OSU Optometry

An OLERF proposal was submitted in 2009 to fund collection of pilot data for the VIP CHIP (Vision In Preschoolers: Correction of Hyperopia In Preschoolers) Cross-sectional Pilot Study (NIH ARRA Challenge grant proposal submitted in 2009). A budget of $14,976 was requested and $8,036 was funded.

The primary goal of the VIP CHIP Cross-sectional pilot study was to demonstrate the feasibility of the educational testing as a primary outcome measure for the planned VIP CHIP randomized clinical trial to evaluate the effect of correction for hyperopia. Recent legislative initiatives at the state level have increased the number of states requiring preschool vision screening or examination prior to school entry. More preschool children than ever before are going to be receiving eye examinations, either as a result of having failed a vision screening, or as a result of increased public awareness of the importance of vision care for young children. Significant refractive error, such as hyperopia is frequently associated with referral on preschool vision screening tests and refractive error is the most frequent and easily corrected vision disorder. The decision of whether to prescribe eyeglasses for preschool children with hyperopia can be challenging, because children with vision problems often do not complain about their eyes or vision because they do not know their experiences are abnormal; but some research suggests that uncorrected refractive errors may interfere with the child’s ability to learn and develop optimally. The following is an overview of the study design:

· Screen 4- to 5-year-old children with non-cycloplegic retinoscopy or autorefraction, using the associated referral criterion for hyperopia in the targeted range in order to identify children with uncorrected hyperopia (3D to < 6D) and age–matched children from the same school who have no significant refractive error (hyperopia < 1.00 D, astigmatism < 1.00 D, anisometropia < 1.00 D, and myopia < 1.00 D).

o The targeted range of hyperopia was chosen to 1) encompass the range over which there is maximal controversy in prescribing for hyperopia and 2) begin at the upper limit of the normal range of hyperopia for preschool children.

· Recruit, consent and enroll identified children. Assess visual acuity (Lea Symbols) and ocular alignment (cover test). Exclude any children with strabismus.

· Schedule enrolled children for testing on a battery of educational tests including the Test of Preschool Early Literacy (TOPEL) (primary outcome measure), Leiter-R (sustained attention subtest), and the Beery Developmental Test of Visual Motor Integration (VMI-5).(Test battery designed by VIP CHIP educational consultant.)

· Perform the educational testing at child’s school or at the Clinical Center.

· Compare educational scores (based on established age-specific norms) from children with uncorrected hyperopia to those from children with emmetropia using an independent t-test.

Due to the ARRA budget caps the study was designed to assess refractive status via screening; however, the ARRA proposal was unfunded. Based in part upon reviewers’ comments, we have modified the study design to include 1) confirmation of refractive status by cycloplegic examination to eliminate potential misclassification and 2) assessment of accommodative response to determine whether any differences between groups are due to differences in accommodative response.

Over the past year, OLERF funding allowed completion of the following 1) purchase of educational tests (TOPEL, Leiter-R, and VMI plus 2 tests of behavior, attention, and/or development [Conner’s Early Childhood and Parents’ Evaluation of Developmental Status]), 2) submission of an R-01 based upon the revised study design (pending review), 3) funding of 15% percent coordinator time, 4) partial funding towards the purchase of a WAM-5500 Grand Seiko open field autorefractor for binocular accommodative response testing, and 5) commencement of recruitment for the revised pilot study.

Project Title: The role of the Bacterial Lipopolysaccharide (LPS, endotoxin) Receptor, MD-2, in Corneal Inflammation and Infection

The ocular surface is continuously exposed to various pathogenic microbes including Pseudomonas aeruginosa, which is a leading cause of microbial infection of the cornea worldwide, and is a major risk factor for contact lens users. Lipopolysaccharide (LPS, endotoxin) is the major cell wall component of P. aeruginosa that activates the host inflammatory response and induces corneal inflammation and bacterial killing. The host response to LPS is a critical first reaction to infection, rapidly recruiting neutrophils to the potential source of infection to kill invading bacteria. Recognition and signaling of this critical pathway requires participation of specific receptors called Toll like Receptor 4 (TLR4) and Myeloid Differentiation-2 (MD-2). The current proposal will examine the role of MD-2 in LPS and Pseudomonas keratitis. Understanding the role of these pathways in corneal inflammation and corneal infection will identify specific molecules that modulate the response, and these molecules can be targets for therapeutic intervention in these important causes of visual impairment and blindness.

Microbial keratitis is a major cause of visual impairment and blindness worldwide and incidence of bacterial keratitis has steadily increased over past few decades. P. aeruginosa is one of the most commonly encountered organisms in infectious keratitis and causes rapid destruction of the human cornea characterized by severe corneal ulceration and stromal destruction (1). Because of its rapid progression and the notorious resistance of P. aeruginosa to antibiotics, the infection is difficult to treat and leads to blindness with the need for corneal transplantation. Thus, we must engage in research to better understand the mediators of host-pathogen interactions. Bacterial pathogens and their products activate resident cells within the tissue and initiate production of pro-inflammatory cytokines that induce a cellular infiltration. Microorganisms are recognized by pattern recognition receptors, including Toll-like receptors (TLRs). Upon recognition of a specific ligand, signaling cascades are activated through several adaptor molecules. TLRs are important in regulating the response to infectious agents in cornea (2). LPS is a major cell wall component of Gram-negative bacteria and induces ocular pathology following corneal injury. LPS signals downstream through TLR4 and initiates innate immune responses in the cornea and produces cytokines that might clear the pathogens. MD-2 is an essential accessory molecule required for LPS recognition and TLR4 signaling. This proposal will utilize a mouse corneal infection model (3) and human corneal epithelial cells (HCEC) in culture (both hTCEpi and 10.014 pRSV-T cell lines) to address the pathogenesis of bacterial keratitis. To have a better understanding regarding the role of inflammatory and infectious mediators of corneal pathogenesis due to bacterial keratitis, this proposal will specifically address the mentioned aim.

The mammalian cornea was for many years thought to be devoid of myeloid lineage cells. However, recent investigations have revealed the presence of heterogeneous populations of macrophages and dendritic cells in the healthy mammalian cornea (4,5). The corneal epithelium contains populations of epithelial cells along with MHC class II+ CD11c+ dendritic cells and CD11b+ macrophages. The corneal stroma also contains dendritic cells (6,7). This network of first responder cells is responsible for the initiation of inflammation in all forms of keratitis. At the cellular level, the initiation of such an inflammatory response, in the absence of tissue damage, relies upon recognition of the invading microbe as foreign. This is accomplished through the binding of host-cell Pathogen Recognition Receptors (PRRs), such as the TLR, to microbial products, with the subsequent activation of signaling cascades that mediate cytokine/chemokine production and inflammation (8). Our group and others reported that TLR2, TLR3, TLR4, TLR5 and TLR9 are expressed in the corneal epithelium (2, 9, 10, 11,). Fig.1 illustrates TLR signaling in the corneal epithelium. P. aeruginosa is the major cause of keratitis and it has been shown that it can activate both TLR4 (via LPS) and TLR5 (flagella) in cornea. Although macrophages play an essential role in LPS induced TLR4 signaling in cornea (12), epithelial cells might also have a role in bacterial defense as seen with epithelial cells from other sources (13). The ability of epithelial cells to recognize pathogen or pathogen associated products depends on the expression of TLRs via recognition of pathogen-associated molecular patterns (14).

MD-2, a 25kDa secreted glycoprotein, is essential for TLR4 mediated recognition of LPS (15). In addition, MD-2 is essential for functional cellular distribution of TLR4. There are reports regarding unresponsiveness of HCEC to LPS due to lack MD-2 expression by these cell lines (16). Recent studies have shown that MD-2 expression can be enhanced by various pro-inflammatory cytokines in different cell lines like intestinal epithelial cells (17), conjunctival epithelial cells (18) or monocytes (19). The minimal expression of MD-2 in both intestinal and conjunctival epithelial cells has been up-regulated by IFNγ treatment. Other cytokines like IL-10 (19) and IL-6 (20) have also been shown to augment MD-2 expression in various cell lines. The promising nature of these results and lack of in vivo demonstration of a role of MD-2 during LPS treatment of the cornea will be addressed in AIM of this proposal.

Implications for the proposed studies: Pseudomonas aeruginosa is a predominant Gram-negative opportunistic bacterium that infects the cornea. Corneal infection caused by P. aeruginosa develops rapidly, triggering an inflammatory response that may lead to vision loss. The most common predisposing factor for Pseudomonas keratitis is use of extended-wear contact lenses or ocular trauma (24). For many years, keratitis caused by Pseudomonas has been commonly associated with the wearing of soft contact lenses (25). The clinical manifestations associated with contact lens wear include contact lens associated red eye (CLARE), contact lens peripheral ulcers (CLPU) and other corneal infiltrative events (CIE), which cause pain, redness, blurred vision and severe discomfort. Since there are high number of contact lens wearer in USA (34 million) and worldwide (140 million), even a small percentage with side effects translates into a large number of affected individuals (2). The severity of keratitis and consequent vision loss necessitates the investigation into the infection and inflammatory process leading to tissue destruction during keratitis by P. aeruginosa.

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