Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system.

Introduction: Systemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets. Methods: Single-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers. Results: Using the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/CD57- T cells, CD4+/CD57+ T cells, CD8+/CD161- T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4-/CD8- T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3- non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

The role of M1/M2 macrophage polarization in primary Sjogren's syndrome.

BACKGROUND: The purpose of this study was to investigate the role of macrophage polarization in the pathogenesis of primary Sjogren's syndrome (pSS). METHODS: Peripheral venous blood samples were collected from 30 patients with pSS and 30 healthy controls. Minor salivary gland samples were abtainted from 10 of these patients and 10 non-pSS controls whose minor salivary gland didn't fulfill the classification criteria for pSS. Enzyme-linked immuno sorbent assay was used to examine the serum concentration of M1/M2 macrophage related cytokines (TNF-a, IL-6, IL-23, IL-4, IL-10 and TGF-ß). Flow cytometry was used to examine the numbers of CD86+ M1 macrophages and CD206+ M2 macrophages in peripheral blood mononuclear cells (PBMCs). Immunofluorescence was used to test the infiltration of macrophages in minor salivary glands. RESULTS: This study observed a significant increase in pSS patients both in the numbers of M1 macrophages in peripheral blood and serum levels of M1-related pro-inflammatory cytokines (IL-6, IL-23 and TNF-α). Conversely, M2 macrophages were downregulated in the peripheral blood of pSS patients. Similarly, in the minor salivary glands of pSS patients, the expression of M1 macrophages was increased, and that of M2 macrophages was decreased. Furthermore, a significantly positive correlation was found between the proportions of M1 macrophages in PBMCs and serum levels of IgG and RF. CONCLUSIONS: This study reveals the presence of an significant imbalance in M1/M2 macrophages in pSS patients. The M1 polarization of macrophages may play an central role in the pathogenesis of pSS.

Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

Harmonized analysis of PBMC by mass cytometry.

Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data.

Cepharanthine synergistically promotes methylprednisolone pharmacodynamics against human peripheral blood mononuclear cells possibly via regulation of P-glycoprotein/glucocorticoid receptor translocation.

BACKGROUND: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. METHODS: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. RESULTS: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 µM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 µM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 µM of CEP did not influence P-glycoprotein expression. 0.03~0.3 µM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 µM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP.

Single-cell transcriptome reveals highly complement activated microglia cells in association with pediatric tuberculous meningitis.

Background: Tuberculous meningitis (TBM) is a devastating form of tuberculosis (TB) causing high mortality and disability. TBM arises due to immune dysregulation, but the underlying immune mechanisms are unclear. Methods: We performed single-cell RNA sequencing on peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells isolated from children (n=6) with TBM using 10 xGenomics platform. We used unsupervised clustering of cells and cluster visualization based on the gene expression profiles, and validated the protein and cytokines by ELISA analysis. Results: We revealed for the first time 33 monocyte populations across the CSF cells and PBMCs of children with TBM. Within these populations, we saw that CD4_C04 cells with Th17 and Th1 phenotypes and Macro_C01 cells with a microglia phenotype, were enriched in the CSF. Lineage tracking analysis of monocyte populations revealed myeloid cell populations, as well as subsets of CD4 and CD8 T-cell populations with distinct effector functions. Importantly, we discovered that complement-activated microglial Macro_C01 cells are associated with a neuroinflammatory response that leads to persistent meningitis. Consistently, we saw an increase in complement protein (C1Q), inflammatory markers (CRP) and inflammatory factor (TNF-α and IL-6) in CSF cells but not blood. Finally, we inferred that Macro_C01 cells recruit CD4_C04 cells through CXCL16/CXCR6. Discussion: We proposed that the microglial Macro_C01 subset activates complement and interacts with the CD4_C04 cell subset to amplify inflammatory signals, which could potentially contribute to augment inflammatory signals, resulting in hyperinflammation and an immune response elicited by Mtb-infected tissues.

Detection of circulating tumor cells by means of machine learning using Smart-Seq2 sequencing.

Circulating tumor cells (CTCs) are tumor cells that separate from the solid tumor and enter the bloodstream, which can cause metastasis. Detection and enumeration of CTCs show promising potential as a predictor for prognosis in cancer patients. Furthermore, single-cells sequencing is a technique that provides genetic information from individual cells and allows to classify them precisely and reliably. Sequencing data typically comprises thousands of gene expression reads per cell, which artificial intelligence algorithms can accurately analyze. This work presents machine-learning-based classifiers that differentiate CTCs from peripheral blood mononuclear cells (PBMCs) based on single cell RNA sequencing data. We developed four tree-based models and we trained and tested them on a dataset consisting of Smart-Seq2 sequenced data from primary tumor sections of breast cancer patients and PBMCs and on a public dataset with manually annotated CTC expression profiles from 34 metastatic breast patients, including triple-negative breast cancer. Our best models achieved about 95% balanced accuracy on the CTC test set on per cell basis, correctly detecting 133 out of 138 CTCs and CTC-PBMC clusters. Considering the non-invasive character of the liquid biopsy examination and our accurate results, we can conclude that our work has potential application value.

Improved ELISPOT protocol for monitoring Th1/Th17 T-cell response following T.gondii infection.

In the monitoring of human Toxoplasma gondii infection, it is crucial to confirm the development of a specific Th1/Th17 immune response memory. The use of a simple, specific, and sensitive assay to follow the T-cell activation is thus required. Current protocols are not always specific as stimulation with peptides is Human Leukocyte Antigen (HLA)-dependent, while stimulation with total-lysis antigens tends to stimulate seronegative donors resulting to false positives. Here, an improved ELISPOT protocol is reported, using peripheral blood mononuclear cells (PBMC) of T.gondii-infected donors, incubated with the inactivated parasite. The results showed that, contrary to standard protocols, a pre-incubation step at high cell density in presence of the inactivated parasite allowed a specific Th1/Th17 response with the secretion of IFN-γ, IL-2, IL-12 and IL-17 cytokines. This protocol allows to evaluate precisely the immune response after a T.gondii infection.

Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

Distinct cellular immune responses in children en route to type 1 diabetes with different first-appearing autoantibodies.

Previous studies have revealed heterogeneity in the progression to clinical type 1 diabetes in children who develop islet-specific antibodies either to insulin (IAA) or glutamic acid decarboxylase (GADA) as the first autoantibodies. Here, we test the hypothesis that children who later develop clinical disease have different early immune responses, depending on the type of the first autoantibody to appear (GADA-first or IAA-first). We use mass cytometry for deep immune profiling of peripheral blood mononuclear cell samples longitudinally collected from children who later progressed to clinical disease (IAA-first, GADA-first, ≥2 autoantibodies first groups) and matched for age, sex, and HLA controls who did not, as part of the Type 1 Diabetes Prediction and Prevention study. We identify differences in immune cell composition of children who later develop disease depending on the type of autoantibodies that appear first. Notably, we observe an increase in CD161 expression in natural killer cells of children with ≥2 autoantibodies and validate this in an independent cohort. The results highlight the importance of endotype-specific analyses and are likely to contribute to our understanding of pathogenic mechanisms underlying type 1 diabetes development.

Dynamic molecular signatures of acute myocardial infarction based on transcriptomics and metabolomics.

Acute myocardial infarction (AMI) commonly precedes ventricular remodeling, heart failure. Few dynamic molecular signatures have gained widespread acceptance in mainstream clinical testing despite the discovery of many potential candidates. These unmet needs with respect to biomarker and drug discovery of AMI necessitate a prioritization. We enrolled patients with AMI aged between 30 and 70. RNA-seq analysis was performed on the peripheral blood mononuclear cells collected from the patients at three time points: 1 day, 7 days, and 3 months after AMI. PLC/LC-MS analysis was conducted on the peripheral blood plasma collected from these patients at the same three time points. Differential genes and metabolites between groups were screened by bio-informatics methods to understand the dynamic changes of AMI in different periods. We obtained 15 transcriptional and 95 metabolite expression profiles at three time points after AMI through high-throughput sequencing. AMI-1d: enrichment analysis revealed the biological features of 1 day after AMI primarily included acute inflammatory response, elevated glycerophospholipid metabolism, and decreased protein synthesis capacity. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) might stand promising biomarkers to differentiate post-AMI stage. Anti-inflammatory therapy during the acute phase is an important direction for preventing related pathology. AMI-7d: the biological features of this stage primarily involved the initiation of cardiac fibrosis response and activation of platelet adhesion pathways. Accompanied by upregulated TGF-beta signaling pathway and ECM receptor interaction, GP5 help assess platelet activation, a potential therapeutic target to improve haemostasis. AMI-3m: the biological features of 3 months after AMI primarily showed a vascular regeneration response with VEGF signaling pathway, NOS3 and SHC2 widely activated, which holds promise for providing new therapeutic approaches for AMI. Our analysis highlights transcriptional and metabolomics signatures at different time points after MI, which deepens our understanding of the dynamic biological responses and associated molecular mechanisms that occur during cardiac repair.

Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

The role of the immune system in early-onset schizophrenia: identifying immune characteristic genes and cells from peripheral blood.

BACKGROUND: Early-onset schizophrenia (EOS) is a type of schizophrenia (SCZ) with an age of onset of < 18 years. An abnormal inflammatory immune system may be involved in the occurrence and development of SCZ. We aimed to identify the immune characteristic genes and cells involved in EOS and to further explore the pathogenesis of EOS from the perspective of immunology. METHODS: We obtained microarray data from a whole-genome mRNA expression in peripheral blood mononuclear cells (PBMCs); 19 patients with EOS (age range: 14.79 ± 1.90) and 18 healthy controls (HC) (age range: 15.67 ± 2.40) were involved. We screened for differentially expressed genes (DEGs) using the Limma software package and modular genes using weighted gene co-expression network analysis (WGCNA). In addition, to identify immune characteristic genes and cells, we performed enrichment analysis, immune infiltration analysis, and receiver operating characteristic (ROC) curve analysis; we also used a random forest (RF), a support vector machine (SVM), and the LASSO-Cox algorithm. RESULTS: We selected the following immune characteristic genes: CCL8, PSMD1, AVPR1B and SEMG1. We employed a RF, a SVM, and the LASSO-Cox algorithm. We identified the following immune characteristic cells: activated mast cells, CD4+ memory resting T cells, resting mast cells, neutrophils and CD4+ memory activated T cells. In addition, the AUC values of the immune characteristic genes and cells were all > 0.7. CONCLUSION: Our results indicate that immune system function is altered in SCZ. In addition, CCL8, PSMD1, AVPR1B and SEMG1 may regulate peripheral immune cells in EOS. Further, immune characteristic genes and cells are expected to be diagnostic markers and therapeutic targets of SCZ.

Transcriptional profiles associated with coronary artery disease in type 2 diabetes mellitus.

Background: Coronary artery disease (CAD) is a common complication of Type 2 diabetes mellitus (T2DM). Understanding the pathogenesis of this complication is essential in both diagnosis and management. Thus, this study aimed to characterize the presence of CAD in T2DM using molecular markers and pathway analyses. Methods: The study is a sex- and age-frequency matched case-control design comparing 23 unrelated adult Filipinos with T2DM-CAD to 23 controls (DM with CAD). Healthy controls served as a reference. Total RNA from peripheral blood mononuclear cells (PBMCs) underwent whole transcriptomic profiling using the Illumina HumanHT-12 v4.0 expression beadchip. Differential gene expression with gene ontogeny analyses was performed, with supporting correlational analyses using weighted correlation network analysis (WGCNA). Results: The study observed that 458 genes were differentially expressed between T2DM with and without CAD (FDR<0.05). The 5 top genes the transcription factor 3 (TCF3), allograft inflammatory factor 1 (AIF1), nuclear factor, interleukin 3 regulated (NFIL3), paired immunoglobulin-like type 2 receptor alpha (PILRA), and cytoskeleton-associated protein 4 (CKAP4) with AUCs >89%. Pathway analyses show differences in innate immunity activity, which centers on the myelocytic (neutrophilic/monocytic) theme. SNP-module analyses point to a possible causal dysfunction in innate immunity that triggers the CAD injury in T2DM. Conclusion: The study findings indicate the involvement of innate immunity in the development of T2DM-CAD, and potential immunity markers can reflect the occurrence of this injury. Further studies can verify the mechanistic hypothesis and use of the markers.

Integrin-linked kinase mRNA expression in circulating mononuclear cells as a biomarker of kidney and vascular damage in experimental chronic kidney disease.

BACKGROUND: Traditional biomarkers of chronic kidney disease (CKD) detect the disease in its late stages and hardly predict associated vascular damage. Integrin-linked kinase (ILK) is a scaffolding protein and a serine/threonine protein kinase that plays multiple roles in several pathophysiological processes during renal damage. However, the involvement of ILK as a biomarker of CKD and its associated vascular problems remains to be fully elucidated. METHODS: CKD was induced by an adenine-rich diet for 6 weeks in mice. We used an inducible ILK knockdown mice (cKD-ILK) model to decrease ILK expression. ILK content in mice's peripheral blood mononuclear cells (PBMCs) was determined and correlated with renal function parameters and with the expression of ILK and fibrosis and inflammation markers in renal and aortic tissues. Also, the expression of five miRNAs that target ILK was analyzed in whole blood of mice. RESULTS: The adenine diet increased ILK expression in PBMCs, renal cortex, and aortas, and creatinine and urea nitrogen concentrations in the plasma of WT mice, while these increases were not observed in cKD-ILK mice. Furthermore, ILK content in PBMCs directly correlated with renal function parameters and with the expression of renal and vascular ILK and fibrosis and inflammation markers. Finally, the expression of the five miRNAs increased in the whole blood of adenine-fed mice, although only four correlated with plasma urea nitrogen, and of those, three were downregulated in cKD-ILK mice. CONCLUSIONS: ILK, in circulating mononuclear cells, could be a potential biomarker of CKD and CKD-associated renal and vascular damage.

PDE4D and miR-203 are promising biomarkers for canine atopic dermatitis.

BACKGROUND: Canine atopic dermatitis (CAD) is a common genetically predisposed, inflammatory, and pruritic skin disorder that affects dogs globally. To date, there are no specific biomarkers available to diagnose CAD, and the current diagnosis is based on a combination of criteria including patient history, clinical signs, and exclusion of other relevant differential diagnoses. METHODS AND RESULTS: We examined the gene expression of phosphodiesterase 4D (PDE4D) in peripheral blood mononuclear cells (PBMCs), as well as miR-203 and miR-483 in plasma, in three groups: healthy dogs, CAD dogs, and other inflammatory pruritic skin diseases (OIPSD) such as pemphigus foliaceus, scabies, cutaneous lymphoma, and dermatophytosis. Our results showed that PDE4D gene expression in the CAD group is statistically higher compared to those in the healthy and OIPSD groups, suggesting PDE4D may be a specific marker for CAD. Nevertheless, no correlation was found between PDE4D gene expression levels and the lesion severity gauged by CAD severity index-4 (CADESI-4). We also showed that miR-203 is a generic marker for clinical dermatitis and differentiates both CAD and OIPSD inflammatory conditions from healthy controls. CONCLUSIONS: We show that PDE4D is a potential marker to differentiate CAD from non-atopic healthy and OIPSD while miR-203 may be a potential marker for general dermatologic inflammation. Future study of PDE4D and miR-203 on a larger scale is warranted.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function.

BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown. METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs. RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αß T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb. CONCLUSION: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Unraveling transcriptomic signatures and dysregulated pathways in systemic lupus erythematosus across disease states.

OBJECTIVES: This study aims to elucidate the transcriptomic signatures and dysregulated pathways in patients with Systemic Lupus Erythematosus (SLE), with a particular focus on those persisting during disease remission. METHODS: We conducted bulk RNA-sequencing of peripheral blood mononuclear cells (PBMCs) from a well-defined cohort comprising 26 remission patients meeting the Low Lupus Disease Activity State (LLDAS) criteria, 76 patients experiencing disease flares, and 15 healthy controls. To elucidate immune signature changes associated with varying disease states, we performed extensive analyses, including the identification of differentially expressed genes and pathways, as well as the construction of protein-protein interaction networks. RESULTS: Several transcriptomic features recovered during remission compared to the active disease state, including down-regulation of plasma and cell cycle signatures, as well as up-regulation of lymphocytes. However, specific innate immune response signatures, such as the interferon (IFN) signature, and gene modules involved in chromatin structure modification, persisted across different disease states. Drug repurposing analysis revealed certain drug classes that can target these persistent signatures, potentially preventing disease relapse. CONCLUSION: Our comprehensive transcriptomic study revealed gene expression signatures for SLE in both active and remission states. The discovery of gene expression modules persisting in the remission stage may shed light on the underlying mechanisms of vulnerability to relapse in these patients, providing valuable insights for their treatment.

In-vitro and in-vivo assessment of nirmatrelvir penetration into CSF, central nervous system cells, tissues, and peripheral blood mononuclear cells.

Three years after SARS-CoV-2 emerged as a global infectious threat, the virus has become endemic. The neurological complications such as depression, anxiety, and other CNS complications after COVID-19 disease are increasing. The brain, and CSF have been shown as viral reservoirs for SARS-CoV-2, yielding a potential hypothesis for CNS effects. Thus, we investigated the CNS pharmacology of orally dosed nirmatrelvir/ritonavir (NMR/RTV). Using both an in vitro and an in vivo rodent model, we investigated CNS penetration and potential pharmacodynamic activity of NMR. Through pharmacokinetic modeling, we estimated the median CSF penetration of NMR to be low at 18.11% of plasma with very low accumulation in rodent brain tissue. Based on the multiples of the 90% maximal effective concentration (EC90) for SARS-CoV-2, NMR concentrations in the CSF and brain do not achieve an exposure level similar to that of plasma. A median of only 16% of all the predicted CSF concentrations in rats were > 3xEC90 (unadjusted for protein binding). This may have implications for viral persistence and neurologic post-acute sequelae of COVID-19 if increased NMR penetration in the CNS leads to decreased CNS viral loads and decreased CNS inflammation.