Roderick J. Fullard

Purification of the isoforms of tear specific prealbumin

A chromatographic method for separating tear specific prealbumin (TSP) into six isoelectric forms is described. Size exclusion high performance liquid chromatography (SE-HPLC) was used to isolate TSP from whole tears, followed by chromatofocusing fast protein liquid chromatography (FPLC) of the SE-TSP fraction on a Mono P column. This yielded six fractions varying in isoelectric point (pI) between 5.3 and 4.6. Subsequent anion exchange FPLC (Mono Q column) allowed a slight further purification of each Mono P fraction and removed Polybuffer from the Mono P fractions. Isoelectric focusing (IEF) of the TSP isoforms verified that the heterogeneity was based on pI, and confirmed that the chromatofocusing separation was in many respects the same as an IEF separation. Purity of TSP isoforms was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), IEF, enzyme-linked immunosorbent assay (ELISA) and immunoblotting of samples separated by SDS-PAGE and IEF. Amino acid analysis and N-terminal amino acid sequencing revealed subtle differences between the TSP isoforms. The entire purification procedure was conducted both with and without the addition of reducing agents and protease inhibitors to tear samples and all buffers used in the purification process. Relatively little difference was seen in the TSP isoform profile under these two sets of conditions. However, the tendency of isolated TSP to aggregate and precipitate was dramatically decreased under reducing conditions, resulting in significantly higher protein recoveries. This chromatographic purification procedure provides a basis for further study of the nature of the heterogeneity of TSP and characterization of the properties of this protein.

Multiple Cytokine Analysis in Human Tears: An Optimized Procedure for Cytometric Bead-Based Assay

Purpose: To compare instruments and reagents used for cytometric bead-based assay (CBA) of cytokines in human tears. Methods: Two CBA instruments and reagent kits from five vendors were compared in parallel. Vendor protocols were modified to accommodate tear volumes. Precision, recovery, dilution linearity, and stability were evaluated. Results: Luminex™ and Bio-Rad™ proved optimal for tear CBA. Good correlation for dilution linearity was observed (r > 0.80, p < 0.05) for 1–4-μ l sample volumes. Samples were stable for 30 min at 25°C. Conclusion: Many tear cytokines can be reliably measured by CBA using an optimized protocol. Concentrations remain stable during periods consistent with collection.

Tear Protein Composition and the Effects of Stimulus

Tear protein profiles are significantly affected by stimulus and by the use of invasive tear collection techniques. The importance of stimulus control has been largely overlooked until recent times. This is most likely due to the fact that the main three tear proteins, lactoferrin, tear specific prealbumin (TSP) and lysozyme, show very little change over a wide range of stimulus conditions (Fullard and Snyder, 1990; Fullard and Tucker, 1991). Comparing non-stimulated tears collected under carefully controlled conditions with high flow-rate stimulated tears collected after discarding the initial 20 µl reveals very large differences for many tear proteins. Secretory IgA (both subclasses), IgM and IgG all show a more than 5-fold decrease in stimulated tears (Fullard and Snyder, 1990). Transferrin and albumin show a smaller, but still significant, decrease in stimulated tears. The three main lacrimal gland proteins and peroxidase undergo minimal change. Levels of the enzyme, lactate dehydrogenase (LDH) have also been shown to be significantly lower in stimulated tears (Van Haeringen and Glasius, 1974; Fullard and Carney, 1984).

Unfolded protein response is activated in aged retinas.

An unfolded protein response (UPR) in addition to oxidative stress and the inflammatory response is known to be activated in age-related ocular disorders, such as macular degeneration, diabetic retinopathy, glaucoma, and cataracts. Therefore, we aimed to investigate whether healthy aged retinas display UPR hallmarks, in order to establish a baseline for the activated UPR markers for age-related ocular diseases. Using western blotting, we determined that the hallmarks of the UPR PERK arm, phosphorylated (p) eIF2a, ATF4, and GADD34, were significantly altered in aged vs. young rat retinas. The cleaved pATF6 (50) and CHOP proteins were dramatically upregulated in the aged rodent retinas, indicating the activation of the ATF6 UPR arm. The UPR activation was associated with a drop in rhodopsin expression and in the NRF2 and HO1 levels, suggesting a decline in the anti-oxidant defense in aged retinas. Moreover, we observed down-regulation of anti-inflammatory IL-10 and IL-13 and upregulation of pro-inflammatory RANTES in the healthy aged retinas, as measured using the Bio-plex assay. Our results suggest that cellular homeostasis in normal aged retinas is compromised, resulting in the concomitant activation of the UPR, oxidative stress, and inflammatory signaling. This knowledge brings us closer to understanding the cellular mechanisms of the age-related retinopathies and ocular disorders characterized by an ongoing UPR, and highlight the UPR signaling molecules that should be validated as potential therapeutic targets.

Adaptation of Impression Cytology to Enable Conjunctival Surface Cell Transcriptome Analysis

Abstract Purpose: This study investigates the extent of the human transcriptome that can be quantified from conjunctival impression cytology extracts. The aim is to determine if sufficient RNA can be isolated from a patient’s conjunctival surface to identify differences in gene expression between dry eye and normal patients of (a) an array of 96 inflammatory biomarkers and associated receptors, and (b) if this comparison can be expanded to the entire transcriptome. Materials and Methods: CIC was used to collect conjunctival surface cells from 53 qualifying normal and dry eye patients. Based on prior optimization of all assay steps, RNA was isolated from the samples using a Qiagen RNeasy Plus Mini Kit and qRT-PCR was used to determine gene expression of 96 genes using TaqMan Low Density Array cards. Samples from six normal and six dry eye patients were then assayed on an Illumina Human HT-12 BeadChip. Results: Optimization steps yielded an RNA processing procedure that improved yield from an initial 12 genes through 96, then to the entire human transcriptome. For the HT-12 BeadChip, more than 30 genes differed by a factor of >1.5 between the dry eye and normal groups and seven genes were down-regulated by a factor of >2.0 in the dry eye group: HLA-DRB5, PSCA, FOS, lysozyme, TSC22D1, CAPN13 and CXCL6. Conclusions: Conjunctival impression cytology can be used to collect sufficient RNA from conjunctival surface cells that, when processed optimally, allows successful transcriptome-wide expression analysis. While the current transcriptome analysis used a limited patient group, larger studies of patients with various types and severities of dry eye should reveal significant gene expression trends that can then be targeted to improve dry eye treatment options.

TNFa knockdown in the retina promotes cone survival in a mouse model of autosomal dominant retinitis pigmentosa

Expression of T17M rhodopsin (T17M) in rods activates the Unfolded Protein Response (UPR) and leads to the development of autosomal dominant retinitis pigmentosa (adRP). The rod death occurs in adRP retinas prior to cone photoreceptor death, so the mechanism by which cone photoreceptors die remains unclear. Therefore, the goal of the study was to verify whether UPR in rods induces TNFa-mediated signaling to the cones and to determine whether the TNFa deficit could prevent adRP cone cell death. Primary rod photoreceptors and cone-derived 661W cells transfected with siRNA against TNFa were treated with tunicamycin to mimic activation of UPR in T17M retinas expressing normal and reduced TNFa levels. The 661W cells were then exposed to recombinant TNFa to evaluate cell viability. In vivo, the role of TNFa was assessed in T17M TNFa+/- mice by electroretinography, optical coherence tomography, histology, immunohistochemistry, and a cytokine enzyme-linked immunosorbent assay. Rods overexpressed and secreted TNFa in response to UPR activation. The recombinant TNFa treatment lowered the number of viable cones, inducing cell death through elevation of pro-inflammatory cytokines and caspase-3/7 activity. The TNFa deficiency significantly protected adRP retinas. The photopic ERG amplitudes and the number of surviving cones dramatically increased in T17M TNFa+/- mice. This neuroprotection was associated with a reduced level of pro-inflammatory cytokines. Our results indicate that rod photoreceptors, following UPR activation during adRP progression, secrete TNFa and signal a self-destructive program to the cones, resulting in their cell death. TNFa therefore holds promise as a therapeutic target for treatment of adRP.