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1.
Nature ; 588(7836): 124-129, 2020 12.
Article in English | MEDLINE | ID: mdl-33268865

ABSTRACT

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.


Subject(s)
Aging/genetics , Cellular Reprogramming/genetics , DNA Methylation , Epigenesis, Genetic , Eye , Nerve Regeneration/genetics , Vision, Ocular/genetics , Vision, Ocular/physiology , Aging/physiology , Animals , Axons/physiology , Cell Line, Tumor , Cell Survival , DNA-Binding Proteins/genetics , Dependovirus/genetics , Dioxygenases , Disease Models, Animal , Eye/cytology , Eye/innervation , Eye/pathology , Female , Genetic Vectors/genetics , Glaucoma/genetics , Glaucoma/pathology , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Inbred C57BL , Octamer Transcription Factor-3/genetics , Optic Nerve Injuries/genetics , Proto-Oncogene Proteins/genetics , Retinal Ganglion Cells/cytology , SOXB1 Transcription Factors/genetics , Transcriptome/genetics
2.
J Neuroophthalmol ; 44(1): 16-21, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-37938114

ABSTRACT

BACKGROUND: In 2005, we reported 3 patients with bilateral optic nerve damage early in life. These patients had stable vision for decades but then experienced significant bilateral vision loss with no obvious cause. Our hypothesis, novel at that time, was that the late decline of vision was due to age-related attrition of retinal ganglion cells superimposed on a reduced neuronal population due to the earlier injury. EVIDENCE ACQUISITION: The field of epigenetics provides a new paradigm with which to consider the normal aging process and the impact of neuronal injury, which has been shown to accelerate aging. Late-in-life decline in function after early neuronal injury occurs in multiple sclerosis due to dysregulated inflammation and postpolio syndrome. Recent studies by our group in mice have also demonstrated the possibility of partial reversal of cellular aging and the potential to mitigate anatomical damage after injury and even improve visual function. RESULTS: The results in mice and nonhuman primates published elsewhere have shown enhanced neuronal survival and visual function after partial epigenetic reprogramming. CONCLUSIONS: Injury promotes epigenetic aging , and this finding can be observed in several clinically relevant scenarios. An understanding of the epigenetic mechanisms at play opens the opportunity to restore function in the nervous system and elsewhere with cellular rejuvenation therapies. Our earlier cases exemplify how reconsideration of previously established concepts can motivate inquiry of new paradigms.


Subject(s)
Multiple Sclerosis , Optic Nerve Diseases , Humans , Mice , Animals , Optic Nerve Diseases/genetics , Optic Nerve , Retinal Ganglion Cells , Aging/genetics , Vision Disorders/genetics , Blindness
3.
Immun Ageing ; 20(1): 53, 2023 Oct 14.
Article in English | MEDLINE | ID: mdl-37838654

ABSTRACT

BACKGROUND: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival. RESULTS: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia. CONCLUSIONS: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

4.
Int J Mol Sci ; 24(3)2023 Feb 02.
Article in English | MEDLINE | ID: mdl-36769164

ABSTRACT

Epigenetic DNA modification by 5-hydroxymethylcytosine (5hmC), generated by the Ten-eleven translocation (TET) dioxygenases, regulates diverse biological functions in many organ tissues, including the mammalian eye. For example, 5hmC has been shown to be involved in epigenetic regulation of retinal gene expression. However, a functional role of 5hmC in corneal differentiation has not been investigated to date. Here, we examined 5hmC and TET function in the human cornea. We found 5hmC highly expressed in MUC16-positive terminally differentiated cells that also co-expressed the 5hmC-generating enzyme TET2. TET2 knockdown (KD) in cultured corneal epithelial cells led to significant reductions of 5hmC peak distributions and resulted in transcriptional repression of molecular pathways involved in corneal differentiation, as evidenced by downregulation of MUC4, MUC16, and Keratin 12. Additionally, integrated TET2 KD RNA-seq and genome-wide Reduced Representation Hydroxymethylation Profiling revealed novel epigenetically regulated genes expressed by terminally differentiated cells, including KRT78, MYEOV, and MAL. In aggregate, our findings reveal a novel function of TET2 in the epigenetic regulation of corneal epithelial gene expression and identify novel TET2-controlled genes expressed in differentiated corneal epithelial cells. These results point to potential roles for TET2 induction strategies to enhance treatment of corneal diseases associated with abnormal epithelial maturation.


Subject(s)
Dioxygenases , Epigenesis, Genetic , Humans , 5-Methylcytosine/metabolism , Cell Differentiation/genetics , Cornea/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , DNA Methylation , DNA-Binding Proteins/metabolism , Mammals/metabolism , Proto-Oncogene Proteins/metabolism
5.
Proc Natl Acad Sci U S A ; 116(20): 9989-9998, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31023885

ABSTRACT

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. While microglia have been studied extensively in autoimmune uveitis, their exact function remains uncertain. The objective of the current study was to determine whether resident microglia are necessary and sufficient to initiate and amplify retinal inflammation in autoimmune uveitis. In this study, we clearly demonstrate that microglia are essential for initiating infiltration of immune cells utilizing a murine model of experimental autoimmune uveoretinitis (EAU) and the recently identified microglia-specific marker P2ry12. Initiating disease is the primary function of microglia in EAU, since eliminating microglia during the later stages of EAU had little effect, indicating that the function of circulating leukocytes is to amplify and sustain destructive inflammation once microglia have triggered disease. In the absence of microglia, uveitis does not develop, since leukocytes cannot gain entry through the blood-retinal barrier, illustrating that microglia play a critical role in regulating infiltration of inflammatory cells into the retina.


Subject(s)
Autoimmune Diseases/etiology , Microglia/physiology , Retinal Diseases/immunology , Uveitis/immunology , Animals , Disease Models, Animal , Female , Mice , Organic Chemicals , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors
6.
Mol Cell ; 49(1): 94-108, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23177739

ABSTRACT

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma, and mutations in Gq-linked G protein-coupled receptors have been identified recently in numerous human malignancies. How Gα(q) and its coupled receptors transduce mitogenic signals is still unclear because of the complexity of signaling events perturbed upon Gq activation. Using a synthetic-biology approach and a genome-wide RNAi screen, we found that a highly conserved guanine nucleotide exchange factor, Trio, is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, and thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-ß. Indeed, whereas many biological responses elicited by Gq depend on the transient activation of second-messenger systems, Gq utilizes a hard-wired protein-protein-interaction-based signaling circuitry to achieve the sustained stimulation of proliferative pathways, thereby controlling normal and aberrant cell growth.


Subject(s)
Guanine Nucleotide Exchange Factors/physiology , Mitosis , Protein Serine-Threonine Kinases/physiology , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Transcription Factor AP-1/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Clozapine/analogs & derivatives , Clozapine/pharmacology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Enzyme Activation , Female , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11 , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/metabolism , Mitogens/pharmacology , NIH 3T3 Cells , Neoplasm Transplantation , Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptors, G-Protein-Coupled/genetics
7.
Proc Natl Acad Sci U S A ; 115(27): E6264-E6273, 2018 07 03.
Article in English | MEDLINE | ID: mdl-29915052

ABSTRACT

Retinal detachment (RD) is a sight-threatening complication common in many highly prevalent retinal disorders. RD rapidly leads to photoreceptor cell death beginning within 12 h following detachment. In patients with sustained RD, progressive visual decline due to photoreceptor cell death is common, leading to significant and permanent loss of vision. Microglia are the resident immune cells of the central nervous system, including the retina, and function in the homeostatic maintenance of the neuro-retinal microenvironment. It is known that microglia become activated and change their morphology in retinal diseases. However, the function of activated microglia in RD is incompletely understood, in part because of the lack of microglia-specific markers. Here, using the newly identified microglia marker P2ry12 and microglial depletion strategies, we demonstrate that retinal microglia are rapidly activated in response to RD and migrate into the injured area within 24 h post-RD, where they closely associate with infiltrating macrophages, a population distinct from microglia. Once in the injured photoreceptor layer, activated microglia can be observed to contain autofluorescence within their cell bodies, suggesting they function to phagocytose injured or dying photoreceptors. Depletion of retinal microglia results in increased disease severity and inhibition of macrophage infiltration, suggesting that microglia are involved in regulating neuroinflammation in the retina. Our work identifies that microglia mediate photoreceptor survival in RD and suggests that this effect may be due to microglial regulation of immune cells and photoreceptor phagocytosis.


Subject(s)
Macrophages/immunology , Microglia/immunology , Photoreceptor Cells, Vertebrate/immunology , Receptors, Purinergic P2Y12/immunology , Retinal Detachment/immunology , Animals , Cell Death/genetics , Cell Death/immunology , Cell Survival/genetics , Cell Survival/immunology , Macrophages/pathology , Mice , Mice, Transgenic , Microglia/pathology , Photoreceptor Cells, Vertebrate/pathology , Receptors, Purinergic P2Y12/genetics , Retinal Detachment/genetics , Retinal Detachment/pathology
8.
Nature ; 511(7509): 353-7, 2014 Jul 17.
Article in English | MEDLINE | ID: mdl-25030174

ABSTRACT

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Limbus Corneae/cytology , Limbus Corneae/physiology , Regeneration , Stem Cells/metabolism , Wound Healing , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP-Binding Cassette Transporters/deficiency , Animals , Apoptosis , Biomarkers/metabolism , Cell Differentiation , Cell Proliferation , Female , Humans , Male , Mice , Mice, Knockout , Molecular Sequence Data , Stem Cell Transplantation , Stem Cells/cytology , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism
9.
Exp Eye Res ; 179: 47-54, 2019 02.
Article in English | MEDLINE | ID: mdl-30365944

ABSTRACT

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.


Subject(s)
Debridement , Disease Models, Animal , Epithelium, Corneal/surgery , Fas Ligand Protein/physiology , Keratitis/prevention & control , Adenoviridae/genetics , Animals , Blotting, Western , Corneal Neovascularization/etiology , Corneal Neovascularization/prevention & control , Corneal Stroma/metabolism , Female , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/physiology , Genetic Vectors , Green Fluorescent Proteins/metabolism , Keratitis/etiology , Keratitis/pathology , Lipopolysaccharides/toxicity , Mice , Mice, Inbred C57BL , Plasmids/genetics
10.
J Immunol ; 197(12): 4626-4638, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27849168

ABSTRACT

Glaucoma is a multifactorial disease resulting in the death of retinal ganglion cells (RGCs) and irreversible blindness. Glaucoma-associated RGC death depends on the proapoptotic and proinflammatory activity of membrane-bound Fas ligand (mFasL). In contrast to mFasL, the natural cleavage product, soluble Fas ligand (sFasL) inhibits mFasL-mediated apoptosis and inflammation and, therefore, is an mFasL antagonist. DBA/2J mice spontaneously develop glaucoma and, predictably, RGC destruction is exacerbated by expression of a mutated membrane-only FasL gene that lacks the extracellular cleavage site. Remarkably, one-time intraocular adeno-associated virus-mediated gene delivery of sFasL provides complete and sustained neuroprotection in the chronic DBA/2J and acute microbead-induced models of glaucoma, even in the presence of elevated intraocular pressure. This protection correlated with inhibition of glial activation, reduced production of TNF-α, and decreased apoptosis of RGCs and loss of axons. These data indicate that cleavage of FasL under homeostatic conditions, and the ensuing release of sFasL, normally limits the neurodestructive activity of FasL. The data further support the notion that sFasL, and not mFasL, contributes to the immune-privileged status of the eye.


Subject(s)
Fas Ligand Protein/metabolism , Genetic Therapy , Glaucoma/therapy , Neuroprotection , Retinal Ganglion Cells/physiology , Acute Disease , Animals , Apoptosis , Cells, Cultured , Chronic Disease , Dependovirus/genetics , Disease Models, Animal , Fas Ligand Protein/genetics , Female , Glaucoma/genetics , Glaucoma/immunology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Tumor Necrosis Factor-alpha/metabolism
12.
Res Sq ; 2024 May 08.
Article in English | MEDLINE | ID: mdl-38766158

ABSTRACT

Neovascular age-related macular degeneration (AMD), results from choroidal neovascularization (CNV), retinal edema and loss of photoreceptors. Previous studies suggested that Fas Ligand (FasL) on retinal pigment epithelial cells inhibited CNV by inducing apoptosis of infiltrating Fas+ vascular endothelial cells. However, induction of apoptosis depends on membrane-bound (mFasL) while the FasL cleavage product (sFasL) is neuroprotective. To better understand how FasL regulates the development of CNV, we used a mouse model of laser CNV to evaluate the development of CNV in mice with a FasL cleavage site mutation (ΔCS) and can only express the membrane-bound form of FasL. There was no significant difference in CNV size and area of vascular leakage in homozygous FasLΔCS/ΔCS mice when compared to wild type mice. Unexpectedly, heterozygous FasLΔCS/WT mice developed significantly less vascular leakage and showed accelerated neovessel maturation. However, CNV was not prevented in heterozygous FasLΔCS/WT mice if the Fas receptor was deleted in myeloid cells (FasLΔCS/+ Fasflox/flox CreLysM). Thus, FasL-mediated CNV inhibition depends on the extent of FasL cleavage, and on FasL engagement of Fas+ myeloid cells. Moreover, accelerated neovessel maturation prevents vascular leakage in AMD.

13.
bioRxiv ; 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38585800

ABSTRACT

Autoimmune uveitis is a major cause of blindness in the working-age population of developed countries. Experimental autoimmune uveitis (EAU) depends on activation of interphotoreceptor retinoid-binding protein (IRBP) specific CD4 + effector T cells that migrate systemically and infiltrate into the retina. Following systemic induction of retinal antigen-specific T cells, the development of EAU can be broken down into three phases: early phase when inflammatory cells begin to infiltrate the retina, amplification phase, and peak phase. Although studied extensively, the function of local antigen-presenting cells (APCs) within the retina remains unclear. Two potential types of APCs are present during uveitis, resident microglia and infiltrating CD11c + dendritic cells (DCs). MHC class II (MHC II) is expressed within the retina on both CD11c + DCs and microglia during the amplification phase of EAU. Therefore, we used microglia specific (P2RY12 and TMEM119) and CD11c + DC specific MHC II knockout mice to study the function of APCs within the retina using the conventional and adoptive transfer methods of inducing EAU. Microglia were essential during all phases of EAU development: the early phase when microglia were MHC Il negative, and amplification and peak phases when microglia were MHC II positive. Unexpectedly, retinal infiltrating MHC Il + CD11c + DCs were present within the retina but their antigen-presenting function was not required for all phases of uveitis. Our data indicate microglia are the critical APCs within the retina and an important therapeutic target that can prevent and/or diminish uveitis even in the presence of circulating IRBP-specific CD4 + effector T cells.

14.
Invest Ophthalmol Vis Sci ; 65(8): 37, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-39042403

ABSTRACT

Purpose: Cell lines are being used in preclinical uveal melanoma (UM) research. Because not all cell lines harbor typical GNAQ or GNA11 hotspot mutations, we aimed at better classifying them and determining whether we could find genetic causes to explain the protein and mRNA expression profiles of the cell lines. Methods: We studied protein and mRNA expression of 14 UM cell lines and determined the presence of single nucleotide variants and small insertions and deletions with next-generation sequencing and copy number alterations with a single nucleotide polymorphism array. The lists of differentially expressed proteins and genes were merged, and shared lists were created, keeping only terms with concordant mRNA and protein expression. Enrichment analyses were performed on the shared lists. Results: Cell lines Mel285 and Mel290 are separate from GNA-mutated cell lines and show downregulation of melanosome-related markers. Both lack typical UM mutations but each harbors four putatively deleterious variants in CTNNB1, PPP1R10, LIMCH1, and APC in Mel285 and ARID1A, PPP1R10, SPG11, and RNF43 in Mel290. The upregulated terms in Mel285 and Mel290 did not point to a convincing alternative origin. Mel285 shows loss of chromosomes 1p, 3p, partial 3q, 6, and partial 8p, whereas Mel290 shows loss of 1p and 6. Expression in the other 12 cell lines was related to BAP1 expression. Conclusions: Although Mel285 and Mel290 have copy number alterations that fit UM, multi-omics analyses show that they belong to a separate group compared to the other analyzed UM cell lines. Therefore, they may not be representative models to test potential therapeutic targets for UM.


Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11 , GTP-Binding Protein alpha Subunits , Gene Expression Regulation, Neoplastic , Melanoma , Mutation , RNA, Messenger , Tumor Suppressor Proteins , Ubiquitin Thiolesterase , Uveal Neoplasms , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathology , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Humans , Ubiquitin Thiolesterase/genetics , RNA, Messenger/genetics , GTP-Binding Protein alpha Subunits/genetics , Tumor Suppressor Proteins/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Cell Line, Tumor , DNA Copy Number Variations , Polymorphism, Single Nucleotide , DNA Mutational Analysis
15.
Clin Cancer Res ; 2024 05 20.
Article in English | MEDLINE | ID: mdl-38767611

ABSTRACT

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic disease for which there are no effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T cell-based immunotherapy targeting B7-H3. EXPERIMENTAL DESIGN: B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, metastatic UM patient-derived organotypic tumor spheroids (PDOTS), and in immunodeficient and humanized murine models. RESULTS: B7-H3 is expressed at high levels on >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable anti-tumor response, even upon tumor re-challenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody. CONCLUSIONS: These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.

16.
Infect Immun ; 81(6): 2217-25, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23569113

ABSTRACT

FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.2 allotype is expressed in BALB/c mice and exhibits increased binding affinity to and increased cytotoxic activity against Fas(+) target cells. Therefore, we hypothesized that BALB/c mice would be more resistant to Staphylococcus aureus-induced endophthalmitis. To test this hypothesis, C57BL/6, BALB/c, and BALB(gld) mice received intravitreal injections of 2,500 CFU of S. aureus (RN6390). Clinical examinations, electroretinography (ERG), histology, and bacterial quantification were performed at 24, 48, 72, and 96 h postinjection. The myeloperoxidase (MPO) assay was used to quantitate neutrophil infiltration. At 96 h postinfection, 86% of C57BL/6 mice presented with complete destruction of the eye, compared to only 29% of BALB/c mice with complete destruction. To our surprise, in the absence of Fas ligand, BALB(gld) mice showed no difference in bacterial clearance compared to BALB/c mice. However, histology and ERG analysis revealed increased retinal damage and significant loss of retinal function. MPO analysis revealed equal numbers of neutrophils in BALB(gld) and BALB/c mice at 24 h postinfection. However, at 48 h, the neutrophil numbers remained significantly elevated in BALB(gld) mice, correlating with the increased retinal damage observed in BALB(gld) mice. We conclude that the increased resistance to S. aureus induced endophthalmitis in BALB/c mice is not dependent upon the FasL. However, in contrast to C57BL/6 mice, FasL is required for resolution of inflammation and protecting host tissue from nonspecific damage in BALB/c mice.


Subject(s)
Endophthalmitis/microbiology , Fas Ligand Protein/metabolism , Inflammation/metabolism , Staphylococcal Infections/immunology , Animals , Endophthalmitis/immunology , Endophthalmitis/pathology , Fas Ligand Protein/genetics , Female , Genetic Predisposition to Disease , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peroxidase/genetics , Peroxidase/metabolism , Retina/pathology , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus aureus
17.
J Immunol ; 186(12): 6822-9, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21555531

ABSTRACT

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a "molecular shield" by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.


Subject(s)
Antigens, CD/immunology , B7-1 Antigen/immunology , T-Lymphocytes/immunology , B7-1 Antigen/administration & dosage , B7-1 Antigen/genetics , B7-H1 Antigen , Cell Line, Tumor , Gene Expression/drug effects , Humans , Immunotherapy/methods , Lymphocyte Activation/immunology , Transfection
18.
Cell Reprogram ; 25(6): 288-299, 2023 12.
Article in English | MEDLINE | ID: mdl-38060815

ABSTRACT

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.


Subject(s)
Glaucoma , Neurodegenerative Diseases , Mice , Animals , Intraocular Pressure , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/therapy , Glaucoma/therapy , Glaucoma/drug therapy , Retina/metabolism , Genetic Therapy , Disease Models, Animal
19.
Mol Vis ; 18: 2805-13, 2012.
Article in English | MEDLINE | ID: mdl-23233783

ABSTRACT

PURPOSE: Retinoblastoma (RB), an intraocular tumor of childhood, is commonly associated with mutations in the RB1 gene. RB116 is a novel, early passage RB cell line that has not been previously characterized. In this study, we examined RB116 for the expression of RB1 and tested the hypothesis that RB116 cells would express stem cell markers as well as retinal progenitor cell markers. We compared RB116 cells with other well known RB cell lines, including Y79 and WERI-RB27. METHODS: We evaluated expression of RB1 in RB116 cells by sequencing, multiplex ligation-dependent probe amplification, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western immunoblot, and immunocytochemistry. Next, RB116 cells, along with Y79 and WERI-RB27 cells, were examined for expression of stem cell markers (ABCG2, Nanog, Oct3/4, ALDH1A1) and retinal progenitor markers (PAX6, CHX10) by quantitative immunocytochemistry. Immunocytochemical findings were accompanied by PCR analysis. RESULTS: RB116 cells expressed RB1 at the mRNA and protein levels, with no mutations detected by either sequencing analysis, or gene dosage abnormalities detected by multiplex ligation-dependent probe amplification. The RB1 protein was immunoreactive in RB116 cells with an atypical perinuclear localization. RB116 cells also expressed stem cell markers, with 3%-5% of cells immunopositive for ABCG2, Oct3/4 and ALDH1A1, with at least 18% of cells immunoreactive to Nanog. These findings were confirmed by RT-PCR. Small percentages of RB116 cells also exhibited immunoreactivity to retinal progenitor markers PAX6 (9.8%) and CHX10 (1.2%). Expression of mRNAs for these markers was confirmed by qRT-PCR. CONCLUSIONS: RB116 cells demonstrate RB1 expression accompanied by atypical perinuclear localization. RB116 cells also express primitive stem cell and retinal progenitor cell markers. Further studies on the phenotypes of both RB1-positive and RB1-negative human RB cells may be important in assessing differentiation potential of these cells, as well as designing targeted differentiation therapies.


Subject(s)
Cell Line, Tumor/metabolism , RNA, Messenger/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Stem Cells/metabolism , Biomarkers/metabolism , Cell Differentiation , Cell Line, Tumor/cytology , Founder Effect , Gene Expression , Humans , Immunohistochemistry , RNA, Messenger/genetics , Retinal Neoplasms/drug therapy , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/drug therapy , Retinoblastoma/metabolism , Retinoblastoma/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology
20.
Ocul Surf ; 23: 197-200, 2022 01.
Article in English | MEDLINE | ID: mdl-34653711

ABSTRACT

PURPOSE: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, the specific cellular tropism of SARS-CoV2 is not fully understood. In the current study, we evaluated the expression and regulation of two SARS-CoV2 viral entry proteins, TMPRSS2 and ACE2, in human ocular epithelial cells and stem cells. METHODS: TMPRSS2 and ACE2 expression in ABCB5-positive limbal stem cells (LSCs) were assessed by RNAseq, flow cytometry and immunohistochemistry. PAX6, TMPRSS2, and ACE2 mRNA expression values were obtained from the GSE135455 and DRA002960 RNA-seq datasets. siRNA-mediated PAX6 knockdown (KD) was performed in limbal and conjunctival epithelial cells. TMPRSS2 and ACE2 expression in the PAX6 KD cells was analyzed by qRT-PCR and Western blot. RESULTS: We found that ABCB5-positive LSCs express high levels of TMPRSS2 and ACE2 compared to ABCB5-negative limbal epithelial cells. Mechanistically, gene knockout and overexpression models revealed that the eye transcription factor PAX6 negatively regulates TMPRSS2 expression. Therefore, low levels of PAX6 in ABCB5-positive LSCs promote TMPRSS2 expression, and high levels of TMPRSS2 and ACE2 expression by LSCs indicate enhanced susceptibility to SARS-CoV2 infection in this stem cell population. CONCLUSIONS: Our study points to a need for COVID-19 testing of LSCs derived from donor corneas before transplantation to patients with limbal stem cell deficiency. Furthermore, our findings suggest that expandable human ABCB5+ LSC cultures might represent a relevant novel model system for studying cellular SARS-CoV2 viral entry mechanisms and evaluating related targeting strategies.


Subject(s)
COVID-19 , RNA, Viral , ATP Binding Cassette Transporter, Subfamily B , COVID-19 Testing , Humans , SARS-CoV-2 , Stem Cells , Viral Proteins , Virus Internalization
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