Intraocular mRNA delivery with endogenous MmPEG10-based virus-like particles.

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy.

Deciphering the impact of aging on splenic endothelial cell heterogeneity and immunosenescence through single-cell RNA sequencing analysis.

BACKGROUND: Aging is associated with significant structural and functional changes in the spleen, leading to immunosenescence, yet the detailed effects on splenic vascular endothelial cells (ECs) and their immunomodulatory roles are not fully understood. In this study, a single-cell RNA (scRNA) atlas of EC transcriptomes from young and aged mouse spleens was constructed to reveal age-related molecular changes, including increased inflammation and reduced vascular development and also the potential interaction between splenic endothelial cells and immune cells. RESULTS: Ten clusters of splenic endothelial cells were identified. DEGs analysis across different EC clusters revealed the molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the loss in vascular development function of aged ECs. Notably, four EC clusters with immunological functions were identified, suggesting an Endothelial-to-Immune-like Cell Transition (EndICLT) potentially driven by aging. Pseudotime analysis of the Immunology4 cluster further indicated a possible aging-induced transitional state, potentially initiated by Ctss gene activation. Finally, the effects of aging on cell signaling communication between different EC clusters and immune cells were analyzed. CONCLUSIONS: This comprehensive atlas elucidates the complex interplay between ECs and immune cells in the aging spleen, offering new insights into endothelial heterogeneity, reprogramming, and the mechanisms of immunosenescence.

Enhancement of Large Language Models' Performance in Diabetes Education: Retrieval-Augmented Generation Approach.

BACKGROUND: Large language models (LLMs) demonstrated advanced performance in processing clinical information. However, commercially available LLMs lack specialized medical knowledge and remain susceptible to generating inaccurate information. Given the need for self-management in diabetes, patients commonly seek information online. We introduce the RISE framework and evaluate its performance in enhancing LLMs to provide accurate responses to diabetes-related inquiries. OBJECTIVE: This study aimed to evaluate the potential of RISE framework, an information retrieval and augmentation tool, to improve the LLM's performance to accurately and safely respond to diabetes-related inquiries. METHODS: The RISE, an innovative retrieval augmentation framework, comprises four steps: Rewriting Query, Information Retrieval, Summarization, and Execution. Using a set of 43 common diabetes-related questions, we evaluated three base LLMs (GPT-4, Anthropic Claude 2, Google Bard) and their RISE-enhanced versions. Assessments were conducted by clinicians for accuracy and comprehensiveness, and by patients for understandability. RESULTS: The integration of RISE significantly improved the accuracy and comprehensiveness of responses from all three based LLMs. On average, the percentage of accurate responses increased by 12% (122 - 107/129) with RISE. Specifically, the rates of accurate responses increased by 7% (42 - 39/43) for GPT-4, 19% (39 - 31/43) for Claude 2, and 9% (41 - 37/43) for Google Bard. The framework also enhanced response comprehensiveness, with mean scores improving by 0.44. Understandability was also enhanced by 0.19 on average. Data collection was conducted from Sept. 30, 2023, to Feb. 05, 2024. CONCLUSIONS: RISE significantly improves LLMs' performance in responding to diabetes-related inquiries, enhancing accuracy, comprehensiveness, and understandability. These improvements have crucial implications for RISE's future role in patient education and chronic illness self-management, which contributes to relieving medical resource pressures and raising public awareness of medical knowledge.

High-dimensional single-cell analysis reveals the immune characteristics of COVID-19.

Coronavirus disease 2019 (COVID-19), driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a global pandemic in March 2020. Pathogenic T cells and inflammatory monocytes are regarded as the central drivers of the cytokine storm associated with the severity of COVID-19. In this study, we explored the characteristic peripheral cellular profiles of patients with COVID-19 in both acute and convalescent phases by single-cell mass cytometry (CyTOF). Using a combination of algorithm-guided data analyses, we identified peripheral immune cell subsets in COVID-19 and revealed CD4+ T-cell depletion, T-cell differentiation, plasma cell expansion, and the reduced antigen presentation capacity of innate immunity. Notably, COVID-19 induces a dysregulation in the balance of monocyte populations by the expansion of the monocyte subsets. Collectively, our results represent a high-dimensional, single-cell profile of the peripheral immune response to SARS-CoV-2 infection.

Differential expression of α6 and ß1 integrins reveals epidermal heterogeneity at single-cell resolution.

Skin epidermis is a constantly renewing epithelium that is composed of various cell types and provides an ideal model system for tissue regeneration and heterogeneity studies. Integrins are a family of transmembrane receptors that mediate cell adhesion in the epidermis, and integrin expression spatially reflects epidermal heterogeneity. It remains unclear whether differential expression of integrins can characterize cell types in skin epidermis. This study applied a fluorescence-activated cell sorting (FACS) strategy based on differential expression of α6 and ß1 integrins, and used transcriptome analysis to explore epidermal heterogeneity. First, epidermal cells were acquired from C57BL/6 mice back skin. Nine bulk-cell populations were sorted with differential expressions of α6 and ß1 integrins, and were successfully characterized as the main cell types in the epidermis through RNA-seq and transcriptome analysis. Then, tetOKrt14-H2BGFP mice were used to trace the cell proliferation rate during wound healing with GFP intensity. Epidermal cells were acquired from the re-epithelialized back skin wounds, and a total of 576 single cells were sorted, combining integrin expression and GFP tracing. FACS single-cell RNA-seq enabled high resolution in the classification of subtypes in both interfollicular epidermis and hair follicle, and both quiescent and intermediate cell states of the basal and infundibulum stem cell populations were distinguished. This study proposed a presorted method to investigate the relationship between integrin expression and epidermal heterogeneity. Multiple epidermal cell types and their expression profiles were identified, which provides data resources for dermatology research.

Single-cell chromatin accessibility and transcriptomic characterization of Behcet's disease.

Behect's disease is a chronic vasculitis characterized by complex multi-organ immune aberrations. However, a comprehensive understanding of the gene-regulatory profile of peripheral autoimmunity and the diverse immune responses across distinct cell types in Behcet's disease (BD) is still lacking. Here, we present a multi-omic single-cell study of 424,817 cells in BD patients and non-BD individuals. This study maps chromatin accessibility and gene expression in the same biological samples, unraveling vast cellular heterogeneity. We identify widespread cell-type-specific, disease-associated active and pro-inflammatory immunity in both transcript and epigenomic aspects. Notably, integrative multi-omic analysis reveals putative TF regulators that might contribute to chromatin accessibility and gene expression in BD. Moreover, we predicted gene-regulatory networks within nominated TF activators, including AP-1, NF-kB, and ETS transcript factor families, which may regulate cellular interaction and govern inflammation. Our study illustrates the epigenetic and transcriptional landscape in BD peripheral blood and expands understanding of potential epigenomic immunopathology in this disease.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mouse Corneal Epithelial Cells.

Purpose: Corneal epithelial homeostasis is maintained by coordinated gene expression across distinct cell populations, but the gene regulatory programs underlying this cellular diversity remain to be characterized. Here we applied single-cell multi-omics analysis to delineate the gene regulatory profile of mouse corneal epithelial cells under normal homeostasis. Methods: Single cells isolated from the cornea epithelium (with marginal conjunctiva) of adult mice were subjected to scRNA-seq and scATAC-seq using the 10×Genomics platform. Cell types were clustered by the graph-based visualization method uniform manifold approximation and projection and unbiased computational informatics analysis. The scRNA-seq and scATAC-seq datasets were integrated following the integration pipeline described in ArchR and Seurat. Results: We characterized diverse corneal epithelial cell types based on gene expression signatures and chromatin accessibility. We found that cell type-specific accessibility regions were mainly located at distal regions, suggesting essential roles of distal regulatory elements in determining corneal epithelial cell diversity. Trajectory analyses revealed a continuum of cell state transition and higher coordination between transcription factor (TF) motif accessibility and gene expression during corneal epithelial cell differentiation. By integrating transcriptomic and chromatin accessibility analysis, we identified cell type-specific and shared gene regulation programs. We also uncovered critical TFs driving corneal epithelial cell differentiation, such as nuclear factor I (NFI) family members, Rarg, Elf3. We found that nuclear factor-κB (NF-κB) family members were positive TFs in limbal cells and some superficial cells, but they were involved in regulating distinct biological processes. Conclusions: Our study presents a comprehensive gene regulatory landscape of mouse cornea epithelial cells, and provides valuable foundations for future investigation of corneal epithelial homeostasis in the context of cornea pathologies and regenerative medicine.

Chromatin accessibility analysis reveals regulatory dynamics and therapeutic relevance of Vogt-Koyanagi-Harada disease.

The barrier to curing Vogt-Koyanagi-Harada disease (VKH) is thought to reside in a lack of understanding in the roles and regulations of peripheral inflammatory immune cells. Here we perform a single-cell multi-omic study of 166,149 cells in peripheral blood mononuclear cells from patients with VKH, profile the chromatin accessibility and gene expression in the same blood samples, and uncover prominent cellular heterogeneity. Immune cells in VKH blood are highly activated and pro-inflammatory. Notably, we describe an enrichment of transcription targets for nuclear factor kappa B in conventional dendritic cells (cDCs) that governed inflammation. Integrative analysis of transcriptomic and chromatin maps shows that the RELA in cDCs is related to disease complications and poor prognosis. Ligand-receptor interaction pairs also identify cDC as an important predictor that regulated multiple immune subsets. Our results reveal epigenetic and transcriptional dynamics in auto-inflammation, especially the cDC subtype that might lead to therapeutic strategies in VKH.

Immune Cell Landscape of Patients With Diabetic Macular Edema by Single-Cell RNA Analysis.

Purpose: We performed single-cell RNA sequencing (scRNA-seq), an unbiased and high-throughput single cell technology, to determine phenotype and function of peripheral immune cells in patients with diabetic macular edema (DME). Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from DME patients and healthy controls (HC). The single-cell samples were loaded on the Chromium platform (10x Genomics) for sequencing. R package Seurat v3 was used for data normalizing, clustering, dimensionality reduction, differential expression analysis, and visualization. Results: We constructed a single-cell RNA atlas comprising 57,650 PBMCs (24,919 HC, 32,731 DME). We divided all immune cells into five major immune cell lineages, including monocytes (MC), T cells (TC), NK cells (NK), B cells (BC), and dendritic cells (DC). Our differential expression gene (DEG) analysis showed that MC was enriched of genes participating in the cytokine pathway and inflammation activation. We further subdivided MC into five subsets: resting CD14++ MC, proinflammatory CD14++ MC, intermediate MC, resting CD16++ MC and pro-inflammatory CD16++ MC. Remarkably, we revealed that the proinflammatory CD14++ monocytes predominated in promoting inflammation, mainly by increasingly production of inflammatory cytokines (TNF, IL1B, and NFKBIA) and chemokines (CCL3, CCL3L1, CCL4L2, CXCL2, and CXCL8). Gene Ontology (GO) and pathway analysis of the DEGs demonstrated that the proinflammatory CD14++ monocytes, especially in DME patients, upregulated inflammatory pathways including tumor necrosis factor-mediated signaling pathway, I-kappaB kinase/NF-kappaB signaling, and toll-like receptor signaling pathway. Conclusion: In this study, we construct the first immune landscape of DME patients with T2D and confirmed innate immune dysregulation in peripheral blood based on an unbiased scRNA-seq approach. And these results demonstrate potential target cell population for anti-inflammation treatments.

Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing.

COVID-19, caused by SARS-CoV-2, has recently affected over 1,200,000 people and killed more than 60,000. The key immune cell subsets change and their states during the course of COVID-19 remain unclear. We sought to comprehensively characterize the transcriptional changes in peripheral blood mononuclear cells during the recovery stage of COVID-19 by single-cell RNA sequencing technique. It was found that T cells decreased remarkably, whereas monocytes increased in patients in the early recovery stage (ERS) of COVID-19. There was an increased ratio of classical CD14++ monocytes with high inflammatory gene expression as well as a greater abundance of CD14++IL1ß+ monocytes in the ERS. CD4+ T cells and CD8+ T cells decreased significantly and expressed high levels of inflammatory genes in the ERS. Among the B cells, the plasma cells increased remarkably, whereas the naïve B cells decreased. Several novel B cell-receptor (BCR) changes were identified, such as IGHV3-23 and IGHV3-7, and isotypes (IGHV3-15, IGHV3-30, and IGKV3-11) previously used for virus vaccine development were confirmed. The strongest pairing frequencies, IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2 specificity, which had not been reported yet. Furthermore, integrated analysis predicted that IL-1ß and M-CSF may be novel candidate target genes for inflammatory storm and that TNFSF13, IL-18, IL-2, and IL-4 may be beneficial for the recovery of COVID-19 patients. Our study provides the first evidence of an inflammatory immune signature in the ERS, suggesting COVID-19 patients are still vulnerable after hospital discharge. Identification of novel BCR signaling may lead to the development of vaccines and antibodies for the treatment of COVID-19.

Erratum: Author Correction: Immune cell profiling of COVID-19 patients in the recovery stage by single-cell sequencing.

[This corrects the article DOI: 10.1038/s41421-020-0168-9.].

A human circulating immune cell landscape in aging and COVID-19.

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.