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Representative double immunofluorescent labeling images of inner human retina incubated with monospecific serum against Rab6A (A, B) and HSP27 (C, D). (A–D) Blue color represents DAPI stained nuclei, green color represents Alexa Fluor 488-conjugated antihuman antibodies staining with the CAR patient AAbs; red color represents Alexa Fluor 594-conjugated antibodies staining with specific antibodies; the fourth image represents overlapping images—yellow color indicates common labeling. An arrow in (A) points at the ganglion cells and in (B) at the punctuated staining of the nerve fiber layer; a star in (B) shows intense staining of the ganglion cells stroma; an arrow in (C) points at the ganglion cells and in (D) shows intense staining of the nerve fiber layer, the star shows the ganglion cell body labeled yellow; Boxed areas indicated the enlarged area in (B) and (D). (B, C) magnified overlapped images. IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer.
Source publication
Purpose:
Autoantibodies (AAbs) with different retinal specificities were reported in cancer-associated retinopathy (CAR) and autoimmune retinopathy (AR). The goal was to identify the small retinal proteins of apparent molecular mass of 23-kDa often recognized by patients' AAbs.
Methods:
Sera specific for a 23-kDa retinal protein of 173 patients...
Contexts in source publication
Context 1
... against Rab6A (red color) showed a strong and uniform immunolabeling in human retina localized to the inner plexiform layer and cytoplasm of the ganglion cells (Figs. 3A, 3B). Also, punctate staining of the inner limiting membrane was present. Photoreceptor cells were not intensely labeled, although some immunofluorescent staining was observed in the inner segments (not shown). Coloc- alization studies showed that patient serum (green color) immunolabeled the same cellular structures of ganglion cells as ...
Context 2
... specific to HSP27 also immunola- beled the ganglion cell layer and nerve fiber layer in a human retina (red color). The representative confocal image of double stained retinas (Figs. 3C, 3D) using specific anti-HSP27 antibody and a human patient serum both produced a strong immunofluorescence in ganglion cells and nerve fibers, indicating similarity in immunolabeling of the same antigen (yellow color) in the ...
Similar publications
Autoantibodies (AAbs) against various retinal proteins have been associated with vision loss in paraneoplastic and non-paraneoplastic autoimmune retinopathies (AR). There are two major paraneoplastic syndromes associated anti-retinal AAbs, cancer-associated retinopathy (CAR), and melanoma-associated retinopathy. Some people without a cancer diagnos...
Purpose: To report an anti-recoverin antibody-positive cancer-associated retinopathy (anti-recoverin CAR) patient with remarkable improvements of visual function and outer retinal morphology following spontaneous regression of cancer.
Observations: A 65-year-old woman with small cell lung carcinoma developed progressive, bilateral vision loss with...
Citations
... Rab6 is a photoreceptor that may be involved in CAR pathogenicity [14]. Rhodopsin transport in photoreceptors may be mediated by Rab6; defective trafficking may trigger retinal degeneration [15]. ...
Introduction
Given the recent additions of immune checkpoint inhibitors (ICIs) to various cancer treatments, adverse effects, especially involving the eyes, have been on the rise. Here, we report an acute exacerbation of cancer-associated retinopathy (CAR) triggered by durvalumab treatment of small-cell lung cancer (SCLC).
Case Presentation
An 81-year-old Asian male complained of a scotoma in the left eye after durvalumab administration, to treat SCLC. Humphrey visual field examination revealed a C-shaped temporal scotoma. Spectralis domain optical coherence tomography revealed outer retinal layer atrophy and progressive loss of the ellipsoid zone in the atrophic peripapillary area. Fundus autofluorescence (AF) images evidenced a large C-shaped hypo-AF with enhanced AF at the margin of the atrophic area, thus at the position of the scotoma. We prescribed subtenon triamcinolone injections under suspicion of CAR exacerbation, supported by positive Western blotting results for Rab6 and aldolase, and immunohistochemical staining of photoreceptor cells. The disrupted ellipsoid zone evident on OCT partially recovered, and a visual field test showed that the scotoma had improved.
Conclusion
ICI-triggered exacerbation of CAR should be considered in SCLC patients before ICI treatment commences; an optimal treatment should preserve functional vision.
... AAbs have been found in the blood of all human sera, regardless of age, gender, or the presence or absence of disease [16]. There are hundreds of AAbs published to be associated with autoimmune and inflammatory disorders of the retina found in blood samples as well as vitreous humor using different approaches [17][18][19][20][21][22]. Blood samples (plasma or serum) are easy to collect and are most commonly used as a source of AAbs. ...
... Antibody binding to retinal cells can be also examined by more sensitive methods such as immunofluorescent IHC on frozen retinal slides [23]. Detection of binding is localized by commercially available secondary antibodies labeled with a fluorescent dye and examined in a fluorescent or confocal microscope [22,25]. In both the colorimetric and fluorescent methods, labeling to retinal layers, such as rods and cones in ...
Introduction: Autoimmune retinopathy (AR) is a sight-threating retinal disorder that is mediated by autoantibodies (AAbs) against retinal proteins. The visual paraneoplastic syndromes, including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR) are mediated by anti-retinal AAbs. A number of immunochemical techniques have been used to detect serum anti-retinal autoantibodies in patients to help with autoimmune diagnosis.
Area covered: We review techniques used for serum autoantibody evaluation in patients with suspected autoimmune retinopathy.
Expert opinion: Detection of serum AAbs have served as the standard diagnostic tool for autoimmune retinopathies and for management of retinal disorders. An identification of anti-retinal autoantibody or multiple autoantibodies can be useful for not only for diagnosis of autoimmune retinopathies but also for management of retinal disorders. We propose that the line-blotting technique used in conjunction with immunohistochemistry are the best and most reliable assays for detection of serum anti-retinal AAb in the context of clinical history and findings. Clinician should recognize that the majority of antigenic targets identified to date in retinal autoimmunity are ubiquitously expressed proteins (e.g. enolase), which may be difficult to reconcile with the specific patterns of retinal damage observed in CAR, MAR, or AR.
... Received 23 November 2019; Received in revised form 11 February 2020; Accepted 26 February 2020 in the diagnosis of AIR (Fox et al., 2016). A specific retinal binding pattern characteristic of melanoma-associated paraneoplastic AIR is serum IgG targeting inner retinal bipolar cells (Heckenlively and Ferreyra, 2008), whereas circulating immunoglobulin (IgG) targeting photoreceptors is more characteristic of nonparaneoplastic forms of AIR (Yang et al., 2016). Treatment of AIR commonly involves the combined use of multiple systemic immunosuppressive drugs, including steroids, antimetabolites, T-cell inhibitors, intravenous immunoglobulin and intravitreal triamcinolone acetonide (Fox et al., 2016;Grewal et al., 2014). ...
... Similar to our findings in SARDS, human AIR is also characterized by the presence of circulating immunoglobulins targeting retinal antigens. The pathophysiology of human paraneoplastic AIR is relatively clear: antibodies to shared antigens between the neoplasm and retinal cells contribute to the retinal pathology (Duvoisin et al., 2017;Yang et al., 2016). Circulating antibodies can cross the plasma membrane and access intracellular epitopes (Xiong et al., 2013), and photoreceptors have been shown to uptake antibodies by endocytosis, leading to caspase-mediated apoptosis (Shiraga and Adamus, 2002). ...
Sudden acquired retinal degeneration syndrome (SARDS) in dogs is proposed to have an immune-mediated etiology. However, there is conflicting evidence regarding the presence of antiretinal antibodies, as assessed by western blotting, in the serum of SARDS patients. Because of the possibility that antibodies recognize only conformational epitopes, we hypothesized that a more sensitive method to investigate circulating retinal autoantibodies in SARDS is immunofluorescence. Sera from 14 dogs with early SARDS, and 14 age- and breed-matched healthy control dogs were screened for circulating antiretinal IgG, IgM, IgE and IgA using indirect immunofluorescence on lightly fixed frozen sections of normal canine retina. Controls without canine serum were also performed. A nuclear counterstain was used to identify cellular retinal layers. Images were obtained using a fluorescent microscope, and 2-3 separate masked observers graded retinal layers for fluorescence staining intensity using a 0–3 scale. Total circulating IgG and IgM was assessed by radial immunodiffusion. Statistical analysis was performed using 2-way ANOVA, paired 2-tailed student's t-test and correlation analysis. Intensity of IgG staining of photoreceptor outer segments was significantly higher using serum from dogs with SARDS compared with healthy controls in 2/3 observers (P < 0.05). Intensity of IgM staining throughout the retina was higher in SARDS dogs compared to matched healthy controls (P < 0.0001), although no specific retinal layer was statistically significant. There were no differences in staining intensity for IgE or IgA. Dogs with SARDS had a comparably lower circulating IgG and higher IgM than healthy controls (P = 0.01 and 0.001 respectively) and IgG and IgM were negatively correlated (r = −0.69, P = 0.007). Despite having decreased serum IgG compared with healthy controls, circulating IgG in dogs with SARDS binds photoreceptor outer segments to a greater extent. Dogs with SARDS have a relatively higher circulating IgM than matched healthy controls. The pathogenic nature of these antibodies is unknown.
... Gene HSPH1 mapped on13q12.3 genomic fragment ( 20) in the region of 88-183 and 70-162, respectively ( Figure 3J and K). HSPB1 gene is localized on human chromosome 7 (7q11.23), ...
... Disaggregated proteins either get refolded back into native proteins or degraded by autophagy and/or proteasomal system. In addition, HSP27 recently was reported to be involved in cancerrelated retinopathy, suggesting its role in developing cancer therapeutics (20). HSBP1 gene is localized in the genomic fragment of 16q23.3 on the chromosome 16 (Table 1), encodes for HSBP1 protein, which is 76 amino acids long with HSBP1 (PF06825.12) ...
Background: Human serpin gene SERPINH1 encodes for a non-inhibitory serpin shock protein 47 kDa (HSP47) a client-specific chaperone, which is a hallmark in the collagen biosynthesis. Till today, there is no comprehensive study on the protein network for human HSP47. Thus, the current study aimed at studying the pathway and expression patterns observed in collagen-specific chaperone, HSP47 in relation to cancer.
Methodology: The study used STRING 10 (http://version10a.string-db.org) in finding putative protein interaction partners of human HSP47. Also, database HMMER3 (www.hmmer.org/) with Pfam 32.0 (September 2018) dataset was used in identifying Pfam protein domains from proteins interacting with HSP47. The dbDEPC3.0 was used for the evaluation of expression patterns of HSP47 in different cancer. Further three online resources, namely, human protein atlas (https://www.proteinatlas.org/), genotype-tissue expression (https://gtexportal.org), and the FANTOM5 project (http://fantom.gsc.riken.jp/5/) was used to examine HSP47 expression in normal human tissues.
Results: Upon constructing a protein interactive map of human HSP47, the study found that a set of molecular chaperones as interaction partners of HSP47, which included two copies each of cAMP response element binding proteins, HSP27, HSP40, HSP70, HSP90, ubiquitin proteins and one copy each of cartilage associated protein, HSPH1, HSBP1, FK506-binding protein 4, kruppel-like factor, peptidyl-prolyl isomerase, and Prolyl
4-hydroxylase beta subunit.
Conclusion: The study found a cocktail of different chaperones interacting with HSP47, originated at different time points from prokaryotes to eukaryotes. Overall, the study was successful in finding HSP47 expression patterns among several normal tissues using three different publicly available datasets. It also assessed the expression pattern of HSP47 in human cancer types. These findings will encourage further studies focusing on the role of HSP47 in human diseases.
... A broad spectrum of antibody reactivities was observed in the series of 441 patients with cancer and unexplained vision loss. In the search for a potential target for those AAbs, we identified 12 retinal autoantigens that represented photoreceptor-specific proteins, such as recoverin, retinal arrestin, CRALBP, and the small GTPase Rab6, 2 heat shock proteins HSP27 and P62 (HSP60), CAII, α-tubulin, and 4 glycolytic enzymatic proteins that were also found in the outer segments of photoreceptor cells: GAPDH, aldolase C, α-enolase, and PKM2 [20,21]. The occurrence of CAR AAbs was compared to those revealed in the control group and the results showed that AAbs were more frequent in the CAR group (p = .0025), ...
... In the past, it has been speculated that anti-recoverin AAbs are the sole biomarker of CAR and other associated AAbs may be indirectly or synergistically involved. However, previous and current studies confirmed that anti-recoverin AAbs are rare [21]. Consequently, an absence of AAbs against recoverin does not exclude the diagnosis of paraneoplastic retinopathy in patients with the appropriate clinical profile of this disease. ...
... However, nowadays CAR is thought to be a group of clinically similar paraneoplastic retinopathies with a different molecular background that leads to photoreceptor death. 1 Other antigens associated with CAR are: retinal enolase, Tubby-like protein 1 (TULP1), aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), interphotoreceptor retinoid binding protein (IRBP), photoreceptor cell-specific nuclear receptor (PNR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase, transducing-α, as well as heat shock cognate protein 70 (hsc-70) and 60 (hsc-60). [5][6][7][8][9] Additionally, some authors indicate other possible antigens that can be associated with CAR, including: guanylyl cyclase-activating proteins (GCAPs), heat shock protein 27 (HSP27), Rab6A GTPase (Rab6A), carbonic anhydrase II (CA II), anti-CV2/collapsin response mediator protein (CRMP5), and anti-Hu antibodies. 7,10-13 Furthermore, Adamus et al. showed that CA II unique epitopes can be used to differentiate autoimmune retinopathies (AR) from CAR. Autoimmune retinopathies sera predominantly reacted with the N-terminal epitope 85-90 and CAR sera have been found to be reactive with the peptide 218-222 clustered within the α-helix. ...
... Neoplasms causing CAR can be as follows: smallcell carcinoma of the lung, other neoplasm of the lung, breast cancer, osteosarcoma, cancers of the cervix, ovary, uterus and thymus, Warthin tumor of parotid gland, as well as carcinoma of unknown origin. [1][2][3][5][6][7]11,12 There are single reports of patients with prostate, pancreatic neurocrine and small intestine tumors. 14,15 In the past CAR has been reported in bladder and laryngeal neoplasms as well as in lymphomas (systemic follicular cell lymphoma) or colon adenoma -only as case reports or case series. ...
The aim of this study was to summarize the current knowledge of paraneoplastic syndromes involving eyes. The main interest was the immunopathogenesis of the abovementioned entities. A web search was conducted using Medline, Web of Science and Scopus engines. Key words concerning ocular paraneoplastic syndromes (OPS) such as: "ocular paraneoplastic syndrome", "cancer-associated retinopathy", "cancer-associated cone dysfunction", "melanoma-associated retinopathy", "bilateral diffuse uveal melanocytic proliferation", and "paraneoplastic optic neuritis" were combined with "immunology", "immune response", "antibodies", or "autoantibodies". Numerous papers were found as a result of "ocular paraneoplastic syndrome" search and many of them matched the chosen criteria. We focused on the most recent papers - published in the last 5 years - and eventually, 92 items were found. Only several papers from each detailed category fulfilled both OPS and immunologic criteria. Site-specific paraneoplastic syndromes still remain a difficult clinical challenge. The immunopathogenesis of some of them seems to be more complex than previously thought.
... Anti-recoverin AAbs were also detected in patients with different cancers without visual presentation, but were not reported in healthy individuals without cancer, suggesting that AAbs are mainly generated against the cancer-expressed recoverin (65). Recoverin has been considered a main biomarker for CAR syndrome but anti-recoverin AAb presence is infrequent; in fact, only about 5% of CAR patients possess anti-recoverin antibodies (66). ...
... Presumed targets in retinal degeneration are photoreceptor cells, the outer layer of the retina (Figure 1). Recoverin is a photoreceptor cell protein, in addition to several other photoreceptor antigens found, including arrestin, guanylate cyclase-activating protein, transducin-α and transducin-β, TULP1, Rab6, rhodopsin, and interphotoreceptor retinoid-binding protein (IRBP) (10,22,28,54,55,66,71,72). However, sera of patients with CAR possess AAbs that not only react with photoreceptor cell antigens but also with bipolar and ganglion cells of the retina (62,66,73,74). ...
... Recoverin is a photoreceptor cell protein, in addition to several other photoreceptor antigens found, including arrestin, guanylate cyclase-activating protein, transducin-α and transducin-β, TULP1, Rab6, rhodopsin, and interphotoreceptor retinoid-binding protein (IRBP) (10,22,28,54,55,66,71,72). However, sera of patients with CAR possess AAbs that not only react with photoreceptor cell antigens but also with bipolar and ganglion cells of the retina (62,66,73,74). AAbs against glycolytic enzymes, such as enolase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase M2, dominate in sera of patients with AR (36). ...
Autoantibodies (AAbs) against various retinal proteins have been associated with vision loss in paraneoplastic and non-paraneoplastic autoimmune retinopathies (AR). There are two major paraneoplastic syndromes associated anti-retinal AAbs, cancer-associated retinopathy (CAR), and melanoma-associated retinopathy. Some people without a cancer diagnosis may present symptoms of CAR and have anti-retinal AAbs. The etiology and pathogenesis of those entities are not fully understood. In this review, we provide evidence for the role of AAbs in retinal death and degeneration. Studies of epitope mapping for anti-recoverin, anti-enolase, and anti-carbonic anhydrase II revealed that although patients’ AAbs may recognize the same retinal protein as normal individuals they bind to different molecular domains, which allows distinguishing between normal and diseased AAbs. Given the great diversity of anti-retinal AAbs, it is likely some antibodies have greater pathogenic potential than others. Pathogenic, but not normal antibodies penetrate the target cell, reach their specific antigen, induce apoptosis, and impact retinal pathophysiology. Photoreceptors, dying by apoptosis, induced by other than immunologic mechanisms produce substantial amounts of metabolic debris, which consequently leads to autoimmunization and enhanced permeability of the blood–retinal barrier. AAbs that were made as a part of anti-cancer response are likely to be the cause of retinal degeneration, whereas others, generated against released antigens from damaged retina, contribute to the progression of retinopathy. Altogether, AAbs may trigger retinal degeneration and may also exacerbate the degenerative process in response to the release of sequestered antigens and influence disease progression.
To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.
Antibodies against neural intracellular antigens have been classified in three groups: (1) markers of paraneoplastic neurological syndromes (onconeural antibodies) and therefore the presence of an underlying cancer; (2) markers of non-paraneoplastic neurological syndromes; and (3) markers of autoimmune retinopathies. All antibodies against neural intracellular proteins recognize linear epitopes, are detected by immunoblot, and are considered non-pathogenic. An exception is the amphiphysin antibodies that can potentially reach the antigen that is briefly exposed to the extracellular space during the process of synaptic vesicle recycling. The most common onconeural antibodies are Hu (markers of paraneoplastic neurological syndromes associated with small-cell lung cancer), Yo (paraneoplastic cerebellar degeneration and breast and ovarian cancer), and Ma2 (limbic and brainstem encephalitis with testicular seminomas). The two most common antibodies present in non-paraneoplastic CNS syndromes are glutamic acid decarboxylase (GAD) and glial fibrillary acidic protein (GFAP) antibodies. GAD antibodies are biomarkers of stiff-person syndrome and also occur in some patients with cerebellar ataxia or drug-resistant temporal lobe epilepsy. GFAP antibodies associate with meningoencephalitis and a broad spectrum of symptoms without a clear specific syndrome. Unlike the other antibodies against intracellular antigens that are usually detectable in serum and CSF, GFAP antibodies are predominantly detected in CSF.
Riassunto
Le sindromi neurologiche paraneoplastiche (SNP) sono malattie autoimmuni rare che si verificano in associazione con il cancro e non sono spiegate da una complicanza metabolica, deficitaria o metastatica, infettiva o iatrogena. La scoperta degli autoanticorpi antineuronali ha consentito un importante progresso nella comprensione di queste sindromi, mettendo in evidenza il loro meccanismo autoimmune. È necessario distinguere gli anticorpi diretti contro un antigene intracellulare (AIC) da quelli diretti contro un antigene situato sulla superficie della membrana dei neuroni. Quando si tratta di un anticorpo diretto contro un AIC, l’associazione con il cancro è la regola e gli anticorpi non hanno un ruolo patogeno identificato. In queste sindromi, i segni neurologici sono dovuti alla distruzione neuronale legata all’infiltrazione dei linfociti T CD8⁺ autoattivati. Al contrario, quando l’anticorpo prende di mira un antigene di membrana, il legame dell’anticorpo con il suo antigene può allora essere responsabile di una disfunzione di una via di segnalazione neuronale e causare dei segni clinici. I sintomi sono quindi almeno parzialmente reversibili dopo trattamento immunomodulatore. In tutti i casi, l’eliminazione della stimolazione antigenica rappresentata dal tumore, quando presente, è una tappa fondamentale nella gestione terapeutica.
