Fibronectin and osteopontin expression in control and uveitic vitreous.

<p>Western blot signals for fibronectin (A) and for osteopontin at 30 kDa (B) and at 72 kDa (C) of control (grey boxes, representative band above) and uveitic vitreous samples (white boxes, representative band above) were quantified by densitometry and statistical significance was calculated using the Mann-Whitney test. Data are represented in a box plot and median is marked by a black line in boxes. Expression level of fibronectin (A) was significantly (*p = 0.02) reduced in diseased (n = 4) compared to healthy vitreous (n = 5) with a difference in the median values of 25.8%. Quantification of osteopontin expression at 30 kDa (B) resulted in a significant (*p = 0.02) decrease in uveitic vitreous (n = 4) compared to control samples (n = 5) with the median values of 99.5% (healthy) and 2.8% (diseased). Osteopontin at 72 kDa (C) shows a significantly (*p = 0.02) decreased expression level in uveitic vitreous (n = 4) compared to controls (n = 5). Median value of healthy vitreous was 98% in contrast to 14.3% of uveitic samples.</p

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future

Immunocytochemical staining of cultured equine Müller cells.

<p>Representative image for eqMC-7 cells and primary cultured equine Müller cells stained for fibronectin (magenta), osteopontin (red) and vimentin (green). Fibronectin shows positive immunoreactivity in a spotted pattern predominantly in the nucleus and the surrounding area (A). Osteopontin is expressed in a thread-like distribution (B). Immunoreactivity for the intermediate filament vimentin displays an elongated, thread-like expression pattern throughout the entire cell (C). Overlay image reveals partly co-localisation of fibronectin and osteopontin in the nucleus surrounding area and co-localisation between osteopontin and vimentin stainings within the equine Müller cell (D). Nuclei are stained with DAPI (blue).</p

Expression pattern of fibronectin and osteopontin in healthy and uveitic retinal tissue.

<p>Retinal expression of fibronectin (magenta), osteopontin (red) and vimentin (green) in a representative healthy (left panels) and ERU diseased retina (right panels). Differential interference contrast (DIC) image of a representative control retina (A) and uveitic retina section (B), display tissue destruction and a loss of photoreceptor outer segments in the ERU case. Fibronectin is expressed along the ILM in healthy retina as a continuous band (C) but shows a spotted pattern along the disintegrated ILM and in the outer uveitic retina (D). Osteopontin expression in healthy retina shows a Müller cell like pattern and a positive staining of photoreceptor outer segments (E). This expression almost disappears in ERU diseased retina (F). Müller cells of control section show a distinct immunoreactivity for vimentin throughout the retina (G) while uveitic retina displays characteristic gliotic Müller cell morphology (H). Overlay image of control retinas reveals a close neighbouring of fibronectin and vimentin at the ILM and an overlap of osteopontin and vimentin especially in Müller cell endfeet (I). The disintegrated fibronectin expression is no longer adjacent to Müller cell endfeet membranes and no co-localization of osteopontin and vimentin is detectable in uveitic retina (J). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane.</p

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future

Identification of Autoantigens in Body Fluids by Combining Pull-Downs and Organic Precipitations of Intact Immune Complexes with Quantitative Label-Free Mass Spectrometry

Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody–antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immuno­globulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We confirmed feasibility of target enrichment and identification from highly complex body fluid proteomes by spiking of a predefined antibody–antigen complex at low level of abundance. As a proof of principle, we studied the blinding disease autoimmune uveitis, which is caused by autoreactive T-cells attacking the inner eye and is accompanied by autoantibodies. We identified three novel autoantigens in the spontaneous animal model equine recurrent uveitis (secreted acidic phosphoprotein osteopontin, extracellular matrix protein 1, and metallo­proteinase inhibitor 2) and confirmed the presence of the corresponding autoantibodies in 15–25% of patient samples by enzyme-linked immuno­sorbent assay. Thus, this workflow led to the identification of novel autoantigens in autoimmune uveitis and may provide a versatile and useful tool to identify autoantigens in other autoimmune diseases in the future

Immunohistochemical analysis of Syt1 expression changes in ERU retina.

<p>Left panels: representative healthy retina; right panels: representative ERU case. Differential interference contrast images of healthy (A) and ERU affected (B) retinal specimen demonstrating that in ERU state, normal retinal architecture is disturbed. GRP78 (green color), a marker staining retinal ganglion cells and a population of inner nuclear layer cells in equine retina was equally expressed in physiological (C) and ERU state (D). Synaptotagmin-1 (Syt1, red color) signal in healthy retina (E) was most prominent in retinal ganglion cell somata, their axons in the nerve fiber layer and in somata of a cell population in the inner nuclear layer, with additional staining foci in the outer and inner plexiform layer and photoreceptor outer segments, while ERU affected retinal sections (F), presented with a clearly reduced overall Syt1 signal. Overlay of GRP78 and Syt1 signals (G: healthy, H: ERU) indicated that in the ERU affected section, Syt1 expression is reduced, although structures expressing it in physiological state are still present. Cell nuclei were counterstained with DAPI (blue color).</p

Unequal Syt1 expression in healthy and ERU affected retinal tissues.

<p>Western blot quantification of Syt1 expression in healthy retinas (white bar, n = 5) and ERU (black bar, n = 7). Representative Syt-1 blots for each group are inserted above respective bars (left, healthy and right, ERU). The band at 68 kDa was used for quantification (arrows). Mean signal intensity of control samples was set as a 100%, mean signal intensity in ERU samples was significantly reduced to 24% (±34%) (* = p≤0.05).</p