Woerly
`BM Distribution of
`the cyclosporin binding protein cyclophilin in
`human tissues immunology 199272399404
`14 Kunert KS Tisdale AS Stern ME Smith JA Gipson 1K Effect of
`cyclosporine ophthalmic emulsion on the number of
`inflammatory
`cells and goblet cells in conjunctival biopsies of patients with dry eye
`invest Ophthalmol Vis Sci 19994045771
`Saito
`Takeuchi
`Fujihara
`Conjunctival epithelium
`of HLA-DR in dry eye patients Ophthalmologica 1999
`
`12 Haendler
`
`Hofer-Warbinek
`
`Hofer
`
`Complementary DNA for
`
`Greiner
`
`Haendler
`
`abstract
`
`15 Tsubota
`
`expression
`2131619
`16 Fleiss JL The design and analysis of clinical experiments New York
`Wiley 1986
`17 Wood LC Stadler
`of cytokines
`expression
`
`Liou
`et al Banier disruption increases gene
`and the SSkD TNF receptor
`in murine skin
`
`Exp Dermatol 1997
`Feingold KR Thorfeldt CR Elias PM Optimization of
`18 Man MQ
`invest Dermatol 1996
`lipid mixtures for banier repair
`physiological
`1061096101
`
`The most important
`
`finding of the present study was that nor
`malized IL-6 levels significantly decreased from baseline after
`months of treatment with 0.05% CsA whereas the decreases seen
`treatment with either 0.1% CsA or vehicle were not statisti
`after
`cally significant The decrease in this inflammatory factor by cy
`closporine is consistent with the results of another study conducted
`taken from separate cohort of patients
`on conjunctival biopsies
`trial Kunert et al.4
`that participated
`in this multi-centered clinical
`significant decrease in the immune
`demonstrated
`there was
`that
`activation markers HLA-DR and CD1 la after
`months of treat
`ment with 0.05% CsA whereas these same markers increased in
`the vehicle group Other studies have demonstrated
`that
`increased
`levels of IL-6 and other
`inflammatory markers are associated
`with dry eye disease.575 For example
`study by Pflugfelder et
`al.6 used the same assay as the present study to demonstrate
`and TNF-1 RNA found
`the levels of IL-hi IL-6 IL-8 TNF-ci
`in the conjunctival epithelium of Sjogren syndrome patients were
`significantly greater than those found in control patients However
`the present study and
`study performed on this patient
`the other
`
`that
`
`population4 are the first
`
`to demonstrate that
`
`treatment
`
`for dry eye disease cyclosporin
`decrease several markers of inflammation
`
`topical ophthalmic
`can significantly
`
`In the present study no statistically significant between-group
`differences were seen in IL-6 levels Thus it
`is not possible to rule
`to the mean as the source of the IL-6 decrease.6
`out regression
`However
`more likely explanation may be
`large therapeutic
`effect observed in the vehicle group Disruption of epidermal bar
`inflammatory
`function results
`in increased levels of several
`cytokines including TNF-ci IL-i and IL-6 in the epidermis.7
`
`rier
`
`effect of the oil-in-water vehicle of this cyclosporine
`therapeutic
`emulsion might be expected
`as topical application of certain lipid
`mixtures can accelerate epidermal barrier recovery
`barrier insults in mice8
`very small amount of RNA was obtained from the samples
`for this study Consequently
`highly sensitive competi
`provided
`tive RT-PCR assay was used to evaluate the levels of IL-6 and
`constituently expressed gene G3PDH The assay was designed to
`these mRNAs in the
`determine
`the relative amount of each of
`competitive template mimic that was
`sample when compared to
`the PCR reaction
`added to
`each of
`tubes The validity of
`the levels of G3PDH were
`that
`approach is supported by the fact
`the study medication or the vehicle
`
`after defined
`
`this
`
`not affected by either
`In conclusion the findings of the present study demonstrate that
`topical CsA decreases the levels of IL-6 an inflammatory cytokine
`that is upregulated in the conjunctiva of patients suffering from dry
`eye disease
`
`Cornea Vol 19 No
`
`2000
`
`FAMY CARE - EXHIBIT 1014-0005