Peripheral Blood Mononuclear Cells Cultures Research Articles

An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture.

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition ('humanise') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

Mineralocorticoid Receptor Signaling in Peripheral Blood Cells in Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is associated with alterations in neuroendocrine function, primarily the hypothalamic-pituitary-adrenal axis, including lower expression of the glucocorticoid receptor (GR) and its target genes in peripheral blood mononuclear cells (PBMC) or full blood. We previously found reduced mineralocorticoid receptor (MR) expression in MS patients' peripheral blood. MS is being treated with a widening variety of disease-modifying treatments (DMT), some of which have similar efficacy but different mechanisms of action; body-fluid biomarkers to support the choice of the optimal initial DMT and/or to indicate an unsatisfactory response before clinical activity are unavailable. Using cell culture of volunteers' PBMCs and subsequent gene expression analysis (microarray and qPCR validation), we identified the mRNA expression of OTUD1 to represent MR signaling. The MR and MR target gene expression levels were then measured in full blood samples. In 119 MS (or CIS) patients, the expression of both MR and OTUD1 was lower than in 42 controls. The expression pattern was related to treatment, with the MR expression being particularly low in patients treated with fingolimod. While MR signaling may be involved in the therapeutic effects of some disease-modifying treatments, MR and OTUD1 expression can complement the neuroendocrine assessment of MS disease course. If confirmed, such assessment may support clinical decision-making.

Safety and efficacy indicators of guarana and Brazil nut extract carried in nanoparticles of coenzyme Q10: Evidence from human blood cells and red earthworm experimental model

This study characterized a nanosupplement based on coenzyme Q10 containing guarana (Paullinia cupana) and Brazil nuts oil (Bertholetia excelsa) (G-Nut). Determined cytotoxic and oxi-immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs) and its effect on mortality of red Californian earthworms (Eisenia fetida) and on the immune efficiency of its coelomocytes immune by in vitro exposure to yeast dead microorganism. The cytotoxic and immunomodulatory effects of G-Nut and the GN-Free extract (0.25–3 mg/mL) were determined in PBMC cultures. Apoptotic, oxidative, and inflammatory markers were determined using biochemical, immunological, and molecular protocols. The effects of G-Nut and GN-Free extracts on mortality and immune efficiency were investigated in earthworms. G-Nut and GN-Free did not induce cytotoxic events in PBMCs, triggering the decrease in apoptotic (caspases 3 and 8) gene expression, lipid and protein oxidation levels, or pro-inflammatory cytokine levels. G-Nut and GN-Free did not trigger earthworm mortality and improved coelomocyte immune efficiency by increasing Eisenia neutrophil extracellular DNA traps and brown body formation when exposed to dead yeasts. The G-Nut nanoformulation is safe and can be used as a new form of food supplement by oral or transdermal delivery.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15–1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15–1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

Antibody-secreting cells as a source of NR1-IgGs in N-methyl-D-aspartate receptor-antibody encephalitis

BackgroundThe pathogenicity of NR1-IgGs in N-methyl-D-aspartate receptor (NMDAR)-antibody encephalitis is known, but the immunobiological mechanisms underlying their production remain unclear. MethodsFor the first time, we explore the origin of NR1-IgGs and evaluate the contribution of B-cells to serum NR1-IgGs levels. Peripheral blood mononuclear cells (PBMCs) were obtained from patients and healthy controls (HCs). Naïve, unswitched memory (USM), switched memory B cells (SM), antibody-secreting cells (ASCs), and PBMC depleted of ASCs were obtained by fluorescence-activated cell sorting and cultured in vitro. ResultsFor some patients, PBMCs spontaneously produced NR1-IgGs. Compared to the patients in PBMC negative group, the positive group had higher NR1-IgG titers in cerebrospinal fluid and Modified Rankin scale scores. The proportions of NR1-IgG positive wells in PBMCs cultures were correlated with NR1-IgGs titers in serum and CSF. The purified ASCs, SM, USM B cells produced NR1-IgGs in vitro. Compared to the patients in ASCs negative group, the positive group exhibited a worse response to second-line IT at 3-month follow-up. Naïve B cells also produce NR1-IgGs, implicating that NR1-IgGs originate from naïve B cells and a pre-germinal centres defect in B cell tolerance checkpoint in some patients. For HCs, no NR1-IgG from cultures was observed. PBMC depleted of ASCs almost eliminated the production of NR1-IgGs. ConclusionsThese collective findings suggested that ASCs might mainly contribute to the production of peripheral NR1-IgG in patients with NMDAR-antibody encephalitis in the acute phase. Our study reveals the pathogenesis and helps develop tailored treatments (eg, anti-CD38) for NMDAR-antibody encephalitis.

The Role of Senescent CD8+T Cells in the Pathogenesis of Disseminated Leishmaniasis.

Disseminated leishmaniasis (DL) caused by L. braziliensis is characterized by the presence of 10 to more than 1000 lesions spread on the body. While protection against Leishmania is mediated by macrophages upon activation by IFN-γ produced by CD4+T cells, the pathology of disseminated leishmaniasis (DL) could be mediated by macrophages, NK, and CD8+T cells. Herein, we evaluate the participation of senescent CD8+T cells in the pathogenesis of DL. Methods: Peripheral blood mononuclear cells (PBMCs), biopsies, co-cultures of CD8+T cells with uninfected and infected macrophages (MØ), and PBMC cultures stimulated with soluble L. braziliensis antigen (SLA) for 72 h from patients with cutaneous leishmaniasis (CL) and DL were used to characterize senescent CD8+T cells. Statistical analysis was performed using the Mann-Whitney and Kruskal-Wallis tests, followed by Dunn's. Results: Patients with DL have an increase in the frequency of circulating CD8+T cells that present a memory/senescent phenotype, while lesions from DL patients have an increase in the frequency of infiltrating CD8+T cells with a senescent/degranulation phenotype. In addition, after specific stimuli, DL patients' circulating CD8+T with memory/senescent profile, showing degranulation characteristics, increased upon SLA stimuli, and those specific CD8+T cells from DL patients had an increased degranulation phenotype, causing more apoptosis of infected target cells. Conclusions: DL patients show a higher frequency of cytotoxic senescent CD8+T cells compared to CL patients, and that could promote the lysis of infected cells, although without parasite killing, releasing Leishmania to the extracellular compartment, contributing to the spread of parasites.

Technical pitfalls when collecting, cryopreserving, thawing, and stimulating human T-cells.

The collection, cryopreservation, thawing, and culture of peripheral blood mononuclear cells (PBMCs) can profoundly influence T cell viability and immunogenicity. Gold-standard PBMC processing protocols have been developed by the Office of HIV/AIDS Network Coordination (HANC); however, these protocols are not universally observed. Herein, we have explored the current literature assessing how technical variation during PBMC processing can influence cellular viability and T cell immunogenicity, noting inconsistent findings between many of these studies. Amid the mounting concerns over scientific replicability, there is growing acknowledgement that improved methodological rigour and transparent reporting is required to facilitate independent reproducibility. This review highlights that in human T cell studies, this entails adopting stringent standardised operating procedures (SOPs) for PBMC processing. We specifically propose the use of HANC's Cross-Network PBMC Processing SOP, when collecting and cryopreserving PBMCs, and the HANC member network International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) PBMC Thawing SOP when thawing PBMCs. These stringent and detailed protocols include comprehensive reporting procedures to document unavoidable technical variations, such as delayed processing times. Additionally, we make further standardisation and reporting recommendations to minimise and document variability during this critical experimental period. This review provides a detailed overview of the challenges inherent to a procedure often considered routine, highlighting the importance of carefully considering each aspect of SOPs for PBMC collection, cryopreservation, thawing, and culture to ensure accurate interpretation and comparison between studies.

Molecular Analysis and Ex Vivo Infectivity of Seronegative Occult Hepatitis C Virus: A Study in Single Haemodialysis Centre.

In occult hepatitis C virus infection (OCI), hepatitis C virus ribonucleic acid (HCV RNA) is detectable in peripheral blood mononuclear cells (PBMCs) but is not evident in serum or plasma. Understanding of OCI in patients with seronegative anti-HCV antibodies is limited. In this study, six HCV isolates from haemodialysis (HD) patients with seronegative OCI were identified by molecular assays and phylogenetic analysis. The virus infectivity was assessed ex vivo using a primary naïve PBMC culture system. HCV isolates obtained from the PBMCs of 10 patients with chronic HCV infection (CCI) were characterised concurrently and used as positive controls in the cell culture. Sequence analysis of the 5' untranslated region (UTR) and non-structural 5B (NS5B) region revealed that HCV genotype 3 was the most prevalent virus type in both the OCI and CCI groups. One of the occult HCV isolates was identified as a mixed type. The mean viral load (log10 RNA copies/106 cells) in the PBMC samples of the OCI group (M = 3.4, SD = 0.7) was lower than that of the CCI group (M = 4.6, SD = 1.7). Upon culture, de novo OCI-HCV replicates were detected in five out of six naïve PBMC cultures. Analysis of the replicates showed a single guanine addition in the domain III of 5'-UTR but the overall molecular structure was retained. Seronegative OCI is an active form of infection that replicates at a low level in PBMCs. Seronegative OCI may share the same route of transmission as CCI. The retained viral competency may have an implication for its persistence.

Urinary Mitochondrial DNA Induces an Inflammatory Response in Peripheral Blood Mononuclear Cells

Background: Mitochondria are semiautonomous cell organelles having its own nucleic acid. Mitochondrial DNA (Mt-DNA) remain in hypomethylated (CpG) state and impose an immunogenic response by binding to the toll-like receptor (TLR-9) through the NF-kB pathway. Innate immune cells recognize the hypomethylated pattern of mt-DNA and quickly trigger the innate immune response. The immunomodulatory effects of urinary mt-DNA derived from renal transplant recipients with COVID-19-associated acute kidney injury (AKI) have not been studied. Materials and Methods: Healthy donor peripheral blood mononuclear cell (PBMC) was cultured with the urinary Mt-DNA derived from the renal transplant recipients, who previously developed SARS-CoV-2 infection associated AKI. Cell activation was measured by the flow cytometry. In cell pellets, interleukin IL-6, IL-10, and Myd88, TLR-9 mRNA transcript expression was measured by the reverse transcription polymerase chain reaction. The IL-6 and IL-10 cytokine levels were measured by the enzyme-linked immunosorbent assay in culture supernatants. Results: The urinary mitochondrial DNA (umt-DNA) significantly induces the activation of > 75% of PBMCs. The m-RNA transcript expression of the inflammatory gene in control versus umt-DNA treated PBMCs was for IL-6 (0.99 ± 0.05 vs. 2.18 ± 1.15 au; P = 0.004), MYD88 was (1.00 ± 0.05 vs. 1.55 ± 0.31; P < 0.001), TLR-9 (1.00 ± 0.05 vs. 3.33 ± 1.37 au; P < 0.001) was upregulated, and the IL-10 (1.00 ± 0.13 vs. −1.73 ± 0.58; P < 0.001) level was downregulated. However, in PBMC culture supernatants, IL-6 level in control versus umt-DNA-treated groups were (37.50 ± 13.79 vs. 186.9 ± 15.50 pg/mL; P < 0.001), which was significantly higher in umt-DNA-treated groups and the IL-10 (8.80 ± 2.16 vs. 7.60 ± 3.12 pg/mL; P = 0.32) level was similar between the control- and umt-DNA-treated groups. Conclusions: Urinary Mt-DNA significantly induces the inflammatory cytokine IL-6 secretion from the PBMCs through the Myd88-dependent pathway.

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-1β < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1β, 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.

Cytotoxicity and anti-inflammatory properties of a GLP-1 receptor agonist in a model of human peripheral blood mononuclear cell culture

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Introduction. &amp;lt;/strong&amp;gt;GLP-1R agonists (GLP-1RAs) are a type of anti-hyperglycemic medications used for the management of type 2 diabetes mellitus (T2DM). In addition to glucose-lowering effects, GLP-1R agonists (GLP-1RAs) also provide further advantages by promoting weight loss and controlling blood pressure. Furthermore, GLP-1RAs have been reported for their therapeutic benefits in neurological, cardiovascular, endocrine, and metabolic diseases. Emerging evidence from many clinical and experimental studies, as well as in vitro researches, suggests that GLP-1RA agonists may reduce inflamma tion and modify the immunological response, but the underlying molecu lar mechanisms remain unclear. In this research, we examined the effect of GLP-1RA on the cytotoxicity, proliferative activity, and cytokine production of human peripheral blood mononuclear cells (PBMCs) in vitro.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt; Methods. &amp;lt;/strong&amp;gt;The peripheral blood mononuclear cells (PBMCs) cultures from healthy volunteers were used. GLP-1RA (DMB), in different concentrations, was added to the cell cultures. A flow cytometry assay was used to assess cell viability, while MTT and CFSE dye dilution assays were used to quantify proliferative activity. Cytokine levels were measured using a sandwich en zyme-linked immunosorbent assay (ELISA).&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Results.&amp;lt;/strong&amp;gt; The cytotoxicity data demonstrated that only the highest DMB concentration (1000 nM) decreased the metabolic activity (viability) of PBMCs (p&amp;amp;lt;0.05) in comparison to untreated cultures, while concentra tions of 200 nM (p&amp;amp;lt;0.05) and 1000 nM (p&amp;amp;lt;0.001) of GLP-1RA increased the proportion of late apoptotic and necrotic cells (p&amp;amp;lt;0.001). The prolif eration of PBMCs was significantly decreased in the presence of GLP-1RA at a 100 nM concentration when compared to the control (p&amp;amp;lt;0.05). The same concentration of GLP-1RA significantly reduced the production of IL-6, IL-1&amp;amp;beta;, and TNF-&amp;amp;alpha;.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Conclusion.&amp;lt;/strong&amp;gt; Our results suggested that GLP-1RA (DMB), at a non-toxic concentration of 100 nM, inhibited the proliferation and production of pro-inflammatory cytokines by human PBMCs in vitro. These findings open the perspective to study the immune response in detail in future experiments.&amp;lt;/p&amp;gt;

A potential role of protein extractions from Metagonimus yokogawai in amelionating inflammation in patients with ankylosing spondylitis

Helminth infections and their components has been recognized to have a positive impact on the immune system. This study aimed to investigate the potential of Metagonimus yokogawai-derived proteins (MYp) to provide protection against ankylosing spondylitis (AS) through modulation of immune responses. The cytotoxicity of MYp at various doses was first assessed using MTS and flow cytometry. Peripheral blood mononuclear cells (PBMCs) were collected from AS patients, and the production of inflammatory cytokines was analyzed through flow cytometry. In the experiments with SKG mice, MYp or vehicle was administered and inflammation was evaluated through immunohistochemistry and enzyme-linked immunosorbent assay. The results showed that MYp did not decrease cell viability of PBMCs even after 48 h. Additionally, the frequencies of IFN-γ and IL-17A producing cells were significantly reduced after MYp treatment in the PBMC cultures. Furthermore, MYp treatment significantly suppressed arthritis and enthesitis in the SKG mouse model. The results suggest the first evidence that MYp can effectively alleviate clinical symptoms and restore cytokine balance in patients with AS.

Anti-aquaporin-4 immune complex stimulates complement-dependent Th17 cytokine release in neuromyelitis optica spectrum disorders

Proinflammatory cytokines, such as (IL: interleukin) IL-6 and IL-17A, and complement fixation are critical in the immunopathogenesis of neuromyelitis optica spectrum disorders (NMOSD). Blocking the IL-6 receptor or the C5 complement pathway reduces relapse risk. However, the role of interleukin (IL)-6 and complement in aquaporin-4 (AQP4) autoimmunity remains unclear. To investigate the role of the anti-AQP4 immunoglobulin (AQP4-IgG)/AQP4 immunocomplex on the induction and profile of ex vivo cytokine and surface marker expression in peripheral blood mononuclear cells (PBMC) culture. Isolated PBMCs obtained from 18 patients with AQP4-IgG-seropositive-NMOSD (8 treatment-naive, 10 rituximab-treated) or ten healthy controls were cultured with AQP4-immunocomplex with or without complement. Changes in PBMC surface markers and cytokine expression were profiled using flow cytometry and ELISA. PBMCs derived from treatment-naive NMOSD patients stimulated with a complex mixture of serum complement proteins produced significant elevations of IL-17A and IL-6. Rituximab-treated patients also exhibited higher IL-6 but not IL-17A release. IL-6 and IL-17A elevations are not observed without complement. Co-stimulation of PBMCs with AQP4-IgG/AQP4 immunocomplex and complement prompts a Th17-biased response consistent with the inflammatory paradigm observed in NMOSD. A possible inflammation model is proposed via antigen-specific autoreactive peripheral blood cells, including NK/NKT cells.

TNF blockers alone and associated with Benznidazole impact in vitro cytokine dynamics in chronic Chagas disease.

Studies involving the immune response in Chagas disease suggest an imbalance in the immune response of symptomatic patients, with an inflammatory profile dominating in Chagas heart disease, mainly by tumour necrosis factor (TNF). TNF is considered a key cytokine in immunopathology in chronic carriers in several processes during the immune response. Our work aimed to evaluate regulatory (interleukin [IL]-4 and IL-10) and inflammatory (TNF, interferon-gamma [IFN-γ], IL-2 and IL-6) cytokines in peripheral blood mononuclear cells culture supernatants. of affected patients with undetermined clinical forms-IND (n = 13) mild heart form-CARD1 (n = 13) and severe cardiac form-CARD2 (n = 16), treated in vitro with two TNF blockers, Adalimumab (ADA) and Etanercept (ETA) alone or in association with Benznidazole (BZ). The results indicate that ADA was more competent in blocking TNF (compared to ETA) in all groups but with much lower levels in the CARD2 group. ETA statistically decreased TNF levels only in the CARD2 group. IFN-γ increased in the CARD2 group after treatment with ETA relative to ADA. IL-4 had its levels decreased when treated by both drugs. IL-2 was detected in cells from CARD2 carriers compared to the NEG group after treatment with both drugs. The association with BZ decreased levels of IL-2/TNF and increased IL-4. These data reinforce the participation of TNF in severe Chagas heart disease and bring perspectives on using these blockers in the immunological treatment of Chagas disease since the use of BZ is extremely limited in these patients.

Immunophenotyping, Part I: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures.

Nanoparticles are increasingly used in biomedical applications to influence the way the immune system reacts to tumors and infectious disease-causing agents. Nanoparticles not-intended for immunomodulation can also influence immune responses by affecting immune cell subsets' viability and/or activity. While immunophenotyping is commonly used to assess the effects of drugs and nanoparticles on immune cell subsets, no standardized approach exists due to the breadth of available cell models and instrumentation. In this chapter, we describe a protocol for flow cytometer calibration and reagent qualification prior to its use in the immunophenotyping experiment. The strategies described herein can be adapted to other instruments. The subsequent chapter-immunophenotyping part II (Chap. 25 )-provides detailed instructions for applying this methodology to analyze nanoparticle effects on subsets of immune cells present in peripheral blood.

Prune Consumption Attenuates Proinflammatory Cytokine Secretion and Alters Monocyte Activation in Postmenopausal Women: Secondary Outcome Analysis of a 12-Mo Randomized Controlled Trial: The Prune Study

BackgroundProinflammatory cytokines are implicated in the pathophysiology of postmenopausal bone loss. Clinical studies demonstrate that prunes prevent bone mineral density loss; however, the mechanism underlying this effect is unknown. ObjectiveWe investigated the effect of prune supplementation on immune, inflammatory, and oxidative stress markers. MethodsA secondary analysis was conducted in the Prune Study, a single-center, parallel-arm, 12-mo randomized controlled trial of postmenopausal women (55–75 y old; n = 235 recruited; n = 183 completed) who were assigned to 1 of 3 groups: “no-prune” control, 50 g prune/d and 100 g prune/d groups. At baseline and after 12 mo of intervention, blood samples were collected to measure serum high-sensitivity C-reactive protein (hs-CRP), serum total antioxidant capacity (TAC), plasma 8-isoprostane, proinflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor (TNF)-α] concentrations in plasma and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) culture supernatants, and the percentage and activation of circulating monocytes, as secondary outcomes. ResultsPrune supplementation did not alter hs-CRP, TAC, 8-isoprostane, and plasma cytokine concentrations. However, percent change from baseline in circulating activated monocytes was lower in the 100 g prune/d group compared with the control group (mean ± SD, −1.8% ± 4.0% in 100 g prune/d compared with 0.1% ± 2.9% in control; P < 0.01). Furthermore, in LPS-stimulated PBMC supernatants, the percent change from baseline in TNF-α secretion was lower in the 50 g prune/d group compared with the control group (−4.4% ± 43.0% in 50 g prune/d compared with 24.3% ± 70.7% in control; P < 0.01), and the percent change from baseline in IL-1β, IL-6, and IL-8 secretion was lower in the 100 g prune/d group compared with the control group (−8.9% ± 61.6%, −4.3% ± 75.3%, −14.3% ± 60.8% in 100 g prune/d compared with 46.9% ± 107.4%, 16.9% ± 70.6%, 39.8% ± 90.8% in control for IL-1β, IL-6, and IL-8, respectively; all P < 0.05). ConclusionsDietary supplementation with 50–100 g prunes for 12 mo reduced proinflammatory cytokine secretion from PBMCs and suppressed the circulating levels of activated monocytes in postmenopausal women.This trial was registered at clinicaltrials.gov as NCT02822378.

In Vivo Generation of Functional CD19 CAR-T Cells Using a Serum-Stable CD3-Targeted Lentiviral Vector

The high cost and complexity of CAR-T therapy has spurred efforts to generate CAR-T cells directly within patients via systemic infusion of serum-stable lentiviral vector particles that target T cells in situ for CAR transgene delivery. To ensure effective in vivo delivery, these vectors need to show resistance to neutralization by human serum, demonstrate transduction specificity for T cells, and support subsequent expansion of CAR-T cells. Lentiviral vectors (LV) pseudotyped with VSV G-glycoprotein (G-WT) transduce target cells via the LDL receptor (LDLR), which is ubiquitously expressed at variable levels and is upregulated on activated T cells. G-WT LVs are widely used to generate therapeutic CAR-T cells ex vivo, following purification and ex vivo activation of the T cells. To generate LVs capable of targeting circulating T cells in vivo, we utilized rational engineering of the VSV-G glycoprotein to design a G-CD3. This modified G protein avoids LDLR recognition, incorporates a CD3-specific single-chain antibody for targeted recognition of T cells, and minimizes LV particle inactivation upon exposure to human serum. G-CD3 LVs were shown to target and transduce resting T cells in peripheral blood mononuclear cell (PBMC) cultures with very high efficiency. The G-CD3 LVs powerfully activated T cells, evident from their increased CD25 expression, and the infection was not hindered in the presence of fresh, complement-active human serum (25%). Additionally, G-CD3 LVs efficiently transduced T cells in fresh human whole blood without apparent off-target transduction of non-T cell lineages. In vivo evaluation of G-CD3 LV efficiency was performed in humanized NSG mice by intravenous (IV) or intraperitoneal (IP) delivery of G-CD3 LVs encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3 LV led to in vivo CAR T cell generation with peak levels of 17% of total circulating human CD45+ cells on day 21 post vector administration. The in vivo generated CD19-CAR-T cells were fully functional as evidenced by the disappearance of endogenous human CD19+ B cells from peripheral blood. Tissue analysis of spleen, bone marrow, and liver at the time of sacrifice (day 39 post LV injection), showed anti-CD19 CAR-T in all three tissues accompanied by an absence or dramatic reduction in tissue-resident human CD19+ B cells. In contrast, CAR-T cells were not detected at any time point in humanized NSG mice injected with G-WT LV, regardless of whether the virus was administered IV or IP. In summary, these data highlight the potential applicability of fully retargeted, serum stable LVs for in vivo gene delivery or genome editing, offering promising prospects for future human gene therapy applications.

Leptin favors imbalance of antigen-specific CD4+ T-cells associated with severity of cat allergy.

Obesity can complicate IgE-mediated allergic diseases. In the present study, we aimed to investigate the ability of obesity-related concentrations of leptin to modulate the in vitro effector and regulatory Fel d1-specific CD4+ T-cell subsets in patients allergic to cat, considered the third most common cause of respiratory allergy in humans. For this study, plasma and peripheral blood mononuclear cells (PBMC) from 30 cat-allergic patients with mild, moderate and severe respiratory symptoms were obtained. The PBMC cultures were stimulated with Fel d1 antigen (10 µg/mL) in the presence or absence of obesity-related leptin dose (50 ηg/mL). After 6 days, the levels of cytokines and IgE in the supernatants were evaluated by multiplex and ELISA, respectively. The frequency of different non-follicular (CXCR5-) and follicular (CXCR5+) Fel d1-specific CD4+ T cell subsets was determined by flow cytometry. The plasma levels of leptin and IgE anti-cat titers were evaluated by ELISA and ImmunoCAP, respectively. Fel d1 induced both IgE production and release of cytokines related to Th2, Th9 and Th17 cell phenotypes. Feld1 was more efficient in increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cell subsets. Leptin favored the expansion Th2-like and Th9-like cells and TFHIL-21- cells positive for IL-4, IL-5 and IL-13, but reduced the proportion of conventional (Treg/Tr-1) and follicular (TFR) regulatory CD4+ T-cell subsets expressing or not CD39 marker. Finally, many of the imbalances between Fel d1-specific CD4+ T-cells were also correlated with plasma leptin and anti-Fel d1 IgE titers. In summary, hyperleptinemia should negatively impact on the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4+ T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.

Cell Cultures as a Versatile Tool in the Research and Treatment of Autoimmune Connective Tissue Diseases.

Cell cultures are an important part of the research and treatment of autoimmune connective tissue diseases. By culturing the various cell types involved in ACTDs, researchers are able to broaden the knowledge about these diseases that, in the near future, may lead to finding cures. Fibroblast cultures and chondrocyte cultures allow scientists to study the behavior, physiology and intracellular interactions of these cells. This helps in understanding the underlying mechanisms of ACTDs, including inflammation, immune dysregulation and tissue damage. Through the analysis of gene expression patterns, surface proteins and cytokine profiles in peripheral blood mononuclear cell cultures and endothelial cell cultures researchers can identify potential biomarkers that can help in diagnosing, monitoring disease activity and predicting patient's response to treatment. Moreover, cell culturing of mesenchymal stem cells and skin modelling in ACTD research and treatment help to evaluate the effects of potential drugs or therapeutics on specific cell types relevant to the disease. Culturing cells in 3D allows us to assess safety, efficacy and the mechanisms of action, thereby aiding in the screening of potential drug candidates and the development of novel therapies. Nowadays, personalized medicine is increasingly mentioned as a future way of dealing with complex diseases such as ACTD. By culturing cells from individual patients and studying patient-specific cells, researchers can gain insights into the unique characteristics of the patient's disease, identify personalized treatment targets, and develop tailored therapeutic strategies for better outcomes. Cell culturing can help in the evaluation of the effects of these therapies on patient-specific cell populations, as well as in predicting overall treatment response. By analyzing changes in response or behavior of patient-derived cells to a treatment, researchers can assess the response effectiveness to specific therapies, thus enabling more informed treatment decisions. This literature review was created as a form of guidance for researchers and clinicians, and it was written with the use of the NCBI database.

Differential Immune-Modulating Activities of Cell Walls and Secreted Metabolites from Probiotic Bacillus coagulans JBI-YZ6.3 under Normal versus Inflamed Culture Conditions.

Spore-forming probiotic bacteria, including Bacillus coagulans, are resilient and produce a variety of beneficial metabolites. We evaluated the immune-modulating effects of the novel probiotic strain Bacillus coagulans JBI-YZ6.3, where the germinated spores, metabolite fraction, and cell wall fraction were tested in parallel using human peripheral blood mononuclear cell cultures under both normal and lipopolysaccharide-induced inflamed culture conditions. The expression of CD25 and CD69 activation markers was evaluated via flow cytometry. Supernatants were tested for cytokines, interferons, chemokines, and growth factors using Luminex arrays. The germinated spores were highly immunogenic; both the cell wall and metabolite fractions contributed significantly. Under normal culture conditions, increased levels of immune activation were observed as increased expressions of CD25 and CD69 relative to natural killer cells, suggesting an increased ability to attack virus-infected target cells. On monocytes, a complex effect was observed, where the expression of CD25 increased under normal conditions but decreased under inflamed conditions. This, in combination with increased interleukin-10 (IL-10) and decreased monocyte chemoattractant protein-1 (MCP-1) production under inflamed conditions, points to anti-inflammatory effects. The production of the stem cell-related growth factor granulocyte colony-stimulating Factor (G-CSF) was enhanced. Further research is warranted to characterize the composition of the postbiotic metabolite fraction and document the characteristics of immunomodulating agents secreted by this probiotic strain.