Michael R. Frost

Alterations in protein expression in tree shrew sclera during development of lens-induced myopia and recovery.

PURPOSE During the development of, and recovery from, negative lens-induced myopia there is regulated remodeling of the scleral extracellular matrix (ECM) that controls the extensibility of the sclera. Difference gel electrophoresis (DIGE) was used to identify and categorize proteins whose levels are altered in this process. METHODS Two groups of five tree shrews started monocular lens wear 24 days after eye opening (days of visual experience [VE]). The lens-induced myopia (LIM) group wore a -5 D lens for 4 days. The recovery (REC) group wore a -5 D lens for 11 days and then recovered for 4 days. Two normal groups (28 and 39 days of VE; n = 5 each) were also examined, age-matched to each of the treatment groups. Refractive and A-scan measures confirmed the effect of the treatments. Scleral proteins were isolated and resolved by DIGE. Proteins that differed in abundance were identified by mass spectrometry. Ingenuity pathway analysis was used to investigate potential biological pathway interactions. RESULTS During normal development (28-39 days of VE), eight proteins decreased and one protein increased in relative abundance. LIM-treated eyes were myopic and longer than control eyes; LIM-control eyes were slightly myopic compared with 28N eyes, indicating a yoking effect. In both the LIM-treated and the LIM-control eyes, there was a general downregulation from normal of proteins involved in transcription, cell adhesion, and protein synthesis. Additional proteins involved in cell adhesion, actin cytoskeleton, transcriptional regulation, and ECM structural proteins differed in the LIM-treated eyes versus normal but did not differ in the control eyes versus normal. REC-treated eyes were recovering from the induced myopia. REC-control eye refractions were not significantly different from the 39N eyes, and few proteins differed from age-matched normal eyes. The balance of protein expression in the REC-treated eyes, compared with normal eyes and REC-control eyes, shifted toward upregulation or a return to normal levels of proteins involved in cell adhesion, cell division, cytoskeleton, and ECM structural proteins, including upregulation of several cytoskeleton-related proteins not affected during myopia development. CONCLUSIONS The DIGE procedure revealed new proteins whose abundance is altered during myopia development and recovery. Many of these are involved in cell-matrix adhesions, cytoskeleton, and transcriptional regulation and extend our understanding of the remodeling that controls the extensibility of the sclera. Reductions in these proteins during minus lens wear may produce the increased scleral viscoelasticity that results in faster axial elongation. Recovery is not a mirror image of lens-induced myopia-many protein levels, decreased during LIM, returned to normal, or slightly above normal, and additional cytoskeleton proteins were upregulated. However, no single protein or pathway appeared to be responsible for the scleral changes during myopia development or recovery.

Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery

Gene expression in tree shrew choroid was examined during the development of minus-lens induced myopia (LIM, a GO condition), after completion of minus-lens compensation (a STAY condition), and early in recovery (REC) from induced myopia (a STOP condition). Five groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), one minus-lens group wore a monocular -5 D lens for 2 days (LIM-2), another minus-lens group achieved stable lens compensation while wearing a monocular -5 D lens for 11 days (LIM-11); a recovery group also wore a -5 D lens for 11 days and then received 2 days of recovery starting at 35 DVE (REC-2). Two age-matched normal groups were examined at 26 DVE and 37 DVE. Quantitative PCR was used to measure the relative differences in mRNA levels in the choroid for 77 candidate genes that were selected based on previous studies or because a whole-transcriptome analysis suggested their expression would change during myopia development or recovery. Small myopic changes were observed in the treated eyes of the LIM-2 group (-1.0 ± 0.2 D; mean ± SEM) indicating eyes were early in the process of developing LIM. The LIM-11 group exhibited complete refractive compensation (-5.1 ± 0.2 D) that was stable for five days. The REC-2 group recovered by 1.3 ± 0.3 D from full refractive compensation. Sixty genes showed significant mRNA expression differences during normal development, LIM, or REC conditions. In LIM-2 choroid (GO), 18 genes were significantly down-regulated in the treated eyes relative to the fellow control eyes and 10 genes were significantly up-regulated. In LIM-11 choroid (STAY), 10 genes were significantly down-regulated and 12 genes were significantly up-regulated. Expression patterns in GO and STAY were similar, but not identical. All genes that showed differential expression in GO and STAY were regulated in the same direction in both conditions. In REC-2 choroid (STOP), 4 genes were significantly down-regulated and 18 genes were significantly up-regulated. Thirteen genes showed bi-directional regulation in GO vs. STOP. The pattern of differential gene expression in STOP was very different from that in GO or in STAY. Significant regulation was observed in genes involved in signaling as well as extracellular matrix turnover. These data support an active role for the choroid in the signaling cascade from retina to sclera. Distinctly different treated eye vs. control eye mRNA signatures are present in the choroid in the GO, STAY, and STOP conditions. The STAY signature, present after full compensation has occurred and the GO visual stimulus is no longer present, may participate in maintaining an elongated globe. The 13 genes with bi-directional expression differences in GO and STOP responded in a sign of defocus-dependent manner. Taken together, these data further suggest that a network of choroidal gene expression changes generate the signal that alters scleral fibroblast gene expression and axial elongation rate.

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 µL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development.

Altered gene expression in tree shrew retina and retinal pigment epithelium produced by short periods of minus-lens wear

ABSTRACT Hyperopic refractive error is detected by retinal neurons, which generate GO signals through a direct emmetropization signaling cascade: retinal pigment epithelium (RPE) into choroid and then into sclera, thereby increasing axial elongation. To examine signaling early in this cascade, we measured gene expression in the retina and RPE after short exposure to hyperopia produced by minus‐lens wear. Gene expression in each tissue was compared with gene expression in combined retina + RPE. Starting 24 days after normal eye opening, three groups of juvenile tree shrews (n = 7 each) wore a monocular −5 D lens. The untreated fellow eye served as a control. The “6h” group wore the lens for 6 h; the “24h” group wore the lens for 24 h; each group provided separate retina and RPE tissues. Group “24hC” wore the lens for 24 h and provided combined retina + RPE tissue. Quantitative PCR was used to measure the relative differences (treated eye vs. control eye) in mRNA levels for 66 candidate genes. In the retina after 6 h, mRNA levels for seven genes were significantly regulated: EGR1 and FOS (early intermediate genes) were down‐regulated in the treated eyes. Genes with secreted protein products, BMP2 and CTGF, were down‐regulated, whilst FGF10, IL18, and SST were up‐regulated. After 24h the pattern changed; only one of the seven genes still showed differential expression; BMP2 was still down‐regulated. Two new genes with secreted protein products, IGF2 and VIP, were up‐regulated. In the RPE, consistent with its role in receiving, processing, and transmitting GO signaling, differential expression was found for genes whose protein products are at the cell surface, intracellular, in the nucleus, and are secreted. After 6h, mRNA levels for 17 genes were down‐regulated in the treated eyes, whilst four genes (GJA1, IGF2R, LRP2, and IL18) were up‐regulated. After 24h the pattern was similar; mRNA levels for 14 of the same genes were still down‐regulated; only LRP2 remained up‐regulated. mRNA levels for six genes no longer showed differential expression, whilst nine genes, not differentially expressed at 6 h, now showed differential expression. In the combined retina + RPE after 24 h, mRNA levels for only seven genes were differentially regulated despite the differential expression of many genes in the RPE. Four genes showed the same expression in combined tissue as in retina alone, including up‐regulation of VIP despite significant VIP down‐regulation in RPE. Thus, hyperopia‐induced GO signaling, as measured by differential gene expression, differs in the retina and the RPE. Retinal gene expression changed between 6 h and 24 h of treatment, suggesting evolution of the retinal response. Gene expression in the RPE was similar at both time points, suggesting sustained signaling. The combined retina + RPE does not accurately represent gene expression in either retina or, especially, RPE. When gene expression signatures were compared with those in choroid and sclera, GO signaling, as encoded by differential gene expression, differs in each compartment of the direct emmetropization signaling cascade. HIGHLIGHTSJuvenile tree shrews wore a monocular −5 D lens for 6h or 24h.mRNA expression was measured for 66 genes in retina, RPE, and combined retina + RPE.In retina, differential expression changed between 6h and 24h.In RPE, the pattern of expression was similar at both time points.Abundant differential gene expression in RPE was not detected in retina + RPE.

Effect of prior vaccination on carriage rates of Streptococcus pneumoniae in older adults: A longitudinal surveillance study

INTRODUCTION Pneumococcus is a commensal of the upper respiratory tract and colonization is common in young children. Carriage studies have provided insights on vaccine effects in children and may also be useful for assessing vaccines in adults. However, culture based prevalence studies in older adults describe low colonization rates. Therefore, we assessed cumulative incidence of pneumococcal colonization in older adults using polymerase chain reaction (PCR) targeting the lytA gene and risk factors for carriage. METHODS 100 community-dwelling adults ≥65 years were enrolled the winter of 2015 and followed biweekly for 12 months. Medical, vaccination and illness history as well as nasopharyngeal (NP) and oropharyngeal (OP) samples were collected. Combined OP and NP were incubated in enrichment broth and screened using real-time lytA PCR. Samples from new colonization events (lytA PCR+) were cultured on gentamicin blood agar plates. Isolates identified by colony morphology as S. pneumoniae were serotyped using a multiplex combined immunoassay-PCR platform which classifies 96 serotypes. Cumulative incidence of pneumococcal carriage was calculated and risk factors for carriage assessed. RESULTS The cumulative incidence of colonization was 41% by PCR and 14% by culture. Monthly prevalence ranged from 0 to 17% by PCR and 1 to 4% by culture with peaks in the spring and fall. Demographics were similar between colonized and never colonized subjects although colonized were younger (72.4 vs. 75.0 years, P = 0.06). Vaccination with any pneumococcal vaccine before or during study period was associated with decreased risk of becoming colonized (p < 0.001) as was vaccination with either the 13-valent conjugated pneumococcal vaccine (PCV13) or 23-valent polysaccharide vaccine (PPSV23) (p < 0.001). CONCLUSION Pneumococcal colonization in older adults as detected by lytA PCR is frequent and pneumococcal vaccination appears to be associated with decreased risk of carriage. Further study is needed to understand the biological significance of molecular detection of pneumococcus in adults.