Ignite Creation Date:
2024-05-05 @ 11:54 AM
Last Modification Date:
2024-10-26 @ 9:15 AM
Study NCT ID:
NCT00167050
Status:
UNKNOWN
Last Update Posted:
2005-11-28
First Post:
2005-09-12
Brief Title:
The Pathogenesis of Superior Limbic Keratoconjunctivitis
Sponsor:
National Taiwan University Hospital
Organization:
National Taiwan University Hospital
Study Overview
Official Title:
None
Status:
UNKNOWN
Status Verified Date:
2005-06
Last Known Status:
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:
Superior limbic keratoconjunctivitis SLK was first described in detail as a clinical entity by Frederick Theodore in 1963 The clinical picture of SLK is well documented but the etiology is still unknown This project will be conducted into through two parts one is to investigate the presentation of chemokine receptors on mast cell and matrix metalloproteinases on fibroblasts by immunohistochemistry method from the pathological specimens of SLK patients who received conjunctiva resection as the treatment The other part is to investigate the mRNA level of those chemokine receptors via reverse transcription - polymerase chain reaction from the conjunctiva collecting form SLK patients
Detailed Description:
1 Specimens collection
We will collect the specimens in two different parts One part is to collect all the paraffinized block of the SLK patients to get fifteen copies of 5-μm section of each paraffinized block from the Department of Pathology All the slides will be deparaffinized and rehydration for further immunohistochemistry IHC stain Primary antibodies for IHC stain include chemokine receptors CXCR1 CXCR2 CXCR3 CXCR4 CCR1 CCR3 CCR4 CCR5 CD30L and matrix metalloproteinases MMP-1 -2 -3 -9
Another part of this study is to collect the conjunctiva of SLK patients and normal control patients This part of the clinical study is sent for IRB approval concomitantly We will collect the conjunctiva of SLK patients when they receive conjunctival resection as the treatment in the following one year And the normal control conjunctiva will be obtained while the patients come to our hospital for cataract surgery or retinal surgery with redundant conjunctiva noted after peritomy The fresh conjunctiva will be stored in -80 before RNA isolation All the tissue will be processed into RNA and reverse transcripted into cDNA Polymerase chain reaction will be done with the primers including CXCR1 CXCR2 CXCR3 CXCR4 CCR1 CCR3 CCR4 CCR5 and CD30L
2 Preparation of RNA and cDNA
Total RNA will be extracted from the conjunctiva of SLK and control groups with Trizol reagent Life Gaithersburg MD USA One microgram of total RNA from each sample will be annealed for 5 min at 70 with 500 ng oligodT Fermentas Hanover MD USA and reverse transcribed to cDNA by 200u RevertAid M-MuLV Reverse Transcriptase Fermentas Hanover MD USA per 20 µl reaction for 1 hr at 42 The reaction will be stopped by heating for 10 min at 70
3 Polymerase chain reaction PCR
PCR will be performed on the resultant cDNA from each sample with specific primers for human chemokines chemokine receptors and glyceraldehydes-3-phosphate dehydrogenase GAPDH The amplification will be performed with a thermocycler The 25-μl reaction mixture consists of 2 μl cDNA 1 μl sense and antisense primer 125 μl 2x PCR mix Conditions for amplifying each chemokine and chemokine receptor are as follows denaturation 1 min at 94 and elongation 3 min at 72 The annealing temperature and the cycle of annealing for chemokines and chemokine receptors will fit the requested temperature and the appropriate number of annealing cycles of each specific primer At the end of amplification the reaction mixture will be heated for 10 min at 72 and then cooled to 4 A 10-μl sample of each PCR product will be separated by performing gel electrophoresis on 2 agarose containing ethidium bromide Sigma St Louis MO USA The 2 agarose gel will be analyzed under ultraviolet light against the DNA molecular length markers The internal control of each sample is human GAPDH
4 Immunohistocytometry IHC
A pre-determined optimal dilution of the primary antibody specifically against mast cell tryptase associated chemokines chemokine receptors MMPs and inflammatory cytokines will be used for IHC All IHC staining will be performed at room temperature Sections are then deparaffinized in xylene and made hydrophilic by immersion in a graded series of ethanol dilutions 100 95 80 and 60 All slides are immersed in antigen retrieval solution Dako S3307 SA USA and autoclaved for 10 min Sections are quenched with 3 fresh H2O2 for 10 min to inhibit endogenous tissue peroxidase activity that might interfere with the result of staining and rinsed with 1x PBS for 5 min twice Sections are further incubated in blocking serum solution for 30 min and then incubated at 37 for 1 h with primary antibody in a humid chamber After washing with 1x PBS for 5 min twice the sections are then incubated with biotinylated secondary antibody solution for 30 min The horseradish peroxidase-conjugated streptavidin-biotin complex is subsequently spread evenly over the sections and incubated for 30 min The sections are washed with 1x PBS for 5 min twice then incubated with diaminobenzidine DAB perioxidase substrate solution for 15 min followed by wash with 1x PBS Finally the section are counterstained with an adequate volume of hematoxylin and then mounted
We will collect the specimens in two different parts One part is to collect all the paraffinized block of the SLK patients to get fifteen copies of 5-μm section of each paraffinized block from the Department of Pathology All the slides will be deparaffinized and rehydration for further immunohistochemistry IHC stain Primary antibodies for IHC stain include chemokine receptors CXCR1 CXCR2 CXCR3 CXCR4 CCR1 CCR3 CCR4 CCR5 CD30L and matrix metalloproteinases MMP-1 -2 -3 -9
Another part of this study is to collect the conjunctiva of SLK patients and normal control patients This part of the clinical study is sent for IRB approval concomitantly We will collect the conjunctiva of SLK patients when they receive conjunctival resection as the treatment in the following one year And the normal control conjunctiva will be obtained while the patients come to our hospital for cataract surgery or retinal surgery with redundant conjunctiva noted after peritomy The fresh conjunctiva will be stored in -80 before RNA isolation All the tissue will be processed into RNA and reverse transcripted into cDNA Polymerase chain reaction will be done with the primers including CXCR1 CXCR2 CXCR3 CXCR4 CCR1 CCR3 CCR4 CCR5 and CD30L
2 Preparation of RNA and cDNA
Total RNA will be extracted from the conjunctiva of SLK and control groups with Trizol reagent Life Gaithersburg MD USA One microgram of total RNA from each sample will be annealed for 5 min at 70 with 500 ng oligodT Fermentas Hanover MD USA and reverse transcribed to cDNA by 200u RevertAid M-MuLV Reverse Transcriptase Fermentas Hanover MD USA per 20 µl reaction for 1 hr at 42 The reaction will be stopped by heating for 10 min at 70
3 Polymerase chain reaction PCR
PCR will be performed on the resultant cDNA from each sample with specific primers for human chemokines chemokine receptors and glyceraldehydes-3-phosphate dehydrogenase GAPDH The amplification will be performed with a thermocycler The 25-μl reaction mixture consists of 2 μl cDNA 1 μl sense and antisense primer 125 μl 2x PCR mix Conditions for amplifying each chemokine and chemokine receptor are as follows denaturation 1 min at 94 and elongation 3 min at 72 The annealing temperature and the cycle of annealing for chemokines and chemokine receptors will fit the requested temperature and the appropriate number of annealing cycles of each specific primer At the end of amplification the reaction mixture will be heated for 10 min at 72 and then cooled to 4 A 10-μl sample of each PCR product will be separated by performing gel electrophoresis on 2 agarose containing ethidium bromide Sigma St Louis MO USA The 2 agarose gel will be analyzed under ultraviolet light against the DNA molecular length markers The internal control of each sample is human GAPDH
4 Immunohistocytometry IHC
A pre-determined optimal dilution of the primary antibody specifically against mast cell tryptase associated chemokines chemokine receptors MMPs and inflammatory cytokines will be used for IHC All IHC staining will be performed at room temperature Sections are then deparaffinized in xylene and made hydrophilic by immersion in a graded series of ethanol dilutions 100 95 80 and 60 All slides are immersed in antigen retrieval solution Dako S3307 SA USA and autoclaved for 10 min Sections are quenched with 3 fresh H2O2 for 10 min to inhibit endogenous tissue peroxidase activity that might interfere with the result of staining and rinsed with 1x PBS for 5 min twice Sections are further incubated in blocking serum solution for 30 min and then incubated at 37 for 1 h with primary antibody in a humid chamber After washing with 1x PBS for 5 min twice the sections are then incubated with biotinylated secondary antibody solution for 30 min The horseradish peroxidase-conjugated streptavidin-biotin complex is subsequently spread evenly over the sections and incubated for 30 min The sections are washed with 1x PBS for 5 min twice then incubated with diaminobenzidine DAB perioxidase substrate solution for 15 min followed by wash with 1x PBS Finally the section are counterstained with an adequate volume of hematoxylin and then mounted
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