Function Of Mononuclear Cells Research Articles

Mitochondrial dysfunction of peripheral blood mononuclear cells is associated with lung carcinogenesis

The composition, quantity, and function of peripheral blood mononuclear cells (PBMCs) are closely correlated with tumorigenesis. However, the mechanisms of PBMCs in lung cancer are not clear. Mitochondria are energy factories of cells, and almost all cellular functions rely on their energy metabolism level. The present study aimed to test whether the mitochondrial function of PBMCs directly determines their tumor immune monitoring function. We recruited 211 subjects, including 105 healthy controls and 106 patients with recently diagnosed with lung cancer. The model of lung carcinogenesis induced by BaP was used in animal experiment, and the Bap carcinogenic metabolite, Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), was used in cell experiment. We found that mitochondrial function of PBMCs decreased significantly in patients with new lung cancer, regardless of age. In vivo, BaP caused PBMC mitochondrial dysfunction in mice before the appearance of visible malignant tissue. Moreover, mitochondrial function decreased significantly in mice with lung cancers induced by BaP compared to those without lung cancer after BaP intervention. In vitro, BPDE also induced mitochondrial dysfunction and reduced the aggressiveness of PBMCs toward cancer cells. Furthermore, the changes in mitochondrial energy metabolism gene expression caused by BPDE are involved in this process. Thus, the mitochondrial function of PBMCs is a potential prognostic biomarker or therapeutic target to improve clinical outcomes in patients with lung cancer.

Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

In vitro stimulation with nontuberculous mycobacteria induced a stronger cytokine response in leukocytes isolated from individuals with latent tuberculosis compared to those isolated from active tuberculosis or cystic fibrosis patients

Mycobacterium tuberculosis and opportunistic environmental non-tuberculous mycobacteria (NTM) can cause severe infection. Why latent tuberculosis infection advances to active disease, and why some individuals with cystic fibrosis (CF) develop pulmonary infections with NTM is still poorly understood. The aim of this study was to investigate the effector function of peripheral blood mononuclear cells (PBMC) from individuals with active or latent tuberculosis, individuals with CF with or without pulmonary NTM-infection and healthy controls, by measuring cytokine response to in vitro stimulation with different species of NTMs. The cytokine concentrations of IL-17A, IL-22, IL-23, IL-10, IL12p70 and IFN-γ were measured in PBMC-culture supernatants after stimulation with NTMs. PBMCs from individuals with latent tuberculosis infection showed strong IL-17A, IL-22, and IFN-γ responses compared to individuals with active tuberculosis or CF. IL-10 production was low in both tuberculosis groups compared to the CF groups and controls. This study suggests that IL-17A and IL-22 might be important to keep tuberculosis in a latent phase and that individuals with CF with an ongoing NTM infection seem to have a low cytokine response.

P738 Response to etrasimod treatment in ulcerative colitis is associated with a reduction of circulating B and pro-inflammatory T cells: a single-cell-based post-hoc sub-analysis of the ELEVATE UC 52 trial

Abstract Background Etrasimod (ETR) is an oral, once-daily, selective sphingosine 1-phosphate receptor 1,4,5 (S1PR) modulator for the treatment of moderately to severely active ulcerative colitis (UC). The mechanism of action is based on a retention of lymphocytes in lymphatic tissue hindering circulating lymphocytes from entering the gut and causing inflammation. However, the effect of ETR on immune cell function and composition is not well characterized. In addition, it is unknown whether the response of UC patients to ETR treatment is associated with specific immune cell signatures. Methods Single-cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) and clinical data integration were used to characterize the effects of 12 weeks of treatment with ETR on the immune composition and function of peripheral blood mononuclear cells (PBMCs) collected from 15 UC patients at weeks 0 and 12 during the ELEVATE UC Phase 3 clinical trial. As an exploratory cohort, PBMCs from 5 UC patients reaching the primary endpoint of clinical remission at week 12 (modified Mayo score <2), 5 UC patients not responding to ETR treatment (modified Mayo score >2) and 5 placebo-treated UC patients were included. 30 samples were processed for scRNAseq; additionally, 28 blood samples were ex-vivo stimulated with ionomycin/PMA for 4 h and cytokine production was assessed by CyTOF. To determine the effects of ETR in UC patients, immune cell function and composition were compared intra-individually between weeks 0 and 12. Subsequent comparisons between ETR responders, non-responders and placebo-treated UC patients were performed to discriminate responders from non-responders. Results Significant reductions in T and B cell subsets, along with increases in the abundance of dendritic and monocytic cells, were observed in responders by week 12 of -treatment. Importantly, ETR treatment significantly reduced circulating T cells producing pro-inflammatory cytokines, including TNFα and IFNγ, and S1PR modulation led to a mitigation of naïve and effector memory CD4 and CD8 T cells in ETR-treated UC patients achieving clinical remission. In contrast, patients not responding to ETR treatment had significantly higher frequencies of both naïve and TNFα-producing CD4 T cells by week 12. Conclusion In line with the described role of S1PR in lymphocyte trafficking, single-cell analyses of circulating immune cells from ETR-treated UC patients confirmed a significant reduction in B cells and pathogenic T cell subsets after ETR treatment providing an in-depth cellular characterization of S1PR modulation in UC patients thereby contributing to a better understanding of the key mechanism of action of ETR. However, these findings require validation in larger cohorts.

Subclinical hypothyroidism in pregnancy – assessment of offspring thyroid status and mitochondrial robustness to stress

Subclinical hypothyroidism’s clinical implications on pregnancy are controversial. Consequently, thyrotropin (TSH) cutoff-values for pregnancy are continuously a subject for debate. In subclinical hypothyroidism, altered levels of thyroid hormones may affect mitochondrial function. Objectives were i) to analyze thyroid hormone levels in offspring of women with and without subclinical hypothyroidism ii) to analyze mitochondrial “robustness” in terms of MTG/TMRM ratio in pregnant women and their offspring in relation to thyroid function and iii) to perform differentiate analyses on different TSH thresholds to determine the importance of cutoff-values to results. Pregnant women were included by blood collections prior to a planned cesarean section, and cord samples were collected after delivery. Thyroid status (analyzed by Siemens Healthcare Diagnostics by an electrochemical luminescent immunoassay based on LOCI-technology) grouped the women and their offspring in euthyroid or subclinical hypothyroid, with groups established from previous recommended third-trimester cutoff-value (TSH > 3.0 mIU/L) and the recently recommended cutoff-value in Denmark (TSH > 3.7 mIU/L). Flow cytometric measurements of mitochondrial function in mononuclear blood cells with the fluorophores TetraMethylRhodamine Methyl Ester (TMRM) and Mitotracker Green (MTG) were used to evaluate mitochondrial robustness as the MTG/TMRM ratio. No significant differences in mitochondrial robustness between euthyroid and subclinical hypothyroid cohorts were observed, irrespective of TSH-cutoff applied. Maternal and cord MTG/TMRM ratios were positively correlated. Cord-TSH was elevated in subclinical hypothyroid offspring, independent of TSH cutoff applied. Cord-TSH was associated with maternal TSH-level, maternal smoking and cord arterial-pH.

Canine immune cells express high levels of CB1 and CB2 cannabinoid receptors and cannabinoid-mediated alteration of canine cytokine production is vehicle-dependent

With the increased popularity and societal acceptance of marijuana and cannabidiol (CBD) use in humans, there is an interest in using cannabinoids in veterinary medicine. There have been a few placebo-controlled clinical trials in dogs suggesting that cannabis-containing extracts are beneficial for dogs with inflammatory diseases such as osteoarthritis, and there is growing interest in their immunosuppressive potential for the treatment of immune-mediated diseases. Since cannabinoids exhibit anti-inflammatory and immunosuppressive effects in many species, the purpose of these studies was to examine whether the plant-derived cannabinoids, CBD and Δ9-tetrahydrocannabinol (THC), would also suppress immune function in canine peripheral blood mononuclear cells (PBMCs). Another goal was to characterize expression of the cannabinoid receptors, CB1 and CB2, in canine immune cells. We hypothesized that CBD and THC would suppress stimulated cytokine expression and that both cannabinoid receptors would be expressed in canine immune cells. Surprisingly, cannabinoid suppressive effects in canine PMBCs were quite modest, with the most robust effect occurring at early stimulation times and predominantly by THC. We further showed that cannabinoid-mediated suppression was dog- and vehicle-dependent with CBD and THC delivered in dimethyl sulfoxide (DMSO) producing more immune suppressive effects as compared to ethanol (ETOH). PCR, flow cytometry, and immunohistochemical staining demonstrated that both CB1 and CB2 are expressed in canine immune cells. Together these data show that canine immune cells are sensitive to suppression by cannabinoids, but more detailed studies are needed to further understand the mechanisms and broad effects of these compounds in the dog.

SIRT1 and miR-34a-5p Expression in PBMCs as Potential Biomarkers for Patients With Type 2 Diabetes With Cognitive Impairments.

Patients with type 2 diabetes mellitus (T2DM) are at significantly increased risk of Alzheimer disease (AD). However, no biomarkers are available for early identification of patients with T2DM with cognitive impairment (T2DM-CI). Mitochondrial dysfunction is linked to AD. Silent Information Regulator 1 (SIRT1), which is responsible for regulating mitochondrial biogenesis, and its related miRNAs were also altered in AD. This study aimed to determine whether mitochondrial function in peripheral blood mononuclear cells (PBMCs) of patients with T2DM-CI was altered and if these alterations could be used as biomarkers. A total of 374 subjects were enrolled, including AD, T2DM-CI, T2DM-nCI (T2DM without cognitive impairment), and healthy controls. The mitochondrial function was determined using a commercial assay kit. The mitochondrial DNA (mtDNA) content, the expression of SIRT1, and selected miRNAs in PBMCs were measured by quantitative polymerase chain reaction. The correlations and diagnostic accuracy were assessed using the Spearman correlation coefficient or receiver operating characteristics analysis, respectively. We found significant changes in mitochondrial function in PBMCs of patients with AD compared with controls (all P < .05), which were not found in T2DM-CI. However, mtDNA content and SIRT1 mRNA expression were lower in PBMCs of patients with T2DM-CI, while miR-34a-5p expression was higher than in patients with T2DM-nCI (all P < .05). A combination of SIRT1 and miR-34a-5p demonstrated excellent discrimination between T2DM-CI and T2DM-nCI (area under the curve = 0.793; sensitivity: 80.01%; specificity: 78.46%). Furthermore, correlation analysis revealed a link between miR-34a-5p expression and hyperglycemia in T2DM-CI. Our findings revealed that there was an alteration of mitochondria at the peripheral level in patients with T2DM-CI. SIRT1 combined with miR-34a-5p in PBMCs performed well in identifying patients with T2DM-CI and may be a promising biomarker.

Inhibitory effect of Eimeria maxima IFN-γ inhibitory molecules on the immune function of T cell subsets in chickens

It has been reported that infection of chicken coccidian could inhibit the production of Th1 cytokine IFN-γ, thereby evading clearance by the host immune system. The present study aimed to have a further investigation into the effects of Eimeria maxima IFN-γ inhibitory molecules (EmHPSP-2 and EmHPSP-3) on the immune function of chicken peripheral blood mononuclear cells (PBMC) and various T cell subsets. First, separated PBMC or sorted T cell subsets were used for incubation with recombinant proteins of EmHPSP-2 (rEmHPSP-2) and EmHPSP-3 (rEmHPSP-3). Subsequently, the effects of rEmHPSP-2 and rEmHPSP-3 on proliferative capacity, nitric oxide (NO) release and mRNA levels of cytokines of the above cells were detected. The sorting purity of CD8+, CD4+ CD25-, CD4+, and CD4+ CD25+ T cells was 93.01, 88.88, 87.04, and 81.26%, respectively. The NO release of PBMC was significantly inhibited by rEmHPSP-2 and rEmHPSP-3. The proliferation of PBMC and CD4+ T cells was significantly inhibited by rEmHPSP-2 and rEmHPSP-3, whereas CD8+, CD4+ CD25-, and CD4+ CD25+ T cells was significantly promoted by the 2 proteins. The 2 proteins significantly downregulated interferon-gamma (IFN-γ) mRNA level, upregulated the transcriptional levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) in PBMC. IFN-γ and IL-2 transcriptional levels were markedly inhibited in CD8+ T cells. IFN-γ transcriptional level was significantly inhibited, but IL-4 was promoted by rEmHPSP-2 and rEmHPSP-3 in CD4+ CD25- T cells. Meanwhile, the inhibitory effects of rEmHPSP-2 and rEmHPSP-3 on the transcriptional levels of IFN-γ and IL-2 were more obvious in CD4+ T cells containing CD25+ cells compared with the CD25+ cells depletion group. It was found that IL-10, TGF-β1, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) mRNA levels were significantly upregulated upon stimulation of chicken CD4+ CD25+ T cells by proteins. This study is not only of great significance to clarify the immune evasion mechanism of chicken coccidia, but also provides candidate antigen molecules for development of a novel vaccine against chicken coccidiosis.

The Effects of Sodium Acetate on the Immune Functions of Peripheral Mononuclear Cells and Polymorphonuclear Granulocytes in Postpartum Dairy Cows.

Excessive lipid mobilization will snatch cell membrane lipids in postpartum dairy cows, which may impair the function of immune cells, including peripheral mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). Acetate, as a precursor and the energy source of milk fat synthesis, plays a key role in lipid synthesis and the energy supply of dairy cows. However, there is little information about the effect of sodium acetate (NaAc) on the immune function of PBMC and PMN in postpartum dairy cows. Therefore, this study aimed to evaluate the effects of NaAc on the immune functions of PBMCs and PMNs in postpartum dairy cows. In this experiment, twenty-four postpartum multiparous Holstein cows were randomly selected and divided into a NaAc treatment group and a control group. Our results demonstrated that the dietary addition of NaAc increased (p < 0.05) the number of monocytes and the monocyte ratio, suggesting that these postpartum cows fed with NaAc may have better immunity. These expressions of genes (LAP, XBP1, and TAP) involved in the antimicrobial activity in PBMCs were elevated (p < 0.05), suggesting that postpartum dairy cows supplemented with NaAc had the ability of antimicrobial activity. In addition, the mRNA expression of the monocarboxylate transporters MCT1 and MCT4 in PBMCs was increased (p < 0.05) in diets supplemented with NaAc in comparison to the control. Notably, the expression of the XBP1 gene related to antimicrobial activity in PMN was upregulated with the addition of NaAc. The mRNA expression of genes (TLN1, ITGB2, and SELL) involved in adhesion was profoundly increased (p < 0.05) in the NaAc groups. In conclusion, our study provided a novel resolution strategy in which the use of NaAc can contribute to immunity in postpartum dairy cows by enhancing the ability of antimicrobial and adhesion in PBMCs and PMNs.

Increased dietary intake of ultraprocessed foods and mitochondrial metabolism alterations in pediatric obesity

The increased intake of ultraprocessed foods (UPFs) in the pediatric age paralleled with the risen prevalence of childhood obesity. The Ultraprocessed Foods in Obesity (UFO) Project aimed at investigating the potential mechanisms for the effects of UPFs in facilitating pediatric obesity, focusing on the direct role of advanced glycation end-products (AGEs) on mitochondrial function, the key regulator of obesity pathophysiology. We comparatively investigated the daily dietary intake of UPFs, energy, nutrients, dietary AGEs [Nε -(carboxymethyl)lysine (CML), Nε -(1-carboxyethyl)lysine (CEL), and Nδ -(5-hydro-5- methyl-4-imidazolon-2-yl)-ornithine (MG-H1)] in 53 obese patients and in 100 healthy controls visiting the Tertiary Center for Pediatric Nutrition of the Department of Translational Medical Science at the University of Naples “Federico II”. AGEs skin accumulation and mitochondrial function in peripheral blood mononuclear cells (PBMCs) were also assessed. A higher intake of UPFs and AGEs, energy, protein, fat, and saturated fatty acids was observed in obese patients. Obese children presented significantly higher skin AGEs accumulation and alterations in mitochondrial metabolism. PBMCs from healthy controls exposed to AGEs showed the same mitochondrial alterations observed in patients. These findings support the UPFs role in pediatric obesity, and the need for dietary strategies limiting UPFs exposure for obesity prevention and treatment.

Opsin 3 expression in human Langerhans cell histiocytosis and its mediation of ELD-1 cellular function.

Langerhans cell histiocytosis (LCH) is a type of -histiocytic disorder characterized by aberrant function, differentiation or proliferation of mononuclear phagocyte system cells, however, the pathogenesis is not fully understood. Opsin 3 (OPN3) plays an important role in regulating cell function. We aimed to investigate OPN3 expression in LCH and Langerhans cells and evaluate its possible regulation of cellular function in a Langerhans cell-like cell line (ELD-1). Expression of OPN3 in LCH and paired adjacent healthy skin tissue was determined using microscopic tools (immunohistochemical and immunofluorescence staining) and RNA scope. OPN3 protein and mRNA levels in primary dendritic cells and ELD-1 were measured by real-time quantitative PCR and western blotting, respectively. The effects of reduced or over-expressed OPN3 mRNA level, via a lentiviral vector, were examined on ELD-1 proliferation, migration, cell cycle and apoptosis using the Cell Counting Kit 8, EdU-594 kit, Transwell assays and Cell Cycle Analysis Kit and Annexin V-PE apoptosis kit, respectively. Lastly, the signalling pathway mediating these functions was investigated via RNA sequencing and western blotting. OPN3 was highly expressed in human LCH tissue compared to healthy tissue, and was expressed in primary dendritic cells and ELD-1. Knockdown of OPN3 in ELD-1 inhibited cell proliferation, the cell cycle, and cell migration, while over-expression reversed these processes. These functions correlated with induction of the MAPK (p38/JNK/ERK) signalling pathway. Our results provide insight into the role of OPN3 in LCH which may become a molecular target for the clinical treatment of LCH.

Identification of peripheral vascular function measures and circulating biomarkers of mitochondrial function in patients with mitochondrial disease.

The development of pharmacological therapies for mitochondrial diseases is hampered by the lack of tissue-level and circulating biomarkers reflecting effects of compounds on endothelial and mitochondrial function. This phase 0 study aimed to identify biomarkers differentiating between patients with mitochondrial disease and healthy volunteers (HVs). In this cross-sectional case-control study, eight participants with mitochondrial disease and eight HVs matched on age, sex, and body mass index underwent study assessments consisting of blood collection for evaluation of plasma and serum biomarkers, mitochondrial function in peripheral blood mononuclear cells (PBMCs), and an array of imaging methods for assessment of (micro)circulation. Plasma biomarkers GDF-15, IL-6, NT-proBNP, and cTNI were significantly elevated in patients compared to HVs, as were several clinical chemistry and hematology markers. No differences between groups were found for mitochondrial membrane potential, mitochondrial reactive oxygen production, oxygen consumption rate, or extracellular acidification rate in PBMCs. Imaging revealed significantly higher nicotinamide-adenine-dinucleotide-hydrogen (NADH) content in skin as well as reduced passive leg movement-induced hyperemia in patients. This study confirmed results of earlier studies regarding plasma biomarkers in mitochondrial disease and identified several imaging techniques that could detect functional differences at the tissue level between participants with mitochondrial disease and HVs. However, assays of mitochondrial function in PBMCs did not show differences between participants with mitochondrial disease and HVs, possibly reflecting compensatory mechanisms and heterogeneity in mutational load. In future clinical trials, using a mix of imaging and blood-based biomarkers may be advisable, as well as combining these with an in vivo challenge to disturb homeostasis.

Effect of supplementation of vitamin D3 or vitamin D2 on serum concentrations of free and total 25-hydroxyvitamin D and the expression of genes involved in immune function in peripheral blood mononuclear cells of weaned pigs

ABSTRACT The present study aimed to compare the effects of vitamin D2 and vitamin D3 supplementation on concentrations of total and free 25(OH)D in plasma and the expression of genes involved in the innate immune system in peripheral blood mononuclear cells (PBMC) in weaned pigs. Five groups of pigs (with an initial body weight of around 9 kg) received basal diets supplemented with either 500 (control group), 1000 or 2000 IU vitamin D3/kg diet or 1000 or 2000 IU vitamin D2/kg diet for a period of 4 weeks. Vitamin D supplementation did not influence feed intake, body weight gain, feed conversion ratio, apparent total tract digestibility of calcium and phosphorus, and serum concentrations of calcium, inorganic phosphate and parathyroid hormone. Supplementation of vitamin D3 led to a dose-dependent increase of the concentrations of total and free 25(OH)D in serum. In contrast, pigs supplemented with 1000 or 2000 IU vitamin D2/kg diet did not have higher concentrations of total and free 25(OH)D in serum than the control group. The ratio of free/total 25(OH)D in serum was not influenced by vitamin D3 supplementation, whereas the group supplemented with 2000 IU vitamin D2/kg diet had a higher free/total 25(OH)D ratio than the groups supplemented with 1000 or 2000 IU vitamin D3/kg diet. Genes involved in vitamin D signalling (CYP27B1, VDR), as well as pro-inflammatory and immune regulatory genes (TLR4, TNF, IL1B and TGFB1) and genes encoding porcine protegrins (NPG1, NPG4), proteins belonging to the group of antimicrobial peptides, in PBMC were not different among groups supplemented with vitamin D3 or vitamin D2 and the control group. Therefore, the study indicates that supplementation of vitamin D2 causes much lower levels of total 25(OH)D than supplementation of vitamin D3 and that supplementation of vitamins D2 or D3 at moderate levels does not have an impact on the innate immune function in healthy pigs.

Impact of shipping temperature on cell viability and T cell responses to bacterial antigens

Background: Interferon-γ (IFN-γ) secretion by T cells is a key correlate of immune protection against many pathogens including tuberculosis and the neglected tropical disease melioidosis. Clinical studies in tropical regions of immune responses to pathogens and vaccine monitoring studies require the collection of samples in resource-limited rural areas and subsequent shipment to central laboratories for downstream assays and long-term storage. Here, we studied the impact of two different shipping temperatures on the viability, composition and function of peripheral blood mononuclear cells (PBMC) using multi-colour flow cytometry and IFN-g enzyme-linked immunospot assay (IFN-g ELISpot), in order to provide guidance on sample shipment conditions for future clinical studies. Methods: Paired peripheral blood mononuclear cell (PBMC) samples from recovered melioidosis patients were stored in liquid nitrogen (-196°C) and then shipped from Bangkok, Thailand to Oxford, UK at either -80°C (dry ice) or -196°C (dry shipper). After thawing, cell viability and composition were assessed by flow cytometry and antigen specific responses to Burkholderia pseudomallei (BP) were measured using IFN-g ELISpot. Results: We observed modest lowering of viability in the majority of samples and a reduction in IFN-g responses to BP which correlated to a decrease of monocytes and natural killer cells in samples shipped at -80°C compared to -196°C. Despite being lower in magnitude antigen-specific responses remained detectable in the majority of samples. Conclusions: Here we demonstrate that shipment of cryopreserved PBMC at -196°C has a benefit on cell viability, recovery and T cell responses to bacterial antigens, although useful information can still be obtained from samples shipped at -80°C, thus providing important guidance for sample management in future clinical trials.

Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLA2s.

Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-β and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Association between oxidative stress, mitochondrial function of peripheral blood mononuclear cells and gastrointestinal cancers

BackgroundThe incidence and mortality rate of gastrointestinal cancers are high worldwide. Increasing studies have illustrated that the occurrence, progression, metastasis and prognosis of cancers are intimately linked to the immune system. Mitochondria, as the main source of cellular energy, play an important role in maintaining the physiological function of immune cells. However, the relationship between mitochondrial function of immune cells and tumorigenesis has not yet been systematically investigated.MethodsA total of 150 cases, including 60 healthy donors and 90 primary gastrointestinal cancer patients without anti-tumor treatments (30 with gastric cancer, 30 with liver cancer and 30 with colorectal cancer) were involved in our study. The oxidant/antioxidant and cytokine levels in plasma, the ROS level, mitochondrial function and apoptosis ratio of peripheral blood mononuclear cells (PBMCs) were evaluated.ResultsThe imbalance between oxidant and antioxidant in plasma was discovered in the primary gastrointestinal cancer patients. The levels of cell reactive oxygen species (ROS) and mitochondrial ROS in PBMCs of primary gastrointestinal cancers were significantly increased compared with that in healthy donors. Meanwhile, the ATP content, the mtDNA copy number and the mitochondrial membrane potential (MMP) in PBMCs of patients with primary gastrointestinal cancers were lower than those in control group. The decreased MMP also occurred in immune cells of gastrointestinal cancers, including T cell, B cell, NK cell and monocyte. Furthermore, the PBMCs apoptosis ratio of primary gastrointestinal cancer patients was significantly higher than that of control group. Importantly, an increase of IL-2 and IL-6 and a decrease of IgG in plasma were found in the patients with primary gastrointestinal cancers. These changes of mitochondrial function in immune cells were consistent among primary gastrointestinal cancers without anti-tumor treatments, such as liver cancer, gastric cancer and colorectal cancer.ConclusionOur study demonstrated that the imbalance of oxidation/antioxidation in primary gastrointestinal cancer patients without anti-tumor treatments results in excessive ROS. The oxidative stress was associated to the mitochondrial dysfunction, the apoptosis of immune cells and eventually the abnormal immune function in primary gastrointestinal cancers. The application of immune cell mitochondrial dysfunction into clinical evaluation is anticipated.

Comparison of P-glycoprotein function in peripheral blood mononuclear cells ex vivo in stable Black and White male and female kidney transplant recipients.

Kidney allograft survival remains poorer in Black compared to White recipients due to racial differences in calcineurin inhibitor (CNI) pharmacology. P-glycoprotein (P-gp), an ABC efflux transporter expressed in peripheral blood mononuclear cells (PBMCs), modulates CNI pharmacokinetics and intracellular pharmacology. This study investigated P-gp function in PBMC ex vivo at 0 (trough), 4, 8, and 12 h in stable Black and White male and female kidney transplant recipients (n=67) receiving tacrolimus and mycophenolic acid. Tacrolimus doses were adjusted to troughs of 4-10ng/ml. P-gp function was quantified with flow cytometric measurement of cyclosporine (CYA; 2.5μM)-reversible efflux of P-gp substrate, 3,3'-Diethyloxacarbocyanine iodide by determining the percentage change of mean fluorescent intensity (MFI) with CYA (% ΔMFI). The composite parameter of area under the concentration versus time (AUC)0-12h % ΔMFI estimated P-gp function. Data analysis examined race, sex, and race-sex associations to P-gp function. A secondary aim analyzed ABCB1 genotypes: 1236C>T (rs1128503), 2677G>T/A (rs2032582), 3435C>T (rs1045642), and P-gp function. P-gp function (% ΔMFI) was higher in White patients at troughs (p=0.031) compared to Black counterparts with similar trends at 4 and 8h. Reduced AUC0-12h % ΔMFI was noted in Black recipients (N=32) compared with Whites (N=35, p=0.029) with notable pairwise adjusted differences between Black and White women (p=0.021). Higher AUC0-12h % ΔMFI was associated with ABCB1 2677 TT compared to GG variants (p=0.035). The AUC0-12h % ΔMFI was greater in White than Black subjects. P-gp function was higher at troughs in White subjects and differed between race-sex groups. P-gp function in PBMC may influence intracellular tacrolimus exposure and inter-relating pharmacodynamic responses which may support race and sex pharmacologic differences.

Eradication of HCV by direct antiviral agents restores mitochondrial function and energy homeostasis in peripheral blood mononuclear cells.

Hepatitis C virus (HCV) adopts several immune evasion mechanisms such as interfering with innate immunity or promoting T-cell exhaustion. However, the recent direct-antiviral agents (DAAs) rapidly eliminate the virus, and the repercussions in terms of immune system balance are unknown. Here we compared the PBMCs transcriptomic profile of patients with HCV chronic infection at baseline (T0) and 12 weeks after the end of the therapy (SVR12) with DAAs. 3862 genes were differently modulated, identifying oxidative phosphorylation as the top canonical pathway differentially activated. Therefore, we dissected PBMCs bioenergetic profile by analyzing mitochondrial respiration and glycolysis at 4 timepoints: T0, 4 weeks of therapy, end of therapy (EoT), and SVR12. Maximal and reserve respiratory capacity considerably increased at EoT, persisting until SVR12. Notably, over time a significant increase was observed in respiratory chain (RC) complexes protein levels and the enzymatic activity of complexes I, II, and IV. Mitochondrial-DNA integrity improved over time, and the expression of mitochondrial biogenesis key regulators such as TFAM, Nrf-1, and PPARGC1A significantly increased at SVR12; hence, RC complexes synthesis and mitochondrial respiration were supported after treatment. HCV clearance with DAAS profoundly changed PBMCs bioenergetic profile, suggesting the immunometabolism study as a new approach to the understanding of viral immune evasion mechanisms and host adaptations during infections and therapies.

Abstract 11469: Effects of Metformin and Donepezil on the Prevention of Doxorubicin-Induced Cardiotoxicity in Breast Cancer Patient: A Randomized Controlled Trial

Introduction: Cardiotoxicity from doxorubicin (DOX) is the most concerning adverse event. Although data from preclinical studies showed that metformin and donepezil prevented myocardial injury and preserved mitochondrial function against DOX-induced cardiotoxicity, the beneficial effects of these drugs in clinical trial have not been established. Hypothesis: Oral administration with metformin or donepezil prior receiving DOX prevents DOX-induced cardiotoxicity in breast cancer patients. Methods: In this double-blind, placebo-controlled trial. We randomly assigned 111 female breast cancer patients to receive oral metformin 2000 mg (n=40), donepezil 10 mg (n=31), or placebo (n=40), once daily 1 day prior to DOX and continued during DOX treatment. Blood samples were obtained at baseline, after chemotherapy cycle 2 and 4. The primary outcome was the change of troponin-I (trop-I) from baseline. The secondary outcomes were the change in the N-terminal pro-B-type natriuretic peptide (NT-proBNP) from baseline, and mitochondrial function in the peripheral blood mononuclear cells. Results: Baseline characteristics were similar between the groups, mean age was 51.93±10.45 years. The mean trop-I changed from baseline were 29.97±4.59 pg/ml in placebo group, 31.07±7.28 pg/ml in metformin group (p = 0.90 vs. placebo) and 30.97±5.07 pg/ml in donepezil group (p = 0.88 vs. placebo). The changes of NT-proBNP level and mitochondrial oxidative stress were comparable between placebo vs. metformin or donepezil groups. However, donepezil significantly decreased mitochondrial mass and mitochondrial maximal respiration after chemotherapy (Figure). Conclusion: In breast cancer patients undergoing chemotherapeutic treatment with DOX, metformin and donepezil did not prevent myocardial injury. Treatment with donepezil resulted in the decrease in mitochondrial mass and maximal respiration following chemotherapy that might caution its use in breast cancer.

Mitochondrial Impairments in Peripheral Blood Mononuclear Cells of Multiple Sclerosis Patients.

Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We aimed to compare the mitochondrial function of the peripheral blood mononuclear cells (PBMCs) from MS patients with (M+) and without (M-) lipid-specific oligoclonal immunoglobulin M bands (LS-OCMB), and healthydonors (HD). We conducted an exploratory cross-sectional study with 19 untreated MS patients (M+ = 9 and M- = 10) and 17 HDs. Mitochondrial superoxide anion production and mitochondrial mass in PBMCs were assessed without and with phytohemagglutinin by flow cytometry. The PBMCs' mitochondrial function was analyzed using Seahorse technology. Superoxide anion production corrected by the mitochondrial mass was higher in MS patients compared with HDs (p = 0.011). Mitochondrial function from M+ patients showed some impairments compared with M- patients. Without stimulus, we observed higher proton leak (p = 0.041) but lower coupling efficiency (p = 0.041) in M+ patients; and under stimulation, lower metabolic potential ECAR (p = 0.011), and lower stressed OCR/ECAR in the same patients. Exclusively among M+ patients, we described a higher mitochondrial dysfunction in the oldest ones. The mitochondrial impairments found in the PBMCs from MS patients, specifically in M+ patients, could help to better understand the disease's physiopathology.