The Pathogenesis of Superior Limbic Keratoconjunctivitis

• ClinicalTrials.gov Identifier: NCT00167050
• Recruitment Status: Unknown
• First Posted: September 14, 2005
• Last Update Posted: November 28, 2005
• Sponsor: National Taiwan University Hospital
• Information provided by: National Taiwan University Hospital

Study Description

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.

Condition or disease
Superior Limbic Keratoconjunctivitis

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 oligo(dT) (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, 12.5 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 Design

• Study Type: Observational
• Enrollment: 10 participants
• Observational Model: Defined Population
• Time Perspective: Other
• Study Start Date: July 2005
• Study Completion Date: June 2006

Groups and Cohorts

Outcome Measures

Eligibility Criteria

• Ages Eligible for Study: 20 Years to 80 Years (Adult, Older Adult)
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: Yes

Criteria

Inclusion Criteria:

• Superior limbic keratoconjunctivitis patients who receive conjunctiva resection

Exclusion Criteria:

-

Contacts and Locations

Contacts

Contact: Fung-Rong Hu, MD; 886-2-23123456 ext 5189; fungrong@ha.mc.ntu.edu.tw

Contact: Yi-Chen Sun, MD; 886-968195019; yichen.sun@msa.hinet.net

Locations

Taiwan

National Taiwan University Hospital; Recruiting

Taipei, Taiwan

Contact: Fung-Rong Hu, MD 886-2-23123456 ext 5189 fungrong@ha.mc.ntu.edu.tw

Contact: Yi-Chen Sun, MD 886-968195019 yichen.sun@msa.hinet.net

Principal Investigator: Fung-Rong Hu, MD

Sponsors and Collaborators

National Taiwan University Hospital

Investigators

• Principal Investigator: Fung-Rong Hu, MD; National Taiwan University Hospital

More Information

Publications automatically indexed to this study by ClinicalTrials.gov Identifier (NCT Number):

Sun YC, Hsiao CH, Chen WL, Hu FR. Overexpression of matrix metalloproteinase-1 (MMP-1) and MMP-3 in superior limbic keratoconjunctivitis. Invest Ophthalmol Vis Sci. 2011 Jun 1;52(6):3701-5. doi: 10.1167/iovs.10-6324.

Sun YC, Hsiao CH, Chen WL, Wang IJ, Hou YC, Hu FR. Conjunctival resection combined with tenon layer excision and the involvement of mast cells in superior limbic keratoconjunctivitis. Am J Ophthalmol. 2008 Mar;145(3):445-452. doi: 10.1016/j.ajo.2007.10.025. Epub 2008 Jan 16.

• ClinicalTrials.gov Identifier: NCT00167050
• Other Study ID Numbers: 9461700634
• First Posted: September 14, 2005
• Last Update Posted: November 28, 2005
• Last Verified: June 2005

Additional relevant MeSH terms:

Keratoconjunctivitis; Conjunctivitis; Conjunctival Diseases; Eye Diseases; Keratitis; Corneal Diseases