National Eye Institute

Innate immune cells respond rapidly to environmental cues through signal-regulated transcription factors (SRTFs) that sense changes in the tissue microenvironment. Signal transducer and activator of transcription (STAT) proteins are critical regulators of cytokine signaling and determine polarized immune responses. Herein, we reveal that activated type 2 innate lymphocytes (ILC2s) express STAT4, a SRTF canonically linked to type 1 immunity. STAT4 expression is induced in ILC2s upon activation by the alarmin IL-25 and linked with accumulation of lung inflammatory ILC2s (iILC2s). Despite elevated STAT4 expression, iILC2s do not acquire type 1 features, such as interferon (IFN)-γ production or T-bet expression and do not respond to IL-12 stimulation. Instead, STAT4 is activated by type I IFNs and supports the maintenance of the iILC2 pool. Transcriptomic analysis of Stat4-deficient ILC2s reveals enhanced type I IFN signaling and impaired proliferation, suggesting that STAT4 functions to antagonize IFN-driven suppression. Our data uncover a novel regulatory axis in which IL-25-induced STAT4 expression equips ILC2s to modulate interferon responses and to prevent aberrant autocrine function of type I IFNs, thus sustaining inflammatory effector populations during immune activation. These findings broaden the understanding of ILC2 activation and suggest new avenues for modulating innate lymphocytes in inflammatory diseases.

Bacteriophage-based display has been utilized for a variety of purposes, such as to assemble protein libraries and conduct biopanning. We have created a modified lambda (λ) bacteriophage platform, ideal for the display and delivery of proteins. Our system utilizes counter-selection recombineering for versatile modification, temperature-sensitive induction for timely lysate production, and an arabinose-inducible mechanism for high-titer, stable yield. Here, we investigated the ability of this specialized λ phage display platform to stimulate highly specific antibodies in mice against the displayed cancer-variant cell-surface receptor EGFRvIII, demonstrating its potential in cancer immunotherapy and broader vaccine development. λ display immunogenicity was explored by generating fusion proteins between the λ head protein D and a 13-mer peptide from the N terminus of glioblastoma variant cell-surface receptor, EGFRvIII. The 13-mer peptide was fused to either the N or C terminus of the λD protein while λ remained a dormant lysogen in the bacterial host chromosome. Recombinant phage lysates were then generated with ~420 displayed fusion proteins per phage particle. Mice were injected with purified recombinant λ phage without an adjuvant via both intraperitoneal and intramuscular routes, and sera harvested at various timepoints were profiled for immunogenicity. Analysis of serum samples by ELISA and Western blotting demonstrated the ability of the λD~EGFRvIII phage display, especially in the C-terminal fusion construction, to elicit a robust anti-EGFRvIII humoral response by either injection route. Notably, the antibody response was highly specific to EGFRvIII without exhibiting cross-reactivity to wild-type EGFR. The data generated in this study demonstrate the λ system's immunotherapeutic potential as a high-titer, stable, self-adjuvanting vector for the stimulation of robust antibody titers with defined specificity.

Immune responsiveness at barrier surfaces is tailored to the exposures of each tissue. In the oral mucosa, mechanisms by which a permeable epithelium coexists with diverse microbiota and maintains integrity during inflammatory pathology remain poorly understood. We compile a multiomics spatial map of this exposed mucosal microenvironment and uncover remarkable immune zonation with organization that is preserved even during inflammatory disease. At the tooth interface, we identify a dynamic epithelium underlined by a layer of neutrophils and a zone of antigen-presenting cell-lymphocyte aggregates. During disease, inflammatory zones expand and organize into immature tertiary lymphoid structures, suggesting local antibody production. Location-specific transcriptomes support a role for the stromal compartment in the spatial organization of immunity. This preserved immune zonation meets the demands for continuous protection of this vulnerable interface and suggests unique tissue-specific wiring of immunity at the human oral mucosal barrier.

ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome is a rare multisystem autoinflammatory disorder caused by heterozygous gain-of-function mutations in ALPK1 Initially characterised by these features, its clinical spectrum extends beyond the acronym. We report the first genetically confirmed case in Latin America. The proband, in her mid-20s, presented with progressive retinal dystrophy leading to vision loss, xerostomia, short dental roots, recurrent low-grade fevers and elevated C-reactive protein. Notably, she lacked splenomegaly and anhidrosis; instead, mild hypohidrosis was identified retrospectively following genetic confirmation of the ALPK1 p.Thr237Met variant. Her mother, carrying the same variant, exhibited blindness from advanced retinal degeneration, severe arthritis, recurrent fevers, hypohidrosis and inability to lactate. This family illustrates the variable expressivity of ROSAH syndrome and underscores that absence of classic features should not exclude the diagnosis. Early recognition and genetic testing are essential, as timely initiation of immunomodulatory therapy may mitigate the risk of loss of vision.

ObjectiveOver the years, the Age-Related Eye Disease Study (AREDS) has contributed to the natural history and understanding of progression related to age-related macular degeneration (AMD) and cataracts. This paper summarizes AREDS data elements available for research.DesignData set description for data acquired during AREDS.ParticipantsAdults aged 55 to 80 years with no to advanced AMD in 1 eye but visual acuity >20/32 in eye without advanced AMD.Methodsdatabase of Genotypes and Phenotypes archives and distributes data acquired in clinical studies such as AREDS. This article provides a detailed description of the data available under controlled access under the National Institutes of Health genomic data sharing Policy.Main Outcome MeasuresAge-Related Eye Disease Study evaluated progression to advanced AMD, 15-letter decrease in visual acuity, and progression of lens opacities as primary outcome measures.ResultsThe data set includes 4757 participants enrolled across 11 participating clinical centers representing varying AMD severity categories and lens opacities. The participants' demographics and clinical variables were collected during baseline and follow-up visits, and data elements included ophthalmic images (fundus and lens photographs) with reading center gradings; nutritional estimates captured from food frequency questionnaires; data related to quality of life, hospitalization, morbidity, and mortality; and genetic data (available in a subset of approximately 2400 participants who submitted blood samples).ConclusionsSummary of data available under controlled access acquired as part of AREDS was provided. The AREDS data set offers a valuable resource for advancing our understanding of AMD and cataract progression and for developing novel tools and applications to transform ophthalmic diagnostics and therapeutics.Financial Disclosure(s)Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Commotio retinae (CR) resulting from retinal trauma can lead to focal photoreceptor degeneration and permanent vision loss. Currently no therapies exist for CR-induced retinal degeneration, in part because of the lack of a large-animal model that replicates human injury pathology and allows testing of therapeutics. Severe CR is clinically characterized by subretinal fluid and focal photoreceptor outer nuclear layer thinning. To develop a porcine CR model, we developed a laser-guided projectile apparatus and optimized projectile delivery procedure using porcine cadaveric eyes embedded in a 3D-printed porcine skull. Scleral and corneal impacts resulted in retinal damage consistent with patient injury, but corneal impacts also led to cornea damage and opacification, which precluded follow-up imaging. In live porcine eyes, scleral impacts of 39.5 m/s induced transient blood-retinal barrier breakdown evidenced by subretinal fluid on optical coherence tomography (OCT), leakage observed on fluorescein and indocyanine green angiography, and transient photoreceptor outer segment disruption seen by OCT and multifocal electroretinography. Impacts above 39.5 m/s induced longer-lasting photoreceptor degeneration but only transient blood-retinal barrier breakdown. This porcine model, combined with clinically relevant imaging and diagnostic modalities, will be valuable for testing the safety and efficacy of therapies to restore vision after focal photoreceptor degeneration.

Growing evidence indicates that PKLR, the gene for pyruvate kinase (PK), is a genetic modifier of the sickle cell phenotype. Coinheritance of specific PKLR variants is associated with increased pain-related hospitalization and can trigger sickle cell disease (SCD) phenotypes in asymptomatic carriers. PK deficiency disrupts RBC glycolysis, leading to ATP deficits and accumulation of 2,3-diphosphoglycerate, which exacerbates sickling in SCD. Using CRISPR-Cas9, we generated null mutations in Pklr [Pklr(13ntdel/13ntdel) or Pklr(246ntdel/246ntdel)] specific for the RBC isoform (PKR) in Townes mice that were homozygous (SS) or heterozygous (AS) for the human sickle globin gene, or homozygous for human hemoglobin A (AA, controls), to investigate the effect of PKR deficiency on the sickle phenotype in mice. PKR-deficient AA and AS mice developed severe anemia, reticulocytosis, and substantial spleen and liver iron deposits. Unlike what is observed in humans, PKR deficiency in AS and SS mice surprisingly decreased sickling, but it was also associated with increased extramedullary hematopoiesis and mitochondrial retention in mature RBCs. These results demonstrate the differential effect of Pklr mutations on the phenotype of both AS and SS mouse models, offering insights into the complex role of PKR deficiency in SCD pathology.

Introduction: Pediatric cataracts are unique in that surgical treatment, while visually beneficial, causes instantaneous presbyopia. Choosing an intraocular lens (IOL) poses a distinct challenge in children, including accounting for eye growth and myopic shift. Specialty IOLs such as enhanced monofocal lens (EML) implants may allow a subset of pediatric patients to achieve good visual acuity at a range of distances. Use of these lenses is well-characterized in adults, but data regarding their use in children are sparse. Patient and Clinical Findings: A retrospective case series of 6 eyes of 5 patients ranging in age from 7 to 15 years old, undergoing CE/IOL with a TECNIS Eyehance EML was performed at a single academic medical center. Diagnosis, Interventions, and Outcomes: Of the 5 patients, 4 underwent CE/IOL unilaterally, and the fifth bilaterally and sequentially. All 6 eyes showed improved distance visual acuity; 1 patient required correction of intentional myopia. 3 of 6 eyes had uncorrected near vision within 3 lines or less of their distance acuity; 1 of these eyes had uncorrected near acuity of 20/40. 4 of 5 patients achieved uncorrected stereopsis of 400 arc seconds. The patient with the lowest distance acuity, near acuity, and stereopsis also had significant retinal comorbidities contributing to limited visual outcomes. Conclusions: To the authors' knowledge, this is the first report of Eyehance lens use in children. Enhanced monofocal specialty lenses can be safe and effective in select pediatric patients, yielding good distance and near vision, and preservation of stereopsis.