Human peripheral blood mononuclear cells (PBMC) are key to several diagnostics assays and basic science research. Blood pre-analytical variations that occur before obtaining the PBMC fraction can ...significantly impact the assays results, including viability, composition, integrity, and gene expression changes of immune cells. With this as motivation, we performed a quantitative shotgun proteomics analysis using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ 8plex) labeling to compare PBMC obtained from 24 h-stored blood at room temperature versus freshly isolated. We identified a total of 3195 proteins, of which 245 were differentially abundant (101 upregulated and 144 downregulated). Our results revealed enriched pathways of downregulated proteins related to exocytosis, localization, vesicle-mediated transport, cell activation, and secretion. In contrast, pathways related to exocytosis, neutrophil degranulation and activation, granulocyte activation, leukocyte degranulation, and myeloid leukocyte activation involved in immune response were enriched in upregulated proteins, which may indicate probable granulocyte contamination and activation due to blood storage time and temperature. Examples of upregulated proteins in the 24 h-PBMC samples are CAMP, S100A8, LTA4H, RASAL3, and S100A6, which are involved in an adaptive immune system and antimicrobial activity, proinflammatory mediation, aminopeptidase activities, and naïve T cells survival. Moreover, examples of downregulated proteins are NDUFA5, TAGLN2, H3C1, TUBA8, and CCT2 that are related to the cytoskeleton, cell junction, mitochondrial respiratory chain. In conclusion, the delay in blood-processing time directly impacts the proteomic profile of human PBMC, possibly through granulocyte contamination and activation.
•The delay in blood-processing time directly impacts the proteome of human Peripheral Blood Mononuclear Cells.•Changes in proteomic profile caused by the storage of blood before the isolation of Peripheral Blood Mononuclear Cells are related to possible contamination and activation of granulocytes.•The use of freshly isolated human Peripheral Blood Mononuclear Cells is recommended for proteomics research.
Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes ...to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age.
Senescent CD8+ T cells in peripheral blood can be detected, quantified, and isolated using a fluorogenic and self‐immobilizing substrate of senescence‐associated β‐galactosidase. Characterization of CD8+ T cells with high SA‐βGal activity isolated from healthy donors in their 20s and 60s revealed a significantly greater abundance of SA‐βGal expressing CD8+ T cells with a unique transcriptional signature and features telomere dysfunction‐induced senescence and p16‐mediated senescence in older humans.
Cordyceps sinensis (CS) is an herbal tonic in traditional Chinese medicine and is used to treat a wide range of disorders, including immune, kidney, respiratory, lung and cardiovascular diseases, in ...China. Most studies are focused mainly on nucleotides and polysaccharides from CS and consider them to be the main active ingredients, while other ingredients are often disregarded. Hundreds of sphingolipids have been identified from CS and showed inhibitory effects on mouse splenic lymphocytes.
This study aimed to establish a method for preparing a fraction of sphingolipids from the mycelial powder of CS and evaluate its immunosuppressive activity.
Fraction of sphingolipids (Fr-SPLs) were prepared by silica gel chromatography and reversed-phase chromatography. Its components were identified and quantified by Quadrupole-Orbitrap UHPLC–MS/MS. PBMCs were prepared from human blood, and splenic lymphocytes, B cells, and T cells were prepared from mouse spleens. The inhibitory effect of Fr-SPLs on cell viability was evaluated by CCK-8 assay. PBMC apoptosis and the ratio of CD4+ T cells and CD8+ T cells were quantified by flow cytometry analysis. The expression of IL-2, IL-10, and TNF-α in PBMCs was detected by ELISA kits.
A fraction containing 84.83% of sphingolipids (SPLs) was prepared from the mycelia of CS and named Fr-SPLs. 15 SPLs were identified from the Fr-SPLs. Fr-SPLs significantly inhibited the viability of human peripheral blood mononuclear cells (PBMCs) with an IC50 value of 9.82 μg/mL and promoted PBMC apoptosis in a dose-dependent manner. Moreover, Fr-SPLs inhibited the viability of mouse splenocytes, as well as that of B cells and T cells derived from splenocytes. Furthermore, Fr-SPLs reduced the production of IL-2, IL-10, and TNF-α in PBMCs.
Fr-SPLs show immunosuppressive activity, and this study will be useful for preparing immunosuppressive components from CS and its mycelia for hyperimmune disease.
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1.A fraction containing 15 sphingolipids (SPLs) with a content of 84.83% is prepared from the mycelia of Cordyceps Sinensis and named Fr-SPLs.2.The wild Cordyceps Sinensis contains these 15 sphingolipids.3.The Fr-SPLs exhibits an immunosuppressive activity.4.The Fr-SPLs has the potential to alleviate the symptoms caused by the COVID-19
•Implantation failure is one of the factors contributing to pregnancy rate reduction after IVF.•Local immunological cells and factors at implantation site play an important role in pregnancy ...process.•PBMC-therapy has positive effect, by developing required initial inflammation for implantation.
Infertility is one of the most common problems among couples worldwide. Recurrent pregnancy loss, premature ovarian failure, recurrent implantation failure and etc. are common high prevalence disorders in societies. We will review the definition, causes and treatment of recurrent implantation failure disorder in recent studies. Implantation refers to the attachment of the embryo to the endometrial luminal surface and recurrent implantation failure (RIF) is defined as the failure to achieve a pregnancy after transferring high-grade embryos through at least three in vitro fertilization (IVF) cycles to the endometrium. The embryo factors, maternal age, uterine factors, and multifactorial effectors have been considered as the causes of implantation failure. In this review, we aim to focus on immunological factors and cells such as pro-inflammatory cytokines and dendritic cells, macrophages, decidual and uterine NK cells, as well as Th-1 cells. There are different types of treatment according to the cause of RIF including aspirin and low-molecular-weight heparin therapy, immunosuppressive drugs, intravenous immunoglobulins, and hydroxychloroquine. Among immunological recurrent implantation failure therapy, we will discuss the intrauterine PBMC-therapy in detail.
Biogenic synthesis of nanoparticles used for biomedical application has received much attention nowadays owing to its quick synthesis, cost effectiveness and biocompatible nature. The present study ...focuses on the biosynthesis of silver palladium bimetallic nanoparticles (AgPd NPs) from aqueous fruit extract of Terminalia chebula. Synthesized AgPd NP was assessed for antimicrobial activity and anticancer potential against lung cancer cells (A549). XRD analysis confirmed the formation of face centered cubic crystalline structure with average size of 20 nm, which was affirmed by DLS analysis. Uniform spherical shaped nanoparticles were observed in SEM and TEM analysis. Zeta potential value of −14.4 mV illustrated the stability of AgPd NPs. Anticancer studies illustrated that AgPd NPs induced ROS generation in lung cancer cells, thereby stimulating mitochondrial apoptotic pathway causing cell death. AgPd NPs exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa. In vitro toxicity studies revealed that AgPd NPs exhibited no cyototoxic and hemolytic effect upto its maximum dose (200 μg/ml), ensuring the biocompatibility of nanoparticles. Our findings demonstrated that aqueous extract of T. chebula act as effective reducing and stabilizing agent for green synthesis of biocompatible AgPd NPs, which exhibits potent antimicrobial and anticancer activities.
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•Biogenic synthesis of AgPd NPs using aqueous fruit extract of Terminalia chebula.•AgPd NPs showed potent anticancer potential against lung cancer cell lines A549.•AgPd NPs induced oxidative stress mediated cell death in A549 cells.•Ag Pd NPs exhibited antimicrobial activity against Gram + ve and –ve bacteria.
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•We identified 110 DEGs in PBMCs from cardioembolic patients and healthy controls.•Integrated bioinformatics analyses identified CTNNB1 and MDM2 as central hub genes.•Ischemic ...neuron-exposed neutrophils show Mdm2/p53-mediated β-catenin degradation.•Neutrophils upregulate pro-survival Ctnnb1 to compensate for this degradation.
A better understanding of the mechanism(s) underlying cardioembolic stroke can promote recovery and reduce the risk of recurrent embolisms.
Peripheral blood mononuclear cell (PBMC) gene expression datasets from cardioembolic patients and healthy controls were obtained from the Gene Expression Omnibus (GEO) database (GSE58294). The Limma software package was utilized to identify differentially-expressed genes (DEGs). Protein-protein interaction (PPI) analysis of the DEGs was performed using STRING. A weighted gene co-expression network analysis (WGCNA) was used to build a gene co-expression network. In vitro experiments assessed the effects on neutrophils exposed to oxygen and glucose-deprived (OGD) cortical neurons. An in vivo murine model of thromboembolic stroke was constructed through thrombin injection to examine effects on circulating neutrophils. Mechanistic in vitro studies were conducted using the proteasome inhibitor MG132, the p53-Mdm2 binding inhibitor Nutlin-3a, Mdm2 small-interfering RNA (siRNA), and Ctnnb1 siRNA.
DEG analysis identified 44 upregulated and 66 downregulated genes in cardioembolic stroke PBMCs. PPI analysis of these DEGs yielded one eight-node protein module with β-catenin (CTNNB1) as the central hub protein. Integration of the DEGs with WGCNA-derived hub genes revealed the key hub DEGs CTNNB1 and mouse double minute 2 (MDM2). Follow-up experiments revealed Mdm2, p53, and phospho-β-catenin upregulation in neutrophils exposed to OGD neurons in vitro and following thromboembolic stroke in vivo. Mechanistic studies revealed that neutrophils transcriptionally upregulate Ctnnb1 expression to compensate for Mdm2/p53-mediated β-catenin degradation induced by exposure to OGD neurons, thereby promoting neutrophil survival.
Compensatory Ctnnb1 transcriptional upregulation in neutrophils induced by ischemic neuron exposure may be involved in promoting neutrophil survival following cardioembolic stroke.
Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, ...single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results.
Whole blood samples from 20 healthy adults were collected and incubated at 20–25 °C for 2–48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2–4 h) and longer (8–48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs.
PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased.
PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.
•Delayed PBMC processing can affect PBMC function and downstream assay quality.•We used flow cytometry, scRNA-seq and ELISpot to assess processing delay impact.•Delays of <24 h had minimal impact on PBMC quality and downstream assay outcomes.•Granulocyte removal after processing delay did not greatly impact gene expression.•Clinical trial sites should have on-site PBMC processing or be located close to central laboratories.
It is increasingly recognized that trastuzumab, an antibody approved for treating human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, exerts some of its antitumor effects ...through enhanced T cell responses. Full activation of CD8
+
T cells requires both expression of major histocompatibility complex class I molecules (HLA-ABC) and expression of the T cell costimulatory molecule CD80 or CD86; however, the impact of trastuzumab treatment on the expression of HLA-ABC and CD80 and CD86 has not been investigated in HER2-overexpressing breast cancer cells. In this study, we found that, while there is no direct correlation between the expression of HER2 and HLA-ABC in breast cancer, knockdown of HER2 or inhibition of HER2 kinase by lapatinib downregulated HLA-ABC expression. Trastuzumab had no meaningful effects on HLA-ABC expression in HER2-overexpressing breast cancer cells in monoculture; however, treatment of such cells with trastuzumab in co-culture with human peripheral blood mononuclear cells (PBMC) significantly upregulated not only HLA-ABC expression but also CD86 expression. We showed that this upregulation was mediated by interferon gamma (IFNγ) secreted from the natural killer (NK) cells in PBMC as a result of engagement of NK cells by trastuzumab. We further confirmed this effect of trastuzumab in vivo using a mouse mammary tumor model transduced to overexpress human HER2. Together, our data provide evidence that trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules in HER2-overexpressing breast cancer cells in the presence of PBMC, which supports the view that T-cell-mediated immune responses are involved in trastuzumab-mediated antitumor effects.
Crohn’s disease (CD), as one of the principal form of inflammatory bowel disease (IBD), is characterized by the chronic and recurring inflammatory conditions in the intestine resulting from the ...over-activation of intestinal immunity. Hyposplenism is strongly associated with CD, while the effect of human spleen on the differentiation and development of immune cell subsets in CD patients remains unclear. Isolated congenital asplenia (ICA) is an extremely rare condition characterized by the absence of a spleen at birth without any other developmental defects. Here, we describe the first case of a patient with both ICA and CD, and follow the progression of CD from remission to active stage. Using cytometry by time of flight (CyTOF) analysis, we draw the first single-cell mapping of peripheral blood mononuclear cells (PBMC) from this unique patient, tracing back to the innate or adaptive immune cell subsets and cell surface markers affected by the spleen. Based on our analysis, it is speculated that the spleen contributes to maintaining immune homeostasis, alleviating intestinal inflammation and improving prognosis by influencing the differentiation and development of peripheral immune cell subsets and the expression of cell surface markers in patients with CD.
Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to ...cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection.
Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-β, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium.
Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.
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