Cellular and molecular determinants of bacterial burden in leprosy granulomas revealed by single-cell multimodal omics.

BACKGROUND: Which cell populations that determine the fate of bacteria in infectious granulomas remain unclear. Leprosy, a granulomatous disease with a strong genetic predisposition, caused by Mycobacterium leprae infection, exhibits distinct sub-types with varying bacterial load and is considered an outstanding disease model for studying host-pathogen interactions. METHODS: We performed single-cell RNA and immune repertoire sequencing on 11 healthy controls and 20 patients with leprosy, and integrated single-cell data with genome-wide genetic data on leprosy. Multiplex immunohistochemistry, and in vitro and in vivo infection experiments were conducted to confirm the multimodal omics findings. FINDINGS: Lepromatous leprosy (L-LEP) granulomas with high bacterial burden were characterised by exhausted CD8+ T cells, and high RGS1 expression in CD8+ T cells was associated with L-LEP. By contrast, tuberculoid leprosy (T-LEP) granulomas with low bacterial burden displayed enrichment in resident memory IFNG+ CD8+ T cells (CD8+ Trm) with high GNLY expression. This enrichment was potentially attributable to the communication between IL1B macrophages and CD8+ Trm via CXCL10-CXCR3 signalling. Additionally, IL1B macrophages in L-LEP exhibited anti-inflammatory phenotype, with high APOE expression contributing to high bacterial burden. Conversely, IL1B macrophages in T-LEP were distinguished by interferon-γ induced GBP family genes. INTERPRETATION: The state of IL1B macrophages and functional CD8+ T cells, as well as the relationship between them, is crucial for controlling bacterial persistence within granulomas. These insights may indicate potential targets for host-directed immunotherapy in granulomatous diseases caused by mycobacteria and other intracellular bacteria. FUNDING: The Key research and development program of Shandong Province (2021LCZX07), Natural Science Foundation of Shandong Province (ZR2023MH046), Youth Science Foundation Cultivation Funding Plan of Shandong First Medical University (Shandong Academy of Medical Sciences) (202201-123), National Natural Science Foundation of China (82471800, 82230107, 82273545, 82304039), the China Postdoctoral Science Foundation (2023M742162), Shandong Province Taishan Scholar Project (tspd20230608), Joint Innovation Team for Clinical & Basic Research (202410), Central guidance for local scientific and technological development projects of Shandong Province (YDZX2023058).

Development and evaluation of a rapid diagnostic method for Sporothrix globosa in Asia using quantitative real-time PCR.

BACKGROUND: Sporotrichosis is a chronic granulomatous infection of the skin and subcutaneous tissue that can affect any organ through lymphatic spread. The prevalence of sporotrichosis infections is increasing and its treatment is challenging as there are no unified and standard diagnostic techniques or antifungal medications. Controlling further spread requires a rapid diagnosis. Assessment of clinical symptoms, histological analysis, serological testing, and pathogen culture are all necessary for the diagnosis of sporotrichosis. However, these procedures are unable to identify the species. The development of safe, reliable, and species-specific diagnostic techniques is essential. OBJECTIVE: To establish and evaluate a new quantitative real-time PCR assay for the rapid diagnosis of sporotrichosis and to identify relevant species. METHODS: Polymorphisms in calmodulin (CAL) gene sequences and the internal transcribed spacer (ITS) were used in a quantitative real-time PCR assay to identify S. globosa, S. schenckii, and non-target species. RESULTS: The quantitative real-time PCR assay had 100% sensitivity and specificity. The limit of detection was 6 fg/µl. Thirty-four clinical specimens were verified to be infected with S. globosa with a 100% positive detection rate. CONCLUSIONS: The quantitative PCR technique developed in this study is a quick, accurate, and targeted method of identifying S. globosa based on polymorphisms in CAL sequences and ITS. It can be used for a prompt clinical diagnosis to identify S. globosa in clinical specimens from patients with sporotrichosis.

Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease.

Bullous pemphigoid (BP) is a type 2 inflammation- and immunity-driven skin disease, yet a comprehensive understanding of the immune landscape, particularly immune-stromal crosstalk in BP, remains elusive. Herein, using single-cell RNA sequencing (scRNA-seq) and in vitro functional analyzes, we pinpoint Th2 cells, dendritic cells (DCs), and fibroblasts as crucial cell populations. The IL13-IL13RA1 ligand-receptor pair is identified as the most significant mediator of immune-stromal crosstalk in BP. Notably, fibroblasts and DCs expressing IL13RA1 respond to IL13-secreting Th2 cells, thereby amplifying Th2 cell-mediated cascade responses, which occurs through the specific upregulation of PLA2G2A in fibroblasts and CCL17 in myeloid cells, creating a positive feedback loop integral to immune-stromal crosstalk. Furthermore, PLA2G2A and CCL17 contribute to an increased titer of pathogenic anti-BP180-NC16A autoantibodies in BP patients. Our work provides a comprehensive insight into BP pathogenesis and shows a mechanism governing immune-stromal interactions, providing potential avenues for future therapeutic research.

Targeted serum proteome profiling reveals nicotinamide adenine dinucleotide phosphate (NADPH)-related biomarkers to discriminate linear IgA bullous disorder from dermatitis herpetiformis.

Linear IgA bullous dermatosis (LABD) and dermatitis herpetiformis (DH) represent the major subtypes of IgA mediated autoimmune bullous disorders. We sought to understand the disease etiology by using serum proteomics. We assessed 92 organ damage biomarkers in LABD, DH, and healthy controls using the Olink high-throughput proteomics. The positive proteomic serum biomarkers were used to correlate with clinical features and HLA type. Targeted proteomic analysis of IgA deposition bullous disorders vs. controls showed elevated biomarkers. Further clustering and enrichment analyses identified distinct clusters between LABD and DH, highlighting the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Comparative analysis revealed biomarkers with distinction between LABD and DH and validated in the skin lesion. Finally, qualitative correlation analysis with DEPs suggested six biomarkers (NBN, NCF2, CAPG, FES, BID, and PXN) have better prognosis in DH patients. These findings provide potential biomarkers to differentiate the disease subtype of IgA deposition bullous disease.

Dynamic Interface-Assisted Rapid Self-Assembly of DNA Origami-Framed Anisotropic Nanoparticles.

The ordered arrangement of nanoparticles can generate unique physicochemical properties, rendering it a pivotal direction in the field of nanotechnology. DNA-based chemical encoding has emerged as an unparalleled strategy for orchestrating precise and controlled nanoparticle assemblies. Nonetheless, it is often time-consuming and has limited assembly efficiency. In this study, we developed a strategy for the rapid and ordered assembly of DNA origami-framed nanoparticles assisted by dynamic interfaces. By assembling Au nanoparticles (AuNPs) onto DNA origami with different sticky ends in various directions, we endowed them with anisotropic specific affinities. After assembling DNA origami-framed AuNPs onto supported lipid bilayers with freely diffusing single-stranded DNA via DNA hybridization, we found that DNA origami-framed AuNPs could form larger ordered assemblies than those in 3D solution within equivalent time frames. Furthermore, we also achieved rapid and ordered assembly of liposome nanoparticles by employing the aforementioned strategy. Our work provides a novel avenue for efficient and rapid assembly of nanoparticles across two-dimensional interfaces, which is expected to promote the application of ordered nanoparticle assemblies in sensor and biomimetic system construction.

Pathological Characteristics and Classification of Unstable Coronary Atherosclerotic Plaques.

Important forensic diagnostic indicators of sudden death in coronary atherosclerotic heart disease, such as acute or chronic myocardial ischemic changes, sometimes make it difficult to locate the ischemic site due to the short death process, the lack of tissue reaction time. In some cases, the deceased died of sudden death on the first-episode, resulting in difficulty for medical examiners to make an accurate diagnosis. However, clinical studies on coronary instability plaque revealed the key role of coronary spasm and thrombosis caused by their lesions in sudden coronary death process. This paper mainly summarizes the pathological characteristics of unstable coronary plaque based on clinical medical research, including plaque rupture, plaque erosion and calcified nodules, as well as the influencing factors leading to plaque instability, and briefly describes the research progress and technique of the atherosclerotic plaques, in order to improve the study on the mechanism of sudden coronary death and improve the accuracy of the forensic diagnosis of sudden coronary death by diagnosing different pathologic states of coronary atherosclerotic plaques.

DNA Adjuvant Hydrogel-Optimized Enzymatic Cascade Reaction for Tumor Chemodynamic-Immunotherapy.

Chemodynamic therapy (CDT) shows immense potential in cancer treatment as it not only directly kills tumor cells but also induces anti-tumor immune responses. However, the efficacy of CDT is hampered by challenges in targeting CDT catalysts specifically to tumors using nanomaterials, along with the limitations of low H2 O2 levels and short catalyst duration within the tumor microenvironment. In this study, DNA adjuvant hydrogel to arrange a glucose oxidase-ferrocene cascade for continuously generating reactive oxygen species (ROS) from glucose in situ for tumor CDT combined with immunotherapy is employed. By precisely tuning the catalyst spacing with DNA double helix, ROS production efficiency is elevated by up to nine times compared to free catalysts, resulting in stronger immunogenetic cell death. Upon intratumoral injection, the DNA hydrogel system elicited potent anti-tumor immune responses, thereby effectively inhibiting established tumors and rejecting re-challenged tumors. This work offers a novel platform for integrated CDT and immunotherapy in cancer treatment.

A new mathematical model for evaluating surface changes in the mid-abdominal sagittal plane after two-level pedicle reduction osteotomy in patients with ankylosing spondylitis.

BACKGROUND: The purpose of this study was to create a mathematical model to precalculate the acreage change in the abdominal median sagittal plane (ac-AMSP) of patients with ankylosing spondylitis (AS) for whom two-level pedicle subtraction osteotomy (PSO) was planned. METHODS: A single-centre retrospective review of prospectively collected data was conducted among 11 adults with AS. Acreage of the abdominal median sagittal plane (a-AMSP) was performed. The distances and angles between the osteotomy apexes, anterosuperior edge of T12, xiphoid process, superior edge of the pubis, and anterosuperior corner of the sacrum were measured on preoperative thoracolumbar computed tomography. A mathematical model was created using basic trigonometric functions in accordance with the abdominal parameters. Planned osteotomized vertebra angles (POVAs) were substituted into the mathematical model, and the predictive ac-AMSP (P-AC) was obtained. A paired sample t test was performed to determine the differences between the P-AC and actual ac-AMSP (A-AC) and between the predictive acreage change rate (P-CR) and actual acreage change rate (A-CR). RESULTS: The mean age and GK were 44.4 ± 8.99 years and 102.9° ± 19.17°, respectively. No significant difference exists between A-CR and P-CR via mathematical modeling (p > 0.05). No statistically significant difference existed between POVA and actual osteotomized vertebra angles (AOVA) (p > 0.05). A statistically significant difference was observed between preoperative and postoperative measurements of LL, SVA, and GK variables (p < 0.001). CONCLUSIONS: The novel mathematical model was reliable in predicting the ac-AMSP in AS patients undergoing two-level PSO.

DNA Nanostructures: Self-Adjuvant Carriers for Highly Efficient Subunit Vaccines.

Subunit vaccines based on antigen proteins or epitopes of pathogens or tumors show advantages in immunological precision and high safety, but are often limited by their low immunogenicity. Adjuvants can boost immune responses by stimulating immune cells or promoting antigen uptake by antigen presenting cells (APCs), yet existing clinical adjuvants struggle in simultaneously achieving these dual functions. Additionally, the spatial organization of antigens might be crucial to their immunogenicity. Hence, superior adjuvants should potently stimulate the immune system, precisely arrange antigens, and effectively deliver antigens to APCs. Recently, precisely organizing and delivering antigens with the unique editability of DNA nanostructures has been proposed, presenting unique abilities in significantly improving the immunogenicity of antigens. In this minireview, we will discuss the principles behind using DNA nanostructures as self-adjuvant carriers and review the latest advancements in this field. The potential and challenges associated with self-adjuvant DNA nanostructures will also be discussed.

Click-Chemistry-Mediated Cell Membrane Glycopolymer Engineering to Potentiate Dendritic Cell Vaccines.

Dendritic cell vaccine (DCV) holds great potential in tumor immunotherapy owing to its potent ability in eliciting tumor-specific immune responses. Aiming at engineering enhanced DCV, we report the first effort to construct a glycopolymer-engineered DC vaccine (G-DCV) via metabolicglycoengineering and copper-free click-chemistry. Model G-DCV was prepared by firstly delivering tumor antigens, ovalbumin (OVA) into dendritic cells (DC) with fluoroalkane-grafted polyethyleneimines, followed by conjugating glycopolymers with a terminal group of dibenzocyclooctyne (DBCO) onto dendritic cells. Compared to unmodified DCV, our G-DCV could induce stronger T cell activation due to the enhanced adhesion between DCs and T cells. Notably, such G-DCV could more effectively inhibit the growth of the mouse B16-OVA (expressing OVA antigen) tumor model after adoptive transfer. Moreover, by combination with an immune checkpoint inhibitor, G-DCV showed further increased anti-tumor effects in treating different tumor models. Thus, our work provides a novel strategy to enhance the therapeutic effectiveness of DC vaccines.

CYBB-Mediated Ferroptosis Associated with Immunosuppression in Mycobacterium leprae-Infected Monocyte-Derived Macrophages.

Mycobacterium leprae-infected macrophages preferentially exhibit the regulatory M2 phenotype in vitro, which helps the immune escape unabated growth of M leprae in host cells. The mechanism that triggers macrophage polarization is still unknown. In this study, we performed single-cell RNA sequencing to determine the initial responses of human monocyte-derived macrophages against M leprae infection of 4 healthy individuals and found an increase in a major alternative-activated macrophage type that overexpressed NEAT1, CCL2, and CD163. Importantly, further functional analysis showed that ferroptosis was positively correlated with M2 polarization of macrophages, and in vitro experiments have shown that inhibition of ferroptosis promotes the survival of M leprae within macrophages. In addition, further joint analysis of our results with mutisequencing data from patients with leprosy and in vitro validation identified that CYBB was the pivotal molecule for ferroptosis that could promote the M2 polarization of M leprae-infected macrophages, resulting in the immune escape and unabated growth of pathogenic bacteria. Overall, our results suggest that M leprae facilitated its survival by inducing CYBB-mediated macrophage ferroptosis leading to its alternative activation and might reveal the potential for a new therapeutic strategy of leprosy.

Quartet DNA reference materials and datasets for comprehensively evaluating germline variant calling performance.

BACKGROUND: Genomic DNA reference materials are widely recognized as essential for ensuring data quality in omics research. However, relying solely on reference datasets to evaluate the accuracy of variant calling results is incomplete, as they are limited to benchmark regions. Therefore, it is important to develop DNA reference materials that enable the assessment of variant detection performance across the entire genome. RESULTS: We established a DNA reference material suite from four immortalized cell lines derived from a family of parents and monozygotic twins. Comprehensive reference datasets of 4.2 million small variants and 15,000 structural variants were integrated and certified for evaluating the reliability of germline variant calls inside the benchmark regions. Importantly, the genetic built-in-truth of the Quartet family design enables estimation of the precision of variant calls outside the benchmark regions. Using the Quartet reference materials along with study samples, batch effects are objectively monitored and alleviated by training a machine learning model with the Quartet reference datasets to remove potential artifact calls. Moreover, the matched RNA and protein reference materials and datasets from the Quartet project enables cross-omics validation of variant calls from multiomics data. CONCLUSIONS: The Quartet DNA reference materials and reference datasets provide a unique resource for objectively assessing the quality of germline variant calls throughout the whole-genome regions and improving the reliability of large-scale genomic profiling.

Genome-wide meta-analysis and fine-mapping prioritize potential causal variants and genes related to leprosy.

To date, genome-wide association studies (GWASs) have discovered 35 susceptible loci of leprosy; however, the cumulative effects of these loci can only partially explain the overall risk of leprosy, and the causal variants and genes within these loci remain unknown. Here, we conducted out new GWASs in two independent cohorts of 5007 cases and 4579 controls and then a meta-analysis in these newly generated and multiple previously published (2277 cases and 3159 controls) datasets were performed. Three novel and 15 previously reported risk loci were identified from these datasets, increasing the known leprosy risk loci of explained genetic heritability from 23.0 to 38.5%. A comprehensive fine-mapping analysis was conducted, and 19 causal variants and 14 causal genes were identified. Specifically, manual checking of epigenomic information from the Epimap database revealed that the causal variants were mainly located within the immune-relevant or immune-specific regulatory elements. Furthermore, by using gene-set, tissue, and cell-type enrichment analyses, we highlighted the key roles of immune-related tissues and cells and implicated the PD-1 signaling pathways in the pathogenetic mechanism of leprosy. Collectively, our study identified candidate causal variants and elucidated the potential regulatory and coding mechanisms for genes associated with leprosy.

Clinical Characteristics and Intraoperative Reduction Technique of Thoracolumbar Fractures Combined with Posterior Column Injury.

OBJECTIVE: To analyse the clinical characteristics of thoracolumbar fractures combined with posterior column injury, and explore its intraoperative reduction technique with clinical efficacy. STUDY DESIGN: Descriptive study.                                   Place and Duration of the Study: Department of Orthopaedics, Fuyang People's Hospital, Anhui, China, from December 2017 to 2021. METHODOLOGY: A total of 60 patients met the inclusion criteria, they were divided into two categories according to injury mechanism and imaging characteristics: flexion-distraction injury (FDI) and burst fracture with lamina fracture (BFLF), and their clinical characteristics were analysed. All patients were treated with posterior pedicle screw internal fixation, and different intraoperative reduction methods were adopted for reduction. Measurements of anterior vertebral heights (AVH), local kyphotic angles (LKA), visual analog scale (VAS) and oswestry disability index (ODI) were evaluated preoperative, after operation, and the last follow-up. RESULTS: The two groups of thoracolumbar fractures combined with posterior column injury had different clinical characteristics, and there were significant differences in preoperative imaging related parameters (p<0.05). All patients in the two groups successfully completed the operation, and there were no direct complications related to the operation. The patients were followed up for 12-24 months. Compared with those before the operation, the AVH, LKA, VAS, and ODI immediately after the operation and at the last follow-up were significantly improved (p<0.05). Bone fusion was achieved in all patients. CONCLUSION: Careful and comprehensive preoperative clinical data analysis is the key to diagnosis of thoracolumbar fractures combined with posterior column injury. According to the type of fracture, reasonable selection of intraoperative reduction technique can obtain satisfactory clinical results. KEY WORDS: Burst fracture, Pedicle screw, Internal fixation, Thoracic, Lumbar.

HLA-C*15:02 and epidermal growth factor receptor inhibitor-induced erosive pustular dermatosis of the scalp.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.

Functional DNA as a Molecular Tool in Regulating Immunoreceptor-Ligand Interactions.

During immune responses, activating ligands would trigger dynamic spatiotemporal organization of immunoreceptors at the cell interface, governing the fate and effector functions of immune cells. To understand the biophysical mechanisms of immunoreceptor signaling, diverse tools, including DNA technologies, have been developed to manipulate receptor-ligand interactions during the immune activation process. With great capability in the controllable assembly of biomolecules, functional DNA-based precise arrangement of immune molecules at cell interfaces has provided a powerful means in revealing the principles of immunoreceptor triggering, even at the single-molecule level. In addition, precisely regulating immunoreceptor-ligand interactions with functional DNA has been applied in immunotherapies of major diseases. This Perspective will focus on the recent advances in exploring immunoreceptor signaling with functional DNA as the molecular tool as well as the applications of functional DNA mediated regulation of immunoreceptor activation. We also outline the challenges and opportunities of applying functional DNA in immune modulation and immunotherapy.