Mononuclear cell (MNC) apheresis products serve as the starting material of many cell therapies, and they can be further processed to yield purified peripheral blood mononuclear cells (PBMCs). This ...PBMC purification step eliminates excess red blood cells (RBCs) and platelets, since these can inhibit the culture and/or expansion of specific cell types of interest such as T cells, NK cells, or monocytes. The use of Ficoll density gradient separation efficiently separates PBMCs from RBCs, and this process has been adapted to the automated Sepax C-Pro device (Cytiva) for clinical manufacturing. In contrast to the Sepax, the Curate Cell Processing System uses microfluidic technology to separate PBMCs without the use of Ficoll.
We performed a series of side-by-side PBMC purifications using the Sepax and Curate systems with 6 healthy donor MNC apheresis products. The Curate microfluidic system yielded ≥75% recovery of total nucleated cells (average = 78.7 ± 2.7%), which was a 48% increase compared to Sepax, with a 77% increase in recovery of CD3+ T cells in particular. This improvement in recovery was accompanied by a significant decrease in both RBC and platelet contamination, with a 99.7 ± 0.1% reduction of platelets in the Curate compared to 90.9 ± 0.6% in the Sepax. Characterization of T cells following isolation showed an equivalent T cell differentiation state of naïve vs. effector cells between the two platforms, and current experiments are underway to evaluate performance of isolated PBMCs in a CAR-T manufacturing process. Given the improved recovery and purity of PBMCs isolated from the Curate microfluidic system, this technology demonstrates significant utility particularly for applications where large cell numbers and efficient cell recovery are desired.
Cold allodynia is a common symptom of neuropathic and inflammatory pain following peripheral nerve injury. The mechanisms underlying this disabling sensory alteration are not entirely understood. In ...primary somatosensory neurons, cold sensitivity is mainly determined by a functional counterbalance between cold-activated TRPM8 channels and Shaker-like Kv1.1-1.2 channels underlying the excitability brake current IKD Here we studied the role of IKD in damage-triggered painful hypersensitivity to innocuous cold. We found that cold allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in mice, was related to both an increase in the proportion of cold-sensitive neurons (CSNs) in DRGs contributing to the sciatic nerve, and a decrease in their cold temperature threshold. IKD density was reduced in high-threshold CSNs from CCI mice compared with sham animals, with no differences in cold-induced TRPM8-dependent current density. The electrophysiological properties and neurochemical profile of CSNs revealed an increase of nociceptive-like phenotype among neurons from CCI animals compared with sham mice. These results were validated using a mathematical model of CSNs, including IKD and TRPM8, showing that a reduction in IKD current density shifts the thermal threshold to higher temperatures and that the reduction of this current induces cold sensitivity in former cold-insensitive neurons expressing low levels of TRPM8-like current. Together, our results suggest that cold allodynia is largely due to a functional downregulation of IKD in both high-threshold CSNs and in a subpopulation of polymodal nociceptors expressing TRPM8, providing a general molecular and neural mechanism for this sensory alteration.SIGNIFICANCE STATEMENT This paper unveils the critical role of the brake potassium current IKD in damage-triggered cold allodynia. Using a well-known form of nerve injury and combining behavioral analysis, calcium imaging, patch clamping, and pharmacological tools, validated by mathematical modeling, we determined that the functional expression of IKD is reduced in sensory neurons in response to peripheral nerve damage. This downregulation not only enhances cold sensitivity of high-threshold cold thermoreceptors signaling cold discomfort, but it also transforms a subpopulation of polymodal nociceptors signaling pain into neurons activated by mild temperature drops. Our results suggest that cold allodynia is linked to a reduction of IKD in both high-threshold cold thermoreceptors and nociceptors expressing TRPM8, providing a general model for this form of cold-induced pain.
Background: Paroxetine is one of the well-known antidepressants. Recent studies have focused on paroxetine’s probable immuno-modulatory effects, since findings have indicated inflammation’s role in ...the pathophysiology of depression. Therefore, in the present study, TLR2 and TLR4 mRNA genes expression was assessed in paroxetine-treated peripheral blood mononuclear cells (PBMCs). Methods: Venous blood samples were drawn from five healthy men (20-40 years old). Peripheral blood mononuclear cells (PBMCs) were isolated from samples and were cultured. After the first incubation for 24h, phytohemagglutinin plus lipopolysaccharide were added to the cells and then were incubated for 24h. Thereafter, cells were treated with different concentrations of paroxetine in the presence or absence of inhibitors of 5-HT2 and 5-HT7 receptors. After incubation for 48h, RNA was extracted and cDNA was synthesized. Using the real-Time PCR technique, TLR2 and TLR4 genes mRNA expression were evaluated. Statistical analysis of data were carried out using GraphPad Prism 7. Results: TLR2 and TLR4 mRNA expression were significantly increased in response to paroxetine at all concentrations. Furthermore, the co-culture of cells with the drug and the 5-HT2R and 5HT7R inhibitor simultaneously revealed that paroxetine’s immuno-modulatory effects viaTLR2 are dependent on serotonin, while it is independent of serotonin in the case of TLR4. Conclusion: Considering paroxetine’s effect in modulating immune responses via increasing TLR2 and TLR4 expression, paroxetine could have therapeutic potentials in diseases with a deficiency in these receptors.
Teucrium polium has been used in traditional medicine around the world for centuries in treatment of various conditions and diseases. Many studies have confirmed pharmacological effects of its ...extracts, although the immunomodulatory effect has not been investigated. Therefore, the aim of our study was to examine the immunomodulatory effect of methanolic extract of T. polium (TPE) on peripheral blood mononuclear cells (PBMCs) derived from healthy donors and patients with hepatitis C virus HCV infection. We analyzed the effect of the extract on PBMCs viability using the MTT test. The cell death type was determined using Annexin V-FITC/7-AAD staining. Immunophenotyping using anti-CD8 FITC, anti-CD4 PE, anti-CD3 ECD, anti-CD20 PC5, anti-CD14 FITC and anti-CD25 PC7 was performed by flow cytometry. Results of the MTT test indicate that TPE stimulates proliferation of healthy PBMCs, while the HCV PBMCs viability was slightly reduced. The percentage of apoptotic HCV PBMCs was higher after TPE treatment compared to the control. The proportion of CD25-expressing cells was higher among the untreated HCV PBMCs than in the untreated healthy PBMCs. TPE treatment significantly and gradually increased CD25 expression in healthy PBMCs, whereas CD25 expression on HCV PBMCs increased only at the highest TPE concentration. The upregulation of double-positive CD3+CD25+, CD20+CD25+ and CD14+CD25+ cells was significant in TPE treated healthy PBMCs, while only the highest concentration was effective on HCV PBMCs. In summary, TPE exerts a strong immunomodulatory effect on healthy PBMCs and, only at the highest concentration, on HCV PBMNCs.
INTRODUCTIONSchizophrenia is a severe mental illness causing significant impairment in personal, family, social, educational, occupational, and other important areas of life. While there is no widely ...accepted endophenotype, peripheral blood cells may serve as an accessible model of intracellular changes in schizophrenia.METHODSWe reviewed the literature on the query "peripheral blood mononuclear cells AND schizophrenia" in Medline (Pubmed), selecting studies that searched for specific biomarkers of schizophrenia. We considered both diagnostic biomarkers and biomarkers of therapeutic response, specific schizophrenia disorders or differential diagnostic biomarkers.RESULTSWe retrieved 41 articles matching the search criteria, among which were studies that considered changes in the production of pro-inflammatory and anti-inflammatory markers, proteins, receptors, enzyme activity, and gene expression as potential biomarkers.CONCLUSIONApproaches analysing a biological axis or a group of related biomarkers may hold the greatest promise for identifying schizophrenia. In addition, pharmacological status, smoking status, inflammatory markers and glucose metabolites, the presence of comorbidities should be considered. Certain biomarkers, while not specific for the diagnosis of schizophrenia, may indicate the prognosis and effectiveness of treatment in the established diagnosis.
Cytokine storm (CS) is a major contributor to the fatal outcome of severe infectious diseases, including Covid-19. Treatment with the complement (C) C5 inhibitor eculizumab was beneficial in ...end-stage Covid-19, however, the mechanism of this effect is unknown. To clarify this, we analyzed the relationship between C activation and production of pro-inflammatory cytokines in a PBMC model.
Human PBMC with or without 20 % autologous serum was incubated with C3a, C5a, zymosan or zymosan-pre-activated serum (ZAS) for 24 h with or without eculizumab or the C5a receptor antagonist, DF2593A. C activation (sC5b-9) and 9 inflammatory cytokines were measured by ELISA.
In serum-free unstimulated PBMC only IL-8 release could be measured during incubation. Addition of C5a increased IL-8 secretion only, ZAS induced both IL-2 and IL-8, while zymosan led to significant production of all cytokines, most abundantly IL-8. In the presence of serum the above effects were greatly enhanced, and the zymosan-induced rises of IL-1α, IL-1β IFN-γ and IL-2 were significantly attenuated by eculizumab but not by DF2593a.
These data highlight the complexity of interrelationships between C activation and cytokine secretion under different experimental conditions. The clinically relevant findings include the abundant formation of the chemokine IL-8, which was stimulated by C5a, and the suppression of numerous inflammatory cytokines by eculizumab, which explains its therapeutic efficacy in severe Covid-19. These data strengthen the clinical relevance of the applied PBMC model for drug screening against CS, enabling the separation of complex innate immune cross-talks.
•In serum-free unstimulated human PBMC culture only IL-8 release could be measured.•Serum increased the release of all cytokines measured.•Zymosan increased the release of all cytokines much more in the presence of serum.•Eculizumab suppressed zymosan-induced secretion of IL-1a, IL-1b, IFγ and IL-2.•A differential relationship between C activation and cytokine release was revealed.
Describing spatiotemporal evolution and characteristics of dispersed systems using the population balance equation (PBE), examples including sectional and moment methods are fraught with numerous ...issues. Hence, this study develops an accurate method by combining computational fluid dynamics and population balance‐Monte Carlo method (CFD‐PBMC) with a moderate computational cost. An efficient sub‐model for particle migration was proposed to simulate the convection and diffusion processes of particulate flows. A graphics processing unit (GPU)‐based parallel computation was performed to accelerate the high‐dimensional CFD‐PBMC. Several classical cases with analytical or benchmark solutions were simulated, and a comprehensive comparison was made using the classical weighted random walk method. Good agreements were obtained, except in the case of radial migration, the reasons for which are explained in detail. The measured speedups on the GPU showed a factor of ~450 for pure migration and ~50 for the CFD‐PBMC method when compared with a standard high‐performance computer.
Prostate cancer is the second most common cancer and the fifth leading cause of cancer-associated death in men. Previous studies have revealed a surprising ability for an immature population of ...myeloid cells called myeloid-derived suppressor cells (MDSCs) in the commencement and development of many tumors, including those of prostate cancer. Herein, the molecular and cellular changes of MDSCs in prostate cancer in both human and nonhuman models are reviewed. The suppressive function of MDSCs are also discussed with a particular focus on the role of IL-6 and JAK/STAT3 signaling pathways in the induction of their suppressive activity. Ultimately, a brief review of MDSC-targeting approaches for potential cancer therapy is presented.
Sanhuang Xiexin Decoction (SHXD), consisting of Coptis chinensis Franch., Scutellaria baicalensis Georgi and Rheum palmatum L., is traditionally used for relieving fever, purging fire for removing ...toxins, eliminating phlegm and haemostasis, eliminating the wetness-evil from the upper warmer, clearing away the heat-evil and expelling superficial evils. Each of the three herbs contained in SHXD has been indicated to have anti-inflammatory effects in vivo, but its effects on rat NK-cell phenotypes remain unexplored, and the comprehensive mechanism of this compound SHXD in curing the inflammation induced by lipopolysaccharides (LPS) remains to be revealed.
The study aim was to assess the effect of SHXD on LPS-induced fever and inflammation in a rat model, reduce NLRP3 activation in NK cells expressing specific cell phenotype antibodies and determine the therapeutic value of this approach in vivo.
SHXD extract was prepared and analysed by the developed ultra-performance liquid chromatography (UPLC) method for the simultaneous detection of 14 compounds. The main peaks were firstly identified on an Orbitrap via high resolution tandem mass spectrometry (MS). Then, the extract was used in the rat model of LPS-induced inflammation and fever for pharmacologically study the effects of drug treatment. Peripheral blood lymphocyte cells were isolated from the animals, including those subjected to the SHXD extract treatment, and the cell phenotype was determined prior to cell culture and after treating the cell cultures with the extract. The phenotypes of cells harvested using CD3, CD4, CD8a, CD81, CD161 and CD86 antibodies were used to verify the enhanced memory of the peripheral blood lymphocytes cells (PBMC) that were induced into nature killer (NK) cells.
The SHXD extract was prepared, analysed and identified via quality control equipment and was observed to have pharmacological effects that reduced NLRP3 activation and fever in rats. The production of NK cells and peripheral blood lymphocytes was induced by the SHXD extract, which manifested as increased levels of CD4+, CD8a+, CD81+, CD161+ and CD86+ cells. The levels of CD3+ cells were significantly different between the model group and the drug-treated or control groups (p < 0.01) with dose independence, while the levels of CD4+ cells were not significantly different between the drug-treated and control groups, with a trend towards lower levels in the model group with dose independence. The levels of CD4+ cells was significantly different between the drug-treated group and the model groups with dose independence (p < 0.05). The levels of CD86+ cells were not significantly different between the drug-treated group and the model and control groups. The levels of CD8a + cells was significantly different between the model group and the drug and control groups (p < 0.05, dose 2.0 μg/ml), with higher levels in the drug-treated group. The levels of CD3+, CD4+, CD8a + cells in the drug treated group have dose dependence with SHXD.
This experiment revealed that SHXD reduced NLRP3 activation in the blood of LPS-treated rats, which occurred through the activation of NK cells that expressed CD3, CD8a and CD161. SHXD may possess anti-inflammatory effect via activacting the one of major pharmacology effcet of NK cells that expressed CD3, CD8a and CD161 phenotypes expression. This result demonstrates that SHXD may possess ability to enhance the memory of peripheral blood lymphocytes and natural killer cells.
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Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop ...better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86
classical monocytes and memory T cells expressing CCR6
and CXCR3
, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39
regulatory T cells and CCR4
CCR6
CXCR3
memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.
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