Patients with a fast progression profile in geographic atrophy have increased CD200 expression on circulating monocytes.

IMPORTANCE: Geographic atrophy (GA) is a progressing atrophy of the neuroretina with no treatment option. BACKGROUND: Age-related malfunction of retinal microglia amplifies response towards age-related tissue stress in age-related macular degeneration. Here, we investigated monocyte CD200 expression - the circulating middleman negotiating retinal microglial activity - in a poorly understood subtype of age-related macular degeneration. DESIGN: Prospective case-control study. PARTICIPANTS: Forty-six patients with GA and 26 healthy controls were included. METHODS: All participants were subjected to a structured interview and detailed retinal examination. Controls were recruited from patient's spouses accompanying them in the clinic to match the groups best possibly. Participants had no history of immune disorders or cancer, and did not receive any immune-modulating medication. Patients did not have any history or sign of choroidal neovascularization in either eye. Fresh drawn blood was stained with monoclonal antibodies and prepared for flow cytometry to evaluate CD200 expression in monocytes and their functional subsets. MAIN OUTCOME MEASURES: The percentage of CD200+ monocytes in patients and controls. RESULTS: We found that monocytes were more CD200 positive in patients with GA compared to healthy age-matched controls. Then, we explored the potential relationship between CD200 expression and important fundus autofluorescence patterns that predict disease progression. Patients with a high risk of progression (patients with high degree of hyperautofluorescence) had distinctly increased CD200 expression compared to other patients with GA. CONCLUSIONS AND RELEVANCE: Our data reveals that abnormal monocytic CD200 expression is present in GA, and in particular among those identified as fast progressors.

Induction of Chemokine Secretion and Monocyte Migration by Human Choroidal Melanocytes in Response to Proinflammatory Cytokines.

Purpose: To determine to which extent inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes, and whether HCMs are able to influence the proliferation of activated T cells. Methods: Primary cultures of HCMs were established from eyes of 13 donors. Human choroidal melanocytes were stimulated with IFN-γ and TNF-α or with supernatant from activated T cells (T-cell-conditioned media [TCM]). Gene expression analysis was performed by using microarrays. Protein levels were quantified with ELISA or cytometric bead array. Supernatants of HCMs were assessed for the capability to attract monocytes in a transwell plate. Proliferation of activated T cells was assessed in a direct coculture with HCMs by a [3H]-thymidine incorporation assay. Results: Stimulation of HCMs with TCM or IFN-γ and TNF-α resulted in increased expression and secretion of CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5 and intercellular adhesion molecule 1. Vascular endothelial growth factor and monocyte migration inhibitory factor were constitutively expressed without changes in response to proinflammatory cytokines. Supernatants derived from unstimulated cultures of 10 HCM donors induced a high initial level of monocyte migration, which decreased upon stimulation with either TCM or IFN-γ and TNF-α. The supernatants from three HCM donors initially showed a low level of monocyte attraction, which increased after exposure to proinflammatory cytokines. Direct coculture of HCMs with T cells resulted in inhibition of T-cell proliferation. Conclusions: These results showed that normal and activated HCMs are immunologically active by secreting chemokines, and that HCMs are able to attract monocytes in addition to inhibiting T-cell proliferation.

Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis.

PURPOSE: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes. METHODS: We used an in vitro coculture system in which UM cell lines and T cells were cultured together, but separated by a membrane. Uveal melanoma gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a transwell plate was used. RESULTS: Gene-expression analysis of UM cell lines showed that coculture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, VEGF, intracellular adhesion molecule 1 (ICAM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant toward monocytes. CONCLUSIONS: Cytokines derived from activated T cells shifted the UM cell transcriptome toward a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor-promoting inflammatory microenvironment.

Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells.

PURPOSE: Accumulation of bisretinoids as lipofuscin in retinal pigment epithelial (RPE) cells is implicated in the pathogenesis of some blinding diseases including age-related macular degeneration (AMD). To identify genes whose expression may change under conditions of bisretinoid accumulation, we investigated the differential gene expression in RPE cells that had accumulated the lipofuscin fluorophore A2E and were exposed to blue light (430 nm). METHODS: A2E-laden RPE cells were exposed to blue light (A2E/430 nm) at various time intervals. Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus). Array results for selected genes were confirmed by real-time reverse-transcriptase polymerase chain reaction. RESULTS: Principal component analysis revealed that the A2E-laden RPE cells irradiated with blue light were clearly distinguishable from the control samples. We found differential regulation of genes belonging to the following functional groups: transcription factors, stress response, apoptosis and immune response. Among the last mentioned were downregulation of four genes that coded for proteins that have an inhibitory effect on the complement cascade: (complement factor H, complement factor H-related 1, complement factor I and vitronectin) and of two belonging to the classical pathway (complement component 1, s subcomponent and complement component 1, r subcomponent). CONCLUSION: This study demonstrates that blue light irradiation of A2E-laden RPE cells can alter the transcription of genes belonging to different functional pathways including stress response, apoptosis and the immune response. We suggest that these molecules may be associated to the pathogenesis of AMD and can potentially serve as future therapeutic targets.

Inflammatory cytokines protect retinal pigment epithelial cells from oxidative stress-induced death.

PURPOSE: To investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19. METHODS: Confluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting. RESULTS: Viability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment. CONCLUSION: Oxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.

Chemokine expression in retinal pigment epithelial ARPE-19 cells in response to coculture with activated T cells.

PURPOSE: To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). METHODS: We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting. RESULTS: Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL10 (IP10); CXCL11 (ITAC); and CX3CL1 (fractalkine). CCL7, CXCL9, CXCL10, and CXCL11 were secreted significantly more in the apical direction. Using recombinant human cytokines and neutralizing antibodies, we identified IFNγ and TNFα as the two major T-cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, CXCL10, CXCL11, CXCL16, and CX3CL1, we observed a synergistic effect of IFNγ and TNFα in combination. CCL20, CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ. CONCLUSIONS: RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.

Altered expression of CD46 and CD59 on leukocytes in neovascular age-related macular degeneration.

PURPOSE: To investigate the expression of the complement regulatory proteins CD46, CD55, and CD59 on peripheral leukocytes in neovascular age-related macular degeneration (AMD). DESIGN: Prospective, case-control study. METHODS: Thirty-five unrelated patients with neovascular AMD and 30 control individuals were included in this case-control study. All participants were subjected to a structured interview and detailed imaging (autofluorescence, digital funduscopy, spectral-domain optical coherence tomography, and fluorescein and indocyanine green angiography in patients suspected of having neovascular AMD) was performed. Fresh ethylenediamine-tetraacetic acid blood was obtained and stained with monoclonal antibodies. Using flow cytometry, the percentage of CD14(+) monocytes, CD45(+) lymphocytes, and CD45(+) granulocytes positive for CD46, CD55, and CD59 was determined in patients with neovascular AMD and was compared with that of controls. RESULTS: We found that the expression of CD46 and CD59 was significantly lower on CD14(+) monocytes in patients with neovascular AMD compared with controls (P = .0070). A significantly lower expression of CD46 on lymphocytes was observed in patients with fibrosis compared with patients without fibrosis (P = .010). CONCLUSIONS: Our study suggests that neovascular AMD is associated with an inadequate regulation of the complement system, supporting current evidence on the role of complement dysregulation in the pathogenesis of AMD.

Astrocytoma cells upregulate expression of pro-inflammatory cytokines after co-culture with activated peripheral blood mononuclear cells.

In this study, we investigated the effect of CD3/CD28-activated peripheral blood mononuclear cells (PBMCs) on two human astrocytoma cell lines (1321N1 and CCF-STTG1), with respect to the expression of cytokines and chemokines. We used an in vitro co-culture system in which the astrocytoma cells and PBMCs were separated by a membrane in a transwell system. Proliferation of T cells was quantified by a [(3) H]-thymidine incorporation assay. Differential gene expression of astrocytoma cells cultured alone or with PBMCs was identified using gene arrays. Protein expression of CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1ß was determined by Luminex. Co-culture of activated T cells and astrocytoma cells resulted in inhibition of T cell proliferation. Moreover, astrocytoma cells upregulated a number of pro-inflammatory genes including CCL3, CCL5, CXCL8, CXCL9, CXCL10, IL6, and IL1ß upon co-culture with activated PBMCs. These results were confirmed on the protein level. Astrocytoma cells inhibited the proliferation of T cells, indicating that astrocytes may play an important role in controlling T cell mediated immune responses in the brain. Contrary to this, soluble factors derived from CD3/CD28-activated PBMCs affected the transcriptome of astrocytoma cells, which then upregulated several pro-inflammatory genes and proteins. This could be part of neuroinflammatory events and may play a role in neurodegenerative diseases.

Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells.

In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding of inflammatory ocular diseases such as uveitis and age-related macular degeneration.

Glycosaminoglycans enhance the fibrillation propensity of the ß2-microglobulin cleavage variant--ΔK58-ß2m.

Dialysis related amyloidosis (DRA) is a serious complication to long-term hemodialysis treatment which causes clinical symptoms such as carpal tunnel syndrome and destructive arthropathies. The disease is characterized by the assembly and deposition of ß2-microglobulin (ß2m) predominantly in the musculoskeletal system, but the initiating events leading to ß2m amyloidogenesis and the molecular mechanisms underlying amyloid fibril formation are still unclear. Glycosaminoglycans (GAGs) and metal ions have been shown to be related to the onset of protein aggregation and to promote de novo fiber formation. In this study, we show that fibrillogenesis of a cleavage variant of ß2m, ΔK58-ß2m, which can be found in the circulation of hemodialysis patients and is able to fibrillate at near-physiological pH in vitro, is affected by the presence of copper ions and heparan sulfate. It is found that the fibrils generated when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ΔK58-ß2m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote the de novo formation of ß2m amyloid by a scaffold mechanism.

Deficient SOCS3 and SHP-1 expression in psoriatic T cells.

IFN-alpha and skin-infiltrating activated T lymphocytes have important roles in the pathogenesis of psoriasis. T cells from psoriatic patients display an increased sensitivity to IFN-alpha, but the pathological mechanisms behind the hyperresponsiveness to IFN-alpha remained unknown. In this study, we show that psoriatic T cells display deficient expression of the suppressor of cytokine signaling (SOCS)3 in response to IFN-alpha and a low baseline expression of the SH2-domain-containing protein-tyrosine phosphatase (SHP)-1 when compared with skin T cells from nonpsoriatic donors. Moreover, IFN-alpha-stimulated psoriatic T cells show enhanced activation of JAKs (JAK1 and TYK2) and signal transducers and activators of transcription. Increased expression of SOCS3 proteins resulting from proteasomal blockade partially inhibits IFN-alpha response. Similarly, forced expression of SOCS3 and SHP-1 inhibits IFN-alpha signaling in psoriatic T cells. In conclusion, our data suggest that loss of regulatory control is involved in the aberrant hypersensitivity of psoriatic T cells to IFN-alpha.

A beta2-microglobulin cleavage variant fibrillates at near-physiological pH.

Beta2-microglobulin (beta2m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of beta2m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native beta2m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of beta2m--found in the circulation of many dialysis patients--is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native beta2m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of beta2m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native beta2m. This may explain the apparent lack of detectable amounts of the variant beta2m in extracts of amyloid from DRA patients.

Vitreous humor and albumin augment the proliferation of cultured retinal precursor cells.

Intravitreal injection is an important delivery route for studies involving the transplantation of various types of precursor cells to the retina; however, the effect on these cells of exposure to the vitreous microenvironment has not been specifically investigated. Here vitreous humor was evaluated for the potential to influence the proliferation of rat retinal precursor cells in vitro. Cells were isolated at embryonic day 19 and plated in standard proliferation medium in the presence or absence of fluid expressed from porcine vitreous humor. Cellular proliferation at different concentrations of vitreous fluid supplementation was quantified by using a (3)H-thymidine incorporation assay. Active components of vitreous fluid were partially characterized by gel filtration chromatography (GFC) and UV spectral analysis. The effect of each vitreous fraction on proliferation was determined as well. Results showed that addition of 20% vitreous fluid to primary rat retinal cultures significantly increased (3)H-thymidine incorporation compared with growth medium without vitreous supplementation. A vitreous fraction showing growth-promoting activity was localized to a molecular mass range <1000 Da, consistent with ascorbic acid. Ascorbic acid was confirmed in vitreous fluid by UV spectral analysis. Growth-augmenting activity was present in higher molecular mass vitreous fractions, consistent with protein components. Albumin, the major protein in vitreous fluid, was found to augment proliferation. Because vitreous-associated augmentation of retinal precursor proliferation remains an epidermal growth factor-dependent phenomenon, the proliferative status of transplanted cells in the vitreous cavity is likely determined by a combination of factors.

Subretinal posterior pole injury induces selective proliferation of RPE cells in the periphery in in vivo studies in pigs.

PURPOSE: To study topographical differences in porcine retinal pigment epithelial (RPE) cell proliferation (1) in vivo, after experimental central surgical subretinal injury, and (2) in vitro. METHODS: Domestic pigs underwent either experimental RPE debridement (n = 5), subretinal amniotic membrane transplantation (n= 4), or both (n= 1) in the left eye. RPE cell proliferation was assayed by injection of the thymidine analogue 5-bromodeoxyuridine (5-BrdU) at postoperative day 0 and 1. RPE cells in S-phase were identified by their incorporation of 5-BrdU, as detected by immunohistochemistry. The in vitro proliferation of primary RPE isolates from the peripheral and central retina was assayed by a colorimetric assay and by [(3)H]thymidine incorporation. RESULTS: After subretinal surgery, in vivo incorporation of 5-BrdU was seen in peripheral RPE cells in 8 of 10 surgically treated eyes, but never in central RPE cells. This observation was true of both types of experimental surgery performed. In vitro, RPE isolates from the pre-equatorial region consistently yielded higher cell densities than did RPE cell isolates from more central parts of the epithelium. This was apparent through the three first passages of porcine RPE cells in culture. After 1 and 4 days in culture, pre-equatorial RPE cells had incorporated significantly more [(3)H]thymidine than had the more central RPE cells. CONCLUSIONS: Experimental subretinal surgical injury of the RPE below the central retina is followed within 48 hours by a peripheral, but not a central, proliferation of RPE cells. In vitro, peripheral RPE cells have a higher proliferative capacity than do central RPE cells.

Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors.

A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2 expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica from the amino acid sequence of the Bcl2 protein and its binding affinity for HLA-A*0201 was confirmed using a biochemical binding assay. We here demonstrate that the 9-mer peptide Bcl2(85-93) induces specific CTL reactivity in immunized C57-A2K(b) or -A2D(b) tg mice. These Bcl2(85-93) specific CTLs react with and lyse Bcl2-expressing human colon carcinoma CCL220 cells which have been transfected with a chimeric HLA-A*0201/H2-K(b) DNA construct similar to that expressed in the transgenic mice. Based on these observations, we suggest that Bcl2(85-93) may be a target for immune therapy.

Aqueous humor enhances the proliferation of rat retinal precursor cells in culture, and this effect is partially reproduced by ascorbic acid.

Aqueous humor has been shown to influence the proliferation of various ocular cell types, but the effect on immature retinal cells is not known. Here, the effect of pig aqueous humor on the proliferation of rat retinal precursor cells (RPCs) was investigated. RPCs were prepared from embryonic day 19 Sprague-Dawley rats and cultured in the presence or absence of aqueous humor from healthy pigs along with a medium consisting of Dulbecco's modified Eagle's medium:Ham's F-12 medium, N2 supplement, and epidermal growth factor. Proliferation was quantified by [(3)H]thymidine incorporation under different treatment conditions, and any associated morphological changes were noted. Potential active components of porcine aqueous humor were partially characterized by gel filtration chromatography, and the effect on RPC proliferation was determined. Results showed that adding 20% aqueous humor increased [(3)H]thymidine incorporation by as much as 317%, as compared with controls. Aqueous supplementation also increased both the number and size of RPC spherical aggregates ("spheres") over the first 4 days, consistent with increased proliferative activity. Using gel filtration and the in vitro proliferation assay, the growth-promoting activity of aqueous humor was localized to two different molecular mass ranges, namely, around 30 kDa and less than 1 kDa. Ascorbic acid was present in the lower molecular mass fraction, and use of this molecule reproduced some, but not all, of the proliferative activity present in aqueous humor. These results highlight the potential role of soluble factors present in the cellular microenvironment with respect to modulation of endogenous progenitor cell activity.

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation.

Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis-related amyloidosis and thus is able to attain alternative conformations that lead to irreversible aggregation and precipitation. In this study, we quantitate the increased conformational instability of cleaved beta2m by extracting rate constants and activation energies by simulating the experimental data using a unified theory for dynamic chromatography and dynamic electrophoresis. The results are correlated with the outcome of independent experiments based on mass spectrometric measurement of H/D exchange. This study illustrates that dynamic capillary electrophoresis is suitable for the investigation of the interconversion of protein conformations of amyloidogenic molecules and is not only restricted to ideal model compounds.

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature.

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.