Ignite Creation Date:
2024-05-06 @ 2:56 AM
Last Modification Date:
2024-10-26 @ 11:25 AM
Study NCT ID:
NCT02160327
Status:
UNKNOWN
Last Update Posted:
2014-06-24
First Post:
2014-05-27
Brief Title:
The Role of Cytokines and Mast Cell in the Pathogenesis of SLK Conjunctivochalasis and Dry Eye
Sponsor:
National Taiwan University Hospital
Organization:
National Taiwan University Hospital
Study Overview
Official Title:
The Role of Cytokines and Mast Cell in the Pathogenesis of Ocular Surface Inflammation Diseases Including Superior Limbic Keratoconjunctivitis Conjunctivochalasis and Dry Eye
Status:
UNKNOWN
Status Verified Date:
2014-06
Last Known Status:
ACTIVE_NOT_RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The specific aims of the the investigators studies are as follows
To collect the tear samples from patients with different ocular surface disorders including SLK conjunctivochalasis and keratoconjunctivitis sicca KCS
To evaluate the differential expression of tear cytokines and pH values between different ocular surface disorders
To collect the surgical conjunctival specimens from the patients with SLK and conjunctivochalasis
To evaluate the factors inducing mast cell migration and how mast cell is activated in SLK via surgical specimens and cultivated fibroblast
To collect the tear samples from patients with different ocular surface disorders including SLK conjunctivochalasis and keratoconjunctivitis sicca KCS
To evaluate the differential expression of tear cytokines and pH values between different ocular surface disorders
To collect the surgical conjunctival specimens from the patients with SLK and conjunctivochalasis
To evaluate the factors inducing mast cell migration and how mast cell is activated in SLK via surgical specimens and cultivated fibroblast
Detailed Description:
1 Detection of various tear cytokine levels in patients with superior limbic keratoconjunctivitis conjunctivochalasis and keratoconjunctivitis sicca
- Basal tear samples will be collected atraumatically from the inferolateral tear meniscus by using glass capillary tubes Care is taken to avoid touching the corneal and conjunctival surface Approximately 30 μl of tear samples is obtained and then the samples are centrifuged for 10 minutes at 1500 rpm 225g Tear sample are placed in microtubes Eppendorf and stored at -70C IL-1β IL-17 TNF-α and IFN-γ levels will be measured by enzyme-linked immunosorbent assay kits
2 Immunohistochemistry stain method for conjunctival specimens - The conjunctiva will be collected from SLK and conjunctivochalasis patients who received superior and inferior bulbar conjunctival resection as needed The redundant conjunctiva noted after peritomy during cataract or retinal surgery will be excised to serve as a normal control The conjunctival specimens of all the SLK conjunctivochalasis and control groups are sent to the pathology department for routine paraffin embedding and hematoxylin and eosin staining The surgical specimens are placed into 4 paraformaldehyde at 4 C for one to two days and then stored in 1 sodium phosphate buffer at 4 C They then are embedded in paraffin To enhance tissue adhesion 5-micrometer thick sections are mounted on glass slides pre- treated with Vectabond Vector Laboratories Burlingame California USA The paraffin sections are deparaffinized rehydrated and washed with phosphate-buffered saline PBS pH 74 Antigen retrieval is performed by immerse the sections in 01 trypsin in water bath for 15 mins The endogenous peroxidase activity is quenched by placing the slides in 3 hydrogen peroxide The slides are then blocked with serum followed by incubation with primary antibodies Primary antibodies TSL and SCF are incubated overnight at 4C Thereafter incubation with biotin conjugated secondary antibody is performed at room temperature for 60 min Further incubation with avidin-biotin horseradish peroxidase complex is then performed at room temperature as well for 30 min using Vectastain elite ABC kit Vector labs CA USA The location where the enzyme is bound will be visualized by the addition of the substrate 33-diaminobenzidine DAKO CA USA a chromogen which produces a brown insoluble precipitate The sections are then counterstained with haematoxylin which is followed by dehydration and mounting with Vecta Mount Mounting medium was used to evaluate the slides
- Basal tear samples will be collected atraumatically from the inferolateral tear meniscus by using glass capillary tubes Care is taken to avoid touching the corneal and conjunctival surface Approximately 30 μl of tear samples is obtained and then the samples are centrifuged for 10 minutes at 1500 rpm 225g Tear sample are placed in microtubes Eppendorf and stored at -70C IL-1β IL-17 TNF-α and IFN-γ levels will be measured by enzyme-linked immunosorbent assay kits
2 Immunohistochemistry stain method for conjunctival specimens - The conjunctiva will be collected from SLK and conjunctivochalasis patients who received superior and inferior bulbar conjunctival resection as needed The redundant conjunctiva noted after peritomy during cataract or retinal surgery will be excised to serve as a normal control The conjunctival specimens of all the SLK conjunctivochalasis and control groups are sent to the pathology department for routine paraffin embedding and hematoxylin and eosin staining The surgical specimens are placed into 4 paraformaldehyde at 4 C for one to two days and then stored in 1 sodium phosphate buffer at 4 C They then are embedded in paraffin To enhance tissue adhesion 5-micrometer thick sections are mounted on glass slides pre- treated with Vectabond Vector Laboratories Burlingame California USA The paraffin sections are deparaffinized rehydrated and washed with phosphate-buffered saline PBS pH 74 Antigen retrieval is performed by immerse the sections in 01 trypsin in water bath for 15 mins The endogenous peroxidase activity is quenched by placing the slides in 3 hydrogen peroxide The slides are then blocked with serum followed by incubation with primary antibodies Primary antibodies TSL and SCF are incubated overnight at 4C Thereafter incubation with biotin conjugated secondary antibody is performed at room temperature for 60 min Further incubation with avidin-biotin horseradish peroxidase complex is then performed at room temperature as well for 30 min using Vectastain elite ABC kit Vector labs CA USA The location where the enzyme is bound will be visualized by the addition of the substrate 33-diaminobenzidine DAKO CA USA a chromogen which produces a brown insoluble precipitate The sections are then counterstained with haematoxylin which is followed by dehydration and mounting with Vecta Mount Mounting medium was used to evaluate the slides
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None