Therapeutic intervention in neuroinflammation for neovascular ocular diseases through targeting the cGAS-STING-necroptosis pathway.

The microglia-mediated neuroinflammation have been shown to play a crucial role in the ocular pathological angiogenesis process, but specific immunotherapies for neovascular ocular diseases are still lacking. This study proposed that targeting GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) might be a novel immunotherapy for these angiogenesis diseases. We found a significant upregulation of CGAS and STING genes in the RNA-seq data derived from retinal tissues of the patients with proliferative diabetic retinopathy. In experimental models of ocular angiogenesis including laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), the cGAS-STING pathway was activated as angiogenesis progressed. Either genetic deletion or pharmacological inhibition of STING resulted in a remarkable suppression of neovascularization in both models. Furthermore, cGAS-STING signaling was specifically activated in myeloid cells, triggering the subsequent RIP1-RIP3-MLKL pathway activation and leading to necroptosis-mediated inflammation. Notably, targeted inhibition of the cGAS-STING pathway with C-176 or SN-011 could significantly suppress pathological angiogenesis in CNV and OIR. Additionally, the combination of C-176 or SN-011 with anti-VEGF therapy led to least angiogenesis, markedly enhancing the anti-angiogenic effectiveness. Together, our findings provide compelling evidence for the importance of the cGAS-STING-necroptosis axis in pathological angiogenesis, highlighting its potential as a promising immunotherapeutic target for treating neovascular ocular diseases.

Refractive stability and timing of spectacle prescription following cataract surgery in myopic eyes.

PURPOSE: To investigate the post-operative refractive stabilisation time and provide evidence for the optimal timing of a spectacle prescription in myopic post-cataract surgery patients. METHODS: A total of 116 consecutive myopic cataract patients were recruited from the Zhongshan Ophthalmic Center in this prospective study. Post-operative subjective refraction was assessed after 1 week and 1 month (4-6 weeks), with the interval for the new spectacle acquisition being recorded. Visual Function Index-14 (VF-14) questionnaires were used to assess the vision-related quality of life. RESULTS: There was no significant difference in spherical (p = 0.33), cylindrical (p = 0.65) or spherical equivalent refractions (p = 0.45) obtained 1 week and 1 month post-operatively, indicating that subjects achieved refractive stability within 1 week. In subgroups having differing age and axial lengths, there were also no significant differences between the 1 week and 1 month findings. The spherical equivalent refractive shift between 1 week and 1 month was significantly correlated with the post-operative prediction error (R = 0.35; p < 0.001). Only five (4.3%) out of 116 patients obtained new spectacles 1 week post-surgery. The VF-14 values improved from 85.77 ± 7.24 to 90.45 ± 5.39 after acquiring new spectacles (p < 0.01). CONCLUSIONS: The stabilisation of subjective refraction occurred within 1 week in myopic cataract patients. Shortening the interval before prescribing a new spectacle prescription is recommended for myopic patients following cataract surgery to improve their vision-related quality of life.

Macrophage-induced integrin signaling promotes Schlemm's canal formation to prevent intraocular hypertension and glaucomatous optic neuropathy.

Schlemm's canal (SC) functions to maintain proper intraocular pressure (IOP) by draining aqueous humor and has emerged as a promising therapeutic target for glaucoma, the second-leading cause of irreversible blindness worldwide. However, our current understanding of the mechanisms governing SC development and functionality remains limited. Here, we show that vitronectin (VTN) produced by limbal macrophages promotes SC formation and prevents intraocular hypertension by activating integrin αvß3 signaling. Genetic inactivation of this signaling system inhibited the phosphorylation of AKT and FOXO1 and reduced ß-catenin activity and FOXC2 expression, thereby causing impaired Prox1 expression and deteriorated SC morphogenesis. This ultimately led to increased IOP and glaucomatous optic neuropathy. Intriguingly, we found that aged SC displayed downregulated integrin ß3 in association with dampened Prox1 expression. Conversely, FOXO1 inhibition rejuvenated the aged SC by inducing Prox1 expression and SC regrowth, highlighting a possible strategy by targeting VTN/integrin αvß3 signaling to improve SC functionality.

Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF-macrophage pathway.

Optic neuropathies, characterized by injury of retinal ganglion cell (RGC) axons of the optic nerve, cause incurable blindness worldwide. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) represent a promising "cell-free" therapy for regenerative medicine; however, the therapeutic effect on neural restoration fluctuates, and the underlying mechanism is poorly understood. Here, we illustrated that intraocular administration of MSC-sEVs promoted both RGC survival and axon regeneration in an optic nerve crush mouse model. Mechanistically, MSC-sEVs primarily targeted retinal mural cells to release high levels of colony-stimulating factor 3 (G-CSF) that recruited a neural restorative population of Ly6Clow monocytes/monocyte-derived macrophages (Mo/MΦ). Intravitreal administration of G-CSF, a clinically proven agent for treating neutropenia, or donor Ly6Clow Mo/MΦ markedly improved neurological outcomes in vivo. Together, our data define a unique mechanism of MSC-sEV-induced G-CSF-to-Ly6Clow Mo/MΦ signaling in repairing optic nerve injury and highlight local delivery of MSC-sEVs, G-CSF, and Ly6Clow Mo/MΦ as therapeutic paradigms for the treatment of optic neuropathies.

General requirements for the production of extracellular vesicles derived from human stem cells.

'General requirements for the production of extracellular vesicles derived from human stem cells' is the first guideline for stem cells derived extracellular vesicles in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the general requirements, process requirements, packaging and labelling requirements and storage requirements for preparing extracellular vesicles derived from human stem cells, which is applicable to the research and production of extracellular vesicles derived from stem cells. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardisation of extracellular vesicles derived from human stem cells.

A primate nigrostriatal atlas of neuronal vulnerability and resilience in a model of Parkinson's disease.

The degenerative process in Parkinson's disease (PD) causes a progressive loss of dopaminergic neurons (DaNs) in the nigrostriatal system. Resolving the differences in neuronal susceptibility warrants an amenable PD model that, in comparison to post-mortem human specimens, controls for environmental and genetic differences in PD pathogenesis. Here we generated high-quality profiles for 250,173 cells from the substantia nigra (SN) and putamen (PT) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian macaques and matched controls. Our primate model of parkinsonism recapitulates important pathologic features in nature PD and provides an unbiased view of the axis of neuronal vulnerability and resistance. We identified seven molecularly defined subtypes of nigral DaNs which manifested a gradient of vulnerability and were confirmed by fluorescence-activated nuclei sorting. Neuronal resilience was associated with a FOXP2-centered regulatory pathway shared between PD-resistant DaNs and glutamatergic excitatory neurons, as well as between humans and nonhuman primates. We also discovered activation of immune response common to glial cells of SN and PT, indicating concurrently activated pathways in the nigrostriatal system. Our study provides a unique resource to understand the mechanistic connections between neuronal susceptibility and PD pathophysiology, and to facilitate future biomarker discovery and targeted cell therapy.

HIF-1α Reduction by Lowering Intraocular Pressure Alleviated Retinal Neovascularization.

Hypoxia-induced retinal neovascularization is a leading cause of blindness worldwide. Oxygen-induced retinopathy (OIR) mouse, a well-established angiogenesis model, has been extensively used to evaluate the effect of anti-angiogenic agents through intravitreal injection. Here, we serendipitously found that the needles used for intravitreal injection caused an unexpected "anti-angiogenic" effect in the OIR mice. To evaluate the effects of various intravitreal puncture sizes on retinal neovascularization and explore the potential underlying mechanism, intravitreal punctures using 0.5 mm (25 G), 0.3 mm (30 G), or 0.21 mm (33 G) needles were performed in OIR mice. Compared with 0.3 mm and 0.21 mm puncture, the 0.5 mm puncture remarkably suppressed the formation of pathological angiogenesis, inhibited vascular leakage, and remodeled the retinal vasculature. Mechanistically, the 0.5 mm puncture induced a substantial reduction in intraocular pressure (IOP), leading to an improvement in oxygen partial pressure (pO2) and significant reduction in Hif1a expression, resulting in resolution of angiogenic and inflammatory responses. Furthermore, IOP-lowering drugs, Travatan or Azarga, also promoted the alleviation of hypoxia and exhibited a potent anti-angiogenesis efficacy. Our study revealed an acute and significant reduction in IOP caused by a large puncture, which could remarkably suppress HIF-1α-mediated retinal neovascularization, indicating that lowering IOP may be a promising therapeutic avenue for treating retinal neovascular diseases.

Nonhuman Primate Eyes Display Variable Growth and Aging Rates in Alignment With Human Eyes.

Purpose: To assess age-related biometric changes of the eye in nonhuman primates (NHPs), to and decipher the growth and aging rates and their comparability with humans. Methods: Ocular anatomic measurements were performed on 341 macaca fascicularis aged 0.5 to 23 years via multimodal approaches including IOLMaster 700. Linear or polynomial regression models were simulated to determine the best fitted age-related function. The metrics were compared with human equivalents in published reports. Results: Macaques exhibited a postnatal eye growth pattern similar to humans, characterized by continuous eye extension coordinated with dramatic reshaping of the lens but not the cornea. The age-related growth of lens thickness (LT), anterior chamber depth (ACD), and axis length (AL) exhibited nonlinear and bipolar patterns. The inflection points were 10 to 12 years old for LT and ACD and 13 to 15 years old for AL in macaques, which were comparable in chronological age at a ratio of ∼1: ratio with that in humans. In contrast, the speed of aging, including the increase in lens density and the decrease in retinal nerve fiber layer thickness, was comparable in relative age at a ratio of ∼1:3 according to the differences in lifespan between macaques and humans. Lens density was a robust indicator for the aging process. Conclusions: Macaque eyes recapitulated the age-related process of human eyes to varying extents with different growth and aging rates. Chronological age or relative age should be considered in different scenarios when macaques are included in preclinical studies.

VEGF-B prevents excessive angiogenesis by inhibiting FGF2/FGFR1 pathway.

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Objective Quantification of Lens Opacity in Posterior Subcapsular Cataracts Using IOL Master 700 and CASIA-2.

PURPOSE: To objectively quantify the lens opacity of posterior subcapsular cataracts (PSCs) using the swept-source optical coherence tomography (SS-OCT)-based devices including IOL Master 700 and CASIA-2. DESIGN: Prospective cross-sectional study. METHODS: A total of 101 eyes of 101 patients with PSCs were enrolled in Zhongshan Ophthalmic Center from 2021 to 2022. The IOL Master 700 and CASIA-2 were used to obtain lens images. The average posterior subcapsular density (APSD) and the maximum posterior subcapsular density (MPSD) within the pupil area (radius: 3 or 5 mm) were measured by Image J. Spearman and Pearson correlation analysis were performed to assess the associations. RESULTS: APSD-3mm, APSD-5mm, MPSD-3mm, and MPSD-5mm had positive correlations with best corrected visual acuity (BCVA; r = 0.658, 0.641, 0.583, and 0.572, P < .001, respectively), all of which were higher than the correlation between LOCS-III P score and BCVA (r = 0.548, P < .001). Particularly, the APSD-3mm showed the highest correlation with BCVA. APSD could distinguish severe PSCs (LOCS-III P score ≥ 5) with an area under the receiver operating characteristic curve (AUC) of 0.836 (95% CI 0.743-0.930) for APSD-3mm and with AUC 0.758 (95% CI 0.643-0.873) for APSD-5mm, highlighting the better performance of APSD-3mm. The APSD-3mm of IOL Master 700 correlated strongly with that of CASIA-2 (r = 0.789, P < .001). CONCLUSIONS: This study presented an objective method for quantifying PSCs using IOL Master 700 and CASIA-2. APSD-3mm can be used as a new accurate and objective index for the quantitative assessment of PSCs.

Using an acousto-optic modulator as a fast spatial light modulator.

High-speed spatial light modulators (SLM) are crucial components for free-space communication and structured illumination imaging. Current approaches for dynamical spatial mode generation, such as liquid crystal SLMs or digital micromirror devices, are limited to a maximum pattern refresh rate of 10 kHz and have a low damage threshold. We demonstrate that arbitrary spatial profiles in a laser pulse can be generated by mapping the temporal radio-frequency (RF) waveform sent to an acousto-optic modulator (AOM) onto the optical field. We find that the fidelity of the SLM performance can be improved through numerical optimization of the RF waveform to overcome the nonlinear effect of AOM. An AOM can thus be used as a 1-dimensional SLM, a technique we call acousto-optic spatial light modulator (AO-SLM), which has 50 µm pixel pitch, over 1 MHz update rate, and high damage threshold. We simulate the application of AO-SLM to single-pixel imaging, which can reconstruct a 32×32 pixel complex object at a rate of 11.6 kHz with 98% fidelity.

IL-4 induces reparative phenotype of RPE cells and protects against retinal neurodegeneration via Nrf2 activation.

Retinal degeneration is a kind of neurodegeneration characterized by progressive neuronal death and dysfunction of retinal pigment epithelium (RPE) cells, leading to permanent visual impairment. It still lacks effective therapeutic options and new drugs are highly warranted. In this study, we found the expression of IL-4, a critical regulator of immunity, was reduced in both patients and mouse models. Importantly, exogenous intravitreal IL-4 application could exert a novel neuroprotective effect, characterized by well-preserved RPE layer and neuroretinal structure, as well as amplified wave-amplitudes in ERG. The RNA-seq analysis revealed that IL-4 treatment suppressed the essential oxidative and pro-inflammatory pathways in the degenerative retina. Particularly, IL-4 upregulated the IL-4Rα on RPE cells and induced a reparative phenotype via the activation of Nrf2 both in vitro and in vivo. Furthermore, the Nrf2-/- mice displayed no recovery in response to IL-4 application, highlighting a significant role of Nrf2 in IL-4-mediated protection. Our data provides evidence that IL-4 protects against retinal neurodegeneration by its antioxidant and anti-inflammatory property through IL-4Rα upregulation and Nrf2 activation in RPE cells. The IL-4/IL-4Rα-Nrf2 axis maybe the potential targets for the development of novel therapies for neurodegenerative diseases.

Multicellular immune dynamics implicate PIM1 as a potential therapeutic target for uveitis.

Uveitis is a severe autoimmune disease, and a common cause of blindness; however, its individual cellular dynamics and pathogenic mechanism remain poorly understood. Herein, by performing single-cell RNA sequencing (scRNA-seq) on experimental autoimmune uveitis (EAU), we identify disease-associated alterations in cell composition and transcriptional regulation as the disease progressed, as well as a disease-related molecule, PIM1. Inhibiting PIM1 reduces the Th17 cell proportion and increases the Treg cell proportion, likely due to regulation of PIM1 to the protein kinase B (AKT)/Forkhead box O1 (FOXO1) pathway. Moreover, inhibiting PIM1 reduces Th17 cell pathogenicity and reduces plasma cell differentiation. Importantly, the upregulation of PIM1 in CD4+ T cells and plasma cells is conserved in a human uveitis, Vogt-Koyanagi-Harada disease (VKH), and inhibition of PIM1 reduces CD4+ T and B cell expansion. Collectively, a dynamic immune cellular atlas during uveitis is developed and implicate that PIM1 may be a potential therapeutic target for VKH.

Linking transcriptomes with morphological and functional phenotypes in mammalian retinal ganglion cells.

Retinal ganglion cells (RGCs) are the brain's gateway to the visual world. They can be classified into different types on the basis of their electrophysiological, transcriptomic, or morphological characteristics. Here, we characterize the transcriptomic, morphological, and functional features of 472 high-quality RGCs using Patch sequencing (Patch-seq), providing functional and morphological annotation of many transcriptomic-defined cell types of a previously established RGC atlas. We show a convergence of different modalities in defining the RGC identity and reveal the degree of correspondence for well-characterized cell types across multimodal data. Moreover, we complement some RGC types with detailed morphological and functional properties. We also identify differentially expressed genes among ON, OFF, and ON-OFF RGCs such as Vat1l, Slitrk6, and Lmo7, providing candidate marker genes for functional studies. Our research suggests that the molecularly distinct clusters may also differ in their roles of encoding visual information.

Comparison of Formula-Specific Factors and Artificial Intelligence Formulas with Axial Length Adjustments in Bilateral Cataract Patients with Long Axial Length.

INTRODUCTION: To evaluate and compare the effectiveness for reducing the prediction error (PE) of the second eye using formula-specific factors, artificial intelligence (AI) formulas (PEARL-DGS and Kane), and the Cooke-modified axial length (CMAL) methods in bilateral cataract patients with long axial length (AL). METHODS: A total of 98 patients with long AL who underwent sequential bilateral cataract surgeries were retrospectively enrolled. The second-eye IOL power was calculated by the formula-specific factors, AI formulas, and CMAL methods when the first eye suffered from refraction surprise. The correction factors of eight formulas were calculated by regression analysis. RESULTS: There was a significant correlation between bilateral preoperative biometric parameters (P < 0.05) as well as bilateral PE (P < 0.05). The Kane formula displayed the lowest median absolute error (MedAE) and highest proportion of PE within ± 0.50 and ± 1.00 D compared with other formulas for the first eye. For the second-eye refinement, all three methods could reduce the second-eye MedAE. The formula-specific correction factors were 0.250, 0.331, 0.343, 0.394, 0.409, 0.452, 0.503, and 0.520 for Kane, Barrett Universal II (BUII), PEARL-DGS, Holladay 2, Holladay 1, Haigis, Hoffer Q, and SRK/T, respectively. The new AI-based Kane and PEARL-DGS with or without the CMAL methods could improve the refractive outcomes of the second eye in sequential bilateral cataract patients with long AL. The Kane, BUII, and PEARL-DGS with specific correction factors displayed higher accuracy compared with the other two methods (P < 0.05). CONCLUSIONS: The new AI-based Kane and PEARL-DGS with or without the CMAL methods could improve the refractive outcomes of the second eye in sequential bilateral cataract patients with long AL. Notably, the Kane, PEARL-DGS, and BUII with specific correction factors displayed higher accuracy.

Identification of TPBG-Expressing Amacrine Cells in DAT-tdTomato Mouse.

Purpose: Neurons are the bricks of the neuronal system and experimental access to certain neuron subtypes will be of great help to decipher neuronal circuits. Here, we identified trophoblast glycoprotein (TPBG)-expressing GABAergic amacrine cells (ACs) that were selectively labeled in DAT-tdTomato transgenic mice. Methods: Retina and brain sections were prepared for immunostaining with antibodies against various biomarkers. Patch-sequencing was performed to obtain the transcriptomes of tdTomato-positive cells in DAT-tdTomato mice. Whole-cell recordings were conducted to identify responses to light stimulation. Results: Tyrosine hydroxylase immunoreactive cells were colocalized with tdTomato-positive cells in substantia nigra pars compacta, but not in the retina. Transcriptomes collected from tdTomato-positive cells in retinas via Patch-sequencing exhibited the expression of marker genes of ACs (Pax6 and Slc32a1) and marker genes of GABAergic neurons (Gad1, Gad2, and Slc6a1). Immunostaining with antibodies against relevant proteins (GAD67, GAD65, and GABA) also confirmed transcriptomic results. Furthermore, tdTomato-positive cells in retinas selectively expressed Tpbg, a marker gene for distinct clusters molecularly defined, which was proved with TPBG immunoreactivity in fluorescently labeled cells. Finally, tdTomato-positive cells recorded showed ON-OFF responses to light stimulation. Conclusions: Ectopic expression occurs in the retina but not in the substantia nigra pars compacta in the DAT-tdTomato mouse, and fluorescently labeled cells in the retina are TPBG-expressing GABAergic ACs. This type of transgenic mice has been proved as an ideal tool to achieve efficient labeling of a distinct subset of ACs that selectively express Tpbg.

Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations.

The extent to which neurogenesis occurs in adult primates remains controversial. In this study, using an optimized single-cell RNA sequencing pipeline, we profiled 207,785 cells from the adult macaque hippocampus and identified 34 cell populations comprising all major hippocampal cell types. Analysis of their gene expression, specification trajectories and gene regulatory networks revealed the presence of all key neurogenic precursor cell populations, including a heterogeneous pool of radial glia-like cells (RGLs), intermediate progenitor cells (IPCs) and neuroblasts. We identified HMGB2 as a novel IPC marker. Comparison with mouse single-cell transcriptomic data revealed differences in neurogenic processes between species. We confirmed that neurogenesis is recapitulated in ex vivo neurosphere cultures from adult primates, further supporting the existence of neural precursor cells (NPCs) that are able to proliferate and differentiate. Our large-scale dataset provides a comprehensive adult neurogenesis atlas for primates.

Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis.

Photoreceptor connecting cilium (CC) is structurally analogous to the transition zone (TZ) of primary cilia and gates the molecular trafficking between the inner and the outer segment (OS). Retinal dystrophies with underlying CC defects are manifested in a broad array of syndromic conditions known as ciliopathies as well as nonsyndromic retinal degenerations. Despite extensive studies, many questions remain in the mechanism of protein trafficking across the photoreceptor CC. Here, we genetically inactivated mouse Tmem138, a gene encoding a putative transmembrane protein localized to the ciliary TZ and linked to ciliopathies. Germline deletion of Tmem138 abolished OS morphogenesis, followed by rapid photoreceptor degeneration. Tmem138 was found localized to the photoreceptor CC and was required for localization of Ahi1 to the distal subdomain of the CC. Among the examined set of OS proteins, rhodopsin was mislocalized throughout the mutant cell body prior to OS morphogenesis. Ablation of Tmem138 in mature rods recapitulated the molecular changes in the germline mutants, causing failure of disc renewal and disintegration of the OS. Furthermore, Tmem138 interacts reciprocally with rhodopsin and a related protein Tmem231, and the ciliary localization of the latter was also altered in the mutant photoreceptors. Taken together, these results suggest a crucial role of Tmem138 in the functional organization of the CC, which is essential for rhodopsin localization and OS biogenesis.

miR-204-containing exosomes ameliorate GVHD-associated dry eye disease.

Graft-versus-host disease (GVHD)­associated dry eye disease is characterized by extensive inflammatory destruction in the ocular surface and causes unbearable pain and visual impairment. Current treatments provide limited benefits. Here, we report that exosomes from mesenchymal stromal cells (MSC-exo) administered as eye drops notably alleviate GVHD-associated dry eye disease by suppressing inflammation and improving epithelial recovery in mice and humans. In a prospective clinical trial, 28 eyes with refractory GVHD­dry eye disease exhibited substantial relief after MSC-exo treatment, showing reduced fluorescein scores, longer tear-film breakup time, increased tear secretion, and lower OSDI scores. Mechanistically, MSC-exo reprogramed proinflammatory M1 macrophages toward the immunosuppressive M2 via miR-204­mediated targeting of the IL-6/IL-6R/Stat3 pathway. Blockade of miR-204 abolished the effects of MSC-exo, while overloading L929-exo with miR-204 markedly attenuated dry eye. Thus, this study suggests that MSC-exo are efficacious in treating GVHD-associated dry eye disease and highlights miR-204 as a potential therapeutic agent.