Conjunctival gene expression profile by Mini-Oligo array for cytokines & fibrogenic factors in subjects with trichiasis

Lead Research Organisation: MRC Unit, The Gambia

Abstract

The development of vaccines and treatments for chlamydial disease and the worldwide burden of blindness and infertility it causes depend on a better understanding of the immunity to and immunopathology that infection causes. Protective immunity without harmful side effects is effective in most individuals. However, in a significant number of individuals, dependent on the prevalence of infection in the community, infection is asymptomatic and results in chronic disease. Traditionally prospects for a long lasting and affordable intervention in the form of the development of a chlamydial vaccine focus almost entirely on stimulating immunity which is effective at preventing or transmitting infection. Thus far after many years of research such a vaccination intervention is lacking. This approach seeks to identify not the elements necessarily involved in preventing infection but host factors which are involved in the chronic disease process. It should therefore be possible to design an invention or vaccination against the development of the chronic disease process that leads to scarring of the mucosal surfaces of either the conjunctiva genital tract. One way to begin research in this area is to identify which host genes are involved in the chronic disease process and this can be done by studying the affected tissue. The expression of many hundreds of genes can now be tested from very small amounts of biological material collected with minimal discomfort using a min-array and RNA isolated from swabs. Once the major genes and the biological pathways that they control have been identified current interventions which block these pathways can be tested for effectiveness to halt or prevent tissue damage. Or vaccination could be tuned to stimulate pathways which block or oppose the action of the harmful disease causing pathways rather than aiming at infection blocking pathways.

Technical Summary

Trachomatous Triachiasis is end result of severe conjunctival infections with C. trachomatis over many years. The underlying causes of disease are the immunologically driven responses which lead to conjunctival fibrosis. The relationship between the immune response and the fibrotic process is poorly defined in trachoma but in other infectious diseases the process is being unraveled. Understanding these processes offers the opportunity to develop an intervention which is based on blocking the development of pathology rather than one which focuses on sterilizing immunity which could itself lead to pathology. Using mini-Oligo DNA gene arrays for extracellular matrix proteins and adhesion molecules along with a cytokine array gives us the opportunity to test ~200 genes. By taking this focused approach we should identify the major genes and pathways involved in conjunctival fibrosis.||Methodology and Equipment: We will recruit subjects with trachomatous trichiasis (TT) and age, sex and location matched controls. From a sub-group of these subjects, 10 TT cases and 10 controls a conjunctival swab will be taken for the isolation of mRNA. All cases will be offered treatment under standard Gambian National Eye Care Programme (NECP) guidelines and protocols. TT surgery will conducted by local NECP community ophthalmic nurses. Arrays for Human Extracellular Matrix and adhesion molecules, Interleukins and Receptors and Th1/Th2/Th3 (GEArray Q superarray Series). Access to bioinformatic analysis software for reading and interpretation of the array is via the internet from Superarray.||Practical and Ethical considerations: Subjects with trichiasis will be referred for surgery conducted by trained NECP personnel. The presence of trachomatous scarring does not require specific treatment, but subjects are counselled according to NECP practice.||Results: Two low density gene arrays were used which covered 226 genes for extracellular matrix proteins/adhesion molecules and cytokines. This will be followed by quantitative gene expression by real-time RT-PCR on conjunctival RNA samples from a larger number of subjects with trichiasis and controls. Mini-gene array results identified that the transcription factor Jun B proto-oncogene (JunB) was up-regulated in the conjunctiva of TT subjects and implies a differential gene expression of downstream targets which this gene controls.||Conclusions: Interestingly, in JunB deficient mice, delayed wound healing and epidermal hyperproliferation is observed and in vitro experiments show that JunB is involved in the paracrine regulatory cytokine networks that control skin homeostasis and regeneration. The results of quantitative expression of JunB and a number of other genes known to be involved in other immune mediated fibrotic diseases (TNF, IL1?, IL-11, IL-11R?, IL-13 and IL-13R?2) will now be further investigated in immune mediated conjunctival scarring. Understanding the pathological process may lead to the possible application of a pharmacological intervention to the progression of conjunctival scarring.

Publications

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Description Programme grant WT093368MA
Amount £1,320,000 (GBP)
Funding ID WT093368MA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 09/2016
 
Title MMP7 
Description Elevated expression of MMP7 associated with fibrotic scarring in trachoma pathology 
Type Of Material Model of mechanisms or symptoms - human 
Provided To Others? No  
Impact Possible biomarker of advancing ocular fibrosis requires investigation in other populations 
 
Title Serum samples 
Description Serum samples from patients with severe conjunctival pathology after chlamydial infection 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact PMID: 19428908 recogintion by sera of proteins important in chlamydial entry into host cells 
URL http://europepmc.org/abstract/MED/19428908
 
Description Profiling the humoral immune response to C .trachomatis in trachoma using genome proteome arrays. 
Organisation University of Texas at San Antonio
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution Provison of clinical material and data interpretation
Collaborator Contribution Screened serum from trachoma patients against a mini expressino library and presently a full library with almost 50 cases and controls. identification of primary antibody target of the immune response associated with pathologyIdentification of new targets of the pathogenic response to chlamydia trachomatis
Impact PMID: 19428908
Start Year 2006
 
Description Profiling the humoral immune response to C .trachomatis in trachoma using genome proteome arrays. 
Organisation University of Texas at San Antonio
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution Provison of clinical material and data interpretation
Collaborator Contribution Screened serum from trachoma patients against a mini expressino library and presently a full library with almost 50 cases and controls. identification of primary antibody target of the immune response associated with pathologyIdentification of new targets of the pathogenic response to chlamydia trachomatis
Impact PMID: 19428908
Start Year 2006