Analysis of Human Peripheral Blood Mononuclear Cells in Patients with Neurodegenerative Diseases.

The role of immune system in the progression of neurodegenerative diseases has been studied for decades in animal models. However, invasive studies in human subjects remain controversial due to the heterogeneity of the presentation of different diagnostic categories at different stages of the disease. Peripheral blood mononuclear cells (PBMCs) contain immune cells including dendritic cells (DCs), monocytes, macrophages, and T lymphocytes. Isolating PBMCs from whole blood samples collected from patients provides a minimally invasive method for analyzing the immune system's function in patients with neurodegenerative diseases. By isolating single cell types from patients' peripheral blood, in vitro analyses can be conducted including RNA sequencing, immunofluorescence, and phagocytic analysis. In this chapter, we discuss PBMC separation and isolation of macrophages in pure culture in vitro. We also outline methods for performing RNA-seq on cultured macrophages and other techniques for investigating the role of macrophages in neurodegenerative disease pathophysiology.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing.

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

Uterine infusion strategies for infertile patients with recurrent implantation failure: a systematic review and network meta-analysis.

BACKGROUND: Intra-uterine infusion treatments were reported to be beneficial to embryo implantation and pregnancy outcomes, and considered as potential therapies for infertile patients with recurrent implantation failure (RIF). Nevertheless, their efficiencies were controversial and there lack of consensus on which intrauterine treatment is the most effective. METHODS: All prospective trials (in Chinese or English) were searched in Databases PubMed, Cochrane, Web of Science, and CNKI from July 2013 to July 2023. We included studies that investigated various uterine infusions, including chorionic gonadotropin, granulocyte colony-stimulating factor, monocytes, platelet-rich plasma, etc. during IVF treatment and reported subsequent pregnancy outcomes. RESULTS: We finally included 56 researches, including 40 randomized controlled trials, 14 non-randomized controlled trials, and 3 prospective cohort studies. This study included a total of 11 uterine perfusion methods: Placebo, Human Chorionic Gonadotropin (HCG), Granulocyte Colony-Stimulating Factor (G-CSF), platelet-rich plasma (PRP), Peripheral Blood Mononuclear Cell (PBMC), Growth hormone (GH), dexamethasone (DEX), Embryo culture supernatant (ESC), PRP combined with G-CSF (PRP + G-CSF), RPR combined with subcutaneous injection of G-CSF (RPR + G-CSFsc), G-CSF combined with subcutaneous injection of AXaIU (G-CSF + AXaIUsc). Intrauterine infusion of HCG, PBMC, G-CSF, and PRP significantly improves pregnancy outcomes in patients with repeated implantation failure compared with blank controls or placebo, and PRP improved the clinical pregnancy and live birth most. GH and ESC infusion might improve the pregnancy outcomes, but uterine infusion of DEX was shown with high miscarriage. The combination therapy did not show a significant advantage over the mono-therapy. CONCLUSIONS: Intrauterine infusion of HCG, PBMC, G-CSF, and PRP are promising strategies for improving pregnancy outcomes for infertile patients with recurrent implantation failure. Among these treatments, PRP may be the best. More researches are required to explore the effect of drug combinations and less commonly used drugs as well. TRIAL REGISTRATION: Our study was registered in PROSPERO and the ID was CRD42023467188.

Altered skin microbiome, inflammation, and JAK/STAT signaling in Southeast Asian ichthyosis patients.

BACKGROUND: Congenital ichthyosis (CI) is a collective group of rare hereditary skin disorders. Patients present with epidermal scaling, fissuring, chronic inflammation, and increased susceptibility to infections. Recently, there is increased interest in the skin microbiome; therefore, we hypothesized that CI patients likely exhibit an abnormal profile of epidermal microbes because of their various underlying skin barrier defects. Among recruited individuals of Southeast Asian ethnicity, we performed skin meta-genomics (i.e., whole-exome sequencing to capture the entire multi-kingdom profile, including fungi, protists, archaea, bacteria, and viruses), comparing 36 CI patients (representing seven subtypes) with that of 15 CI age-and gender-matched controls who had no family history of CI. RESULTS: This case-control study revealed 20 novel and 31 recurrent pathogenic variants. Microbiome meta-analysis showed distinct microbial populations, decreases in commensal microbiota, and higher colonization by pathogenic species associated with CI; these were correlated with increased production of inflammatory cytokines and Th17- and JAK/STAT-signaling pathways in peripheral blood mononuclear cells. In the wounds of CI patients, we identified specific changes in microbiota and alterations in inflammatory pathways, which are likely responsible for impaired wound healing. CONCLUSIONS: Together, this research enhances our understanding of the microbiological, immunological, and molecular properties of CI and should provide critical information for improving therapeutic management of CI patients.

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen.

High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. We utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen's ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology.

Impact of doxorubicin-loaded ferritin nanocages (FerOX) vs. free doxorubicin on T lymphocytes: a translational clinical study on breast cancer patients undergoing neoadjuvant chemotherapy.

Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity.Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing adverse effects, highlighting the pivotal role played by FerOX in mitigating DOX-induced toxicity towards T-cells, thereby positioning it as a promising DOX formulation. This study contributes valuable insights to modern cancer therapy and immunomodulation.

The transcription factor HMGB2 indirectly regulates APRIL expression and Gd-IgA1 production in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Proliferation-inducing ligand (APRIL) was identified as an important cause of glycosylation deficiency of IgA1 (Gd-IgA1), which can 'trigger' IgAN. Our previous study indicated that high migration group protein B2 (HMGB2) in peripheral blood mononuclear cells from patients with IgAN was associated with disease severity, but the underlying mechanism remains unclear. MATERIALS AND METHODS: The location of HMGB2 was identified by immunofluorescence. qRT-PCR and Western blotting were used to measure HMGB2, HMGA1, and APRIL expression. Gd-IgA1 levels were detected by enzyme-linked immunosorbent assay (ELISA). In addition, we used DNA pull-down, protein profiling, and transcription factor prediction software to identify proteins bound to the promoter region of the APRIL gene. RNA interference and coimmunoprecipitation (Co-IP) were used to verify the relationships among HMGB2, high mobility group AT-hook protein 1 (HMGA1), and APRIL. RESULTS: HMGB2 expression was greater in IgAN patients than in HCs and was positively associated with APRIL expression in B cells. DNA pull-down and protein profiling revealed that HMGB2 and HMGA1 bound to the promoter region of the APRIL gene. The expression levels of HMGA1, APRIL, and Gd-IgA1 were downregulated after HMGB2 knockdown. Co-IP indicated that HMGB2 binds to HMGA1. The Gd-IgA1 concentration in the supernatant was reduced after HMGA1 knockdown. HMGA1 binding sites were predicted in the promoter region of the APRIL gene. CONCLUSION: HMGB2 expression is greater in IgAN patients than in healthy controls; it promotes APRIL expression by interacting with HMGA1, thereby inducing Gd-IgA1 overexpression and leading to IgAN.

Eukaryotic initiation factor 3a promotes the development of diffuse large B-cell lymphoma through regulating cell proliferation.

BACKGROUND: One-third of diffuse large B-cell lymphoma (DLBCL) patients suffer relapse after standard treatment. Eukaryotic initiation factor 3a (eIF3a) is a key player in the initial stage of translation, which has been widely reported to be correlated with tumorigenesis and therapeutic response. This study aimed to explore the biological role of eIF3a, evaluate its prognostic and therapeutic potential in DLBCL. METHODS: RNA-seq datasets from GEO database were utilized to detect the expression and prognostic role of eIF3a in DLBCL patients. Protein level of eIF3a was estimated by western blot and immunohistochemical. Next, DLBCL cells were transfected with lentiviral vector either eIF3a-knockdown or empty to assess the biological role of eIF3a. Then, samples were divided into 2 clusters based on eIF3a expression and differentially expressed genes (DEGs) were identified. Function enrichment and mutation analysis of DEGs were employed to detect potential biological roles. Moreover, we also applied pan-cancer and chemosensitivity analysis for deep exploration. RESULTS: eIF3a expression was found to be higher in DLBCL than healthy controls, which was associated with worse prognosis. The expression of eIF3a protein was significantly increased in DLBCL cell lines compared with peripheral blood mononuclear cells (PBMCs) from healthy donors. eIF3a knockdown inhibited the proliferation of DLBCL cells and the expression of proliferation-related proteins and increase cell apoptosis rate. Besides, 114 DEGs were identified which had a close linkage to cell cycle and tumor immune. eIF3a and DEGs mutations were found to be correlated to chemosensitivity and vital signal pathways. Pan-cancer analysis demonstrated that high eIF3a expression was associated with worse prognosis in several tumors. Moreover, eIF3a expression was found to be related to chemosensitivity of several anti-tumor drugs in DLBCL, including Vincristine and Wee1 inhibitor. CONCLUSIONS: We firstly revealed the high expression and prognostic role of eIF3a in DLBCL, and eIF3a might promote the development of DLBCL through regulating cell proliferation and apoptosis. eIF3a expression was related to immune profile and chemosensitivity in DLBCL. These results suggest that eIF3a could serve as a potential prognostic biomarker and therapeutic target in DLBCL.

Circulating Immune Landscape Profiling in Psoriasis Vulgaris and Psoriatic Arthritis by Mass Cytometry.

Background: Psoriasis, a systemic disorder mediated by the immune system, can appear on the skin, joints, or both. Individuals with cutaneous psoriasis (PsC) have an elevated risk of developing psoriatic arthritis (PsA) during their lifetime. Despite this known association, the cellular and molecular mechanisms underlying this progression remain unclear. Methods: We performed high-dimensional, in-depth immunophenotyping of peripheral blood mononuclear cells (PBMCs) in patients with PsA and psoriasis vulgaris (PsV) by mass cytometry. Blood samples were collected before and after therapy for a longitudinal study. Then three sets of comparisons were made here: active PsA vs. active PsV, untreated PsV vs. treated PsV, and untreated PsA vs. treated PsA. Results: Marked differences were observed in multiple lymphocyte subsets of PsA related to PsV, with expansion of CD4+ T cells, CD16- NK cells, and B cells. Notably, two critical markers, CD28 and CD127, specifically differentiated PsA from PsV. The expression levels of CD28 and CD127 on both Naïve T cells (TN) and central memory CD4+ T cells (TCM) were considerably higher in PsA than PsV. Meanwhile, after treatment, patients with PsV had higher levels of CD28hi CD127hi CD4+ TCM cells, CD28hi CD127hi CD4+ TN cells, and CD16- NK cells. Conclusion: In the circulation of PsA patients, the TN and CD4+ TCM are characterized with more abundant CD28 and CD127, which effectively distinguished PsA from PsV. This may indicate that individuals undergoing PsV could be stratified at high risk of developing PsA based on the circulating levels of CD28 and CD127 on specific cell subsets.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-ß levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-ß was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

Monochrome Multiplex Quantitative PCR Telomere Length Measurement.

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

Exploring secretory proteome and cytokine kinetic of human peripheral blood mononuclear cells exposed to methicillin-resistant Staphylococcus aureus biofilms and planktonic bacteria.

Staphylococcus aureus is a highly successful pathogen infecting various body parts and forming biofilms on natural and artificial surfaces resulting in difficult-to-treat and chronic infections. We investigated the secreted cytokines and proteomes of isolated peripheral blood mononuclear cells (PBMCs) from healthy volunteers exposed to methicillin-resistant S. aureus (MRSA) biofilms or planktonic bacteria. Additionally, the cytokine profiles in sera from patients with community-acquired pneumonia (CAP) caused by S. aureus were investigated. The aim was to gain insights into the immune response involved and differentiate between the planktonic and sessile MRSA forms. We identified 321 and 298 targets that were significantly differently expressed in PBMCs when exposed to planktonic or biofilm-embedded bacteria, respectively. PBMCs exposed to planktonic MRSA cells secreted increased levels of TNF-α, while IL-18 was elevated when exposed to the biofilm. The machine-learning analyses of the cytokine profiles obtained for the in vitro PBMCs and CAP sera distinguished between the two types of bacteria forms based on cytokines IL-18, IL12, and IL-17, and with a lower importance IL-6. Particularly, IL-18 which has not been correlated with S. aureus biofilms so far might represent a suitable marker for monitoring chronification during MRSA infection to individualize the therapy, but this hypothesis must be proved in clinical trials.

Blood MALT1 expression could help predict treatment outcomes in psoriasis patients, especially in those receiving biologics.

INTRODUCTION: Mucosa-associated lymphoid tissue 1 (MALT1) modulates T helper cell differentiation, pro-inflammatory cytokine production, and epidermal hyperplasia to participate in the pathology of psoriasis. This study aimed to explore the correlation of blood MALT1 with treatment outcomes in psoriasis patients. METHODS: MALT1 was detected in peripheral blood mononuclear cells by reverse transcription-quantitative polymerase chain reaction in 210 psoriasis patients before starting or converting to a new therapy, 50 disease controls, and 50 healthy controls. The psoriasis area severity index (PASI) score was evaluated at month (M)1, M3, and M6 in psoriasis patients. RESULTS: MALT1 was increased in psoriasis patients versus disease controls and healthy controls (both p < .001); and positively related to body mass index (p = .019) and PASI score (p < .001) in psoriasis patients. PASI75 rate at M1, M3, and M6 was 22.9%, 46.2%, and 71.0%, respectively; while PASI90 rate at M1, M3, and M6 was 3.8%, 29.0%, and 50.5%, respectively, in psoriasis patients. PASI75/90 rates at M1, M3, and M6 were increased in psoriasis patients receiving biologics versus those without (all p < .05). Pretreatment MALT1 was higher in psoriasis patients who achieved PASI75 (p = .001) and PASI90 (p < .001) at M6 compared to those who did not achieve that. Subgroup analyses discovered that pretreatment MALT1 had a stronger ability to predict PASI75 and 90 realizations in psoriasis patients receiving biologics (area under the curve [AUC]: 0.723 and 0.808) versus those without (AUC: 0.594 and 0.675). CONCLUSION: Blood MALT1 measurement may assist in predicting outcomes in psoriasis patients, especially in those receiving biologics.

Ruxolitinib Improves Immune-Dysregulation Features but not Epigenetic Abnormality in a Patient with STAT1 GOF.

PURPOSE: Patients with STAT1 gain-of-function (GOF) mutations often exhibit autoimmune features. The JAK1/2 inhibitor ruxolitinib can be administered to alleviate autoimmune symptoms; however, it is unclear how immune cells are molecularly changed by ruxolitinib treatment. Then, we aimed to investigate the trnscriptional and epigenetic status of immune cells before and after ruxolitinib treatment in a patient with STAT1 GOF. METHODS: A patient with a heterozygous STAT1 GOF variant (p.Ala267Val), exhibiting autoimmune features, was treated with ruxolitinib, and peripheral blood mononuclear cells (PBMCs) were longitudinally collected. PBMCs were transcriptionally analyzed by single-cell cellular indexing of the transcriptomes and epitopes by sequencing (CITE-seq), and epigenetically analyzed by assay of transposase-accessible chromatin sequencing (ATAC-seq). RESULTS: CITE-seq analysis revealed that before treatment, the patient's PBMCs exhibited aberrantly activated inflammatory features, especially IFN-related features. In particular, monocytes showed high expression levels of a subset of IFN-stimulated genes (ISGs). Ruxolitinib treatment substantially downregulated aberrantly overexpressed ISGs, and improved autoimmune features. However, epigenetic analysis demonstrated that genetic regions of ISGs-e.g., STAT1, IRF1, MX1, and OAS1-were highly accessible even after ruxolitinib treatment. When ruxolitinib was temporarily discontinued, the patient's autoimmune features were aggravated, which is in line with sustained epigenetic abnormality. CONCLUSIONS: In a patient with STAT1 GOF, ruxolitinib treatment improved autoimmune features and downregulated aberrantly overexpressed ISGs, but did not correct epigenetic abnormality of ISGs.

Essential oil supplementation in milk replacers: short- and long-term impacts on feed efficiency, the faecal microbiota and the plasma metabolome in dairy calves.

Early supplementation with oregano essential oil (EO) in milk replacer (MR) may improve growth, immune responses, the microbiota and the metabolome in dairy calves during pre-weaning and in adulthood. Sixteen female dairy calves (3 days of age) were divided in two groups (n = 8/group): the control group (no EO) and the EO group (0.23 ml of EO in MR during 45 days). After weaning, calves were kept in a feedlot and fed ad libitum. The animals were weighed, and blood and faecal samples were collected on days 3 (T0), 45 (T1) and 370 (T2) to measure the biochemical profile and characterise peripheral blood mononuclear cells (PBMCs; CD4+, CD8+, CD14+, CD21+ and WC1+), the metabolome and microbiota composition. The EO group only had greater average daily weight gain during the suckling (EO supplementation) period (P = 0.030). The EO group showed higher average CD14+ population (monocytes) values, a lower abundance of Ruminococcaceae UCG-014, Faecalibacterium, Blautia and Alloprevotella and increased abundances of Allistipes and Akkermansia. The modification of some metabolites in plasma, such as butyric acid, 3-indole-propionic acid and succinic acid, particularly at T1, are consistent with intestinal microbiota changes. The data suggest that early EO supplementation increases feed efficiency only during the suckling period with notable changes in the microbiota and plasma metabolome; however, not all of these changes can be considered desirable from a gut health point of view. Additional research studies is required to demonstrate that EOs are a viable natural alternative to antibiotics for improving calf growth performance and health.

Metacell-based differential expression analysis identifies cell type specific temporal gene response programs in COVID-19 patient PBMCs.

By profiling gene expression in individual cells, single-cell RNA-sequencing (scRNA-seq) can resolve cellular heterogeneity and cell-type gene expression dynamics. Its application to time-series samples can identify temporal gene programs active in different cell types, for example, immune cells' responses to viral infection. However, current scRNA-seq analysis has limitations. One is the low number of genes detected per cell. The second is insufficient replicates (often 1-2) due to high experimental cost. The third lies in the data analysis-treating individual cells as independent measurements leads to inflated statistics. To address these, we explore a new computational framework, specifically whether "metacells" constructed to maintain cellular heterogeneity within individual cell types (or clusters) can be used as "replicates" for increasing statistical rigor. Toward this, we applied SEACells to a time-series scRNA-seq dataset from peripheral blood mononuclear cells (PBMCs) after SARS-CoV-2 infection to construct metacells, and used them in maSigPro for quadratic regression to find significantly differentially expressed genes (DEGs) over time, followed by clustering expression velocity trends. We showed that such metacells retained greater expression variances and produced more biologically meaningful DEGs compared to either metacells generated randomly or from simple pseudobulk methods. More specifically, this approach correctly identified the known ISG15 interferon response program in almost all PBMC cell types and many DEGs enriched in the previously defined SARS-CoV-2 infection response pathway. It also uncovered additional and more cell type-specific temporal gene expression programs. Overall, our results demonstrate that the metacell-pseudoreplicate strategy could potentially overcome the limitation of 1-2 replicates.

mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1ß < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1ß, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.