Activation Of Mononuclear Cells Research Articles

Immunomodulation profile of the biosimilar trastuzumab MYL-1401O in a bioequivalence phase I study

The initial Phase-I single centre, single dose, randomized, double-blind, cross-over study was planned to assess the pharmacokinetic and pharmacodynamic bioequivalence of the trastuzumab biosimilar (MYL-1401O) compared to the reference Herceptin®. Their respective immunomodulation profile presented in this paper involved healthy males receiving a single infusion of both monoclonals, separated by a washout period. Sixty parameters were assessed in total, including serum cytokines, peripheral mononuclear cell (PBMC) subsets, cell activation and response to recall antigens and mitogen, pre- and post- infusion, as well as a cytokine release assay (CRA) at baseline. Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6 h, and a modulation of mononuclear cell subset profile and activation level, notably CD16 + cells. Except for CD8 + T cells, there were no significant differences between Herceptin® and MYL-1401O. In CRA, PBMC stimulated with MYL-1401O or Herceptin® similarly secreted IL-6, TNF-α, IL-1β, GM-CSF, IFN-γ, and IL-10, but no or low level of IL-2. Interestingly, some observed adverse events correlated with IL-2 and IFN-γ in CRA. MYL-1401O exhibited a very similar immunomodulation profile to Herceptin®, strongly supporting its bioequivalence. This approach may thus be included in a proof-of-concept study. CRA may be used as a predictive assay for the evaluation of clinical monoclonals.

Altered Histone Deacetylase Versus Histone Acetyltransferase Activity in Peripheral Blood Mononuclear Cells from Apparently Healthy Young Adults with Prior Exposure to Adverse Childhood Experiences

Introduction: Adverse childhood experiences (ACEs) are severe psychosocial stressors which occur in early developmental periods and promote later life cardiometabolic disease in a dose-dependent manner. ACE-related risk may be mediated in part by epigenetic alterations, particularly in myeloid lineage immune cells. Histone acetylation is a dynamic epigenetic process that regulates gene expression via modification of chromatin structure and is dictated by the opposed actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). An appropriate balance between HDAC and HAT activity is critical for maintaining cellular function, and an imbalance has been implicated in the premature development of human diseases including cardiometabolic disease. Glucocorticoids (e.g., cortisol) are well-described regulators of immune function and can influence histone acetylation through glucocorticoid receptors. Thus, we hypothesized that HDAC:HAT activity would be altered in young adults with prior ACE exposure, and that HDAC:HAT activity would be associated with salivary cortisol concentrations. Purpose: We examined HDAC:HAT activity in peripheral blood mononuclear cells (PBMCs) in apparently healthy young adults with versus without prior exposure to ACEs and explored whether circulating cortisol was associated with HDAC:HAT activity. Methods: PBMCs were isolated from whole blood collected within 1 hour of wakening from 27 young adult women who reported high (≥ 4 ACEs) ACE exposure (ACE4+; mean ± SD; age = 21 ± 3 y, BMI = 25.7 ± 4.5 kg/m2) and from 14 young adult women who reported no ACE exposure (CON; age = 21 ± 3 y, BMI = 26.1 ± 6 kg/m2). Salivary cortisol was also collected within 1 hour of wakening and 8 hours later. Nuclear proteins were isolated from PBMCs and HDAC and HAT activities were quantified by commercially available assay kits. Salivary cortisol was quantified via ELISA. Independent samples t-tests were used to examine differences in HDAC:HAT, HDAC, and HAT activity. Pearson correlation coeffcients were used to examine the relation of HDAC:HAT activity with morning and afternoon cortisol concentrations, and the diurnal cortisol slope in the complete sample. Results: HDAC:HAT activity was reduced in ACE4+ versus CON (140.4 ± 52 vs. 189.6 ± 42 arbitrary units, p = 0.004). This difference was primarily driven by reduced HDAC activity (13,706 ± 4,696 vs. 18,060 ± 3,641 ng/h/mg, p = 0.004) in ACE4+ versus CON, whereas HAT activity was not different (99.7 ± 11 vs. 96.1 ± 9 ng/h/mg, p = 0.30). HDAC:HAT activity was not associated with morning (r=0.27, p = 0.07) or afternoon (r=-0.23, p = 0.16) cortisol, but was associated with the diurnal cortisol slope (r=-0.37, p = 0.02). Discussion: Young adults with prior ACE exposure have an altered balance between HDAC and HAT activity in PBMCs that appears to be driven by lower HDAC activity, and which is negatively related to diurnal cortisol slope. These findings warrant further exploration, particularly regarding the specific HDACs implicated, their role in reduced cellular function as it relates to disease risk, and whether these can be modulated to improve function among those with ACE exposure. Further, our data support future examination of the role of glucocorticoid signaling in regulating histone acetylation in this population. This study was supported by the Center for Integrative Research on Childhood Adversity (Grant #P20GM109097), through the National Institute of General Medical Sciences (to NDMJ and TKT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bighorn Sheep (Ovis canadensis) with Higher Whole Blood Selenium Levels Have Improved Survival and Altered Immune Responses.

Bighorn sheep (Ovis canadensis) are herbivorous ungulates that live in forage-poor areas of the American west. The trace minerals that herbivores derive from forage are important for immune function. Therefore, identifying trace minerals that affect immune function in bighorn sheep could provide important insights into disease susceptibility and population health in threatened populations. We sought to determine whether trace mineral composition in blood or plasma correlates to survival and determine whether immunologic parameters correlate with any trace minerals that affect survival. We used data collected from 2016 to 2018 as part of a large study on bighorn sheep in southeastern Oregon and northern Nevada, US. We measured the survival of 135 bighorn sheep during the 8-mo monitoring period, including general metrics of immune function and trace mineral levels. We found that animals with higher selenium had improved survival over the monitoring period, with higher peripheral blood mononuclear cell activity (lymphocytes and monocytes) and lower bacterial killing ability in an in vitro assay. This suggests that bighorn sheep may have altered immune function when selenium levels are low, making them more likely to die during the 8-mo monitoring period. Future work should consider whether habitat management strategies that increase selenium intake might improve disease resistance and survival in bighorn sheep in selenium-poor areas.

Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients.

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

Comparative toxicological-hygienic assessment, structural-morphological, physicochemical characteristics, and virucidal properties of new nanopowder materials TiO2 and TiO2@Ag

In order to address safety concerns related to the acquisition and utilization of TiO2 and TiO2@Ag nanomaterials, as well as to investigate their disinfectant and biological effects, the structural-morphological, morpho­metry, toxicological, cytotoxic, and virucidal properties of these specified nanomaterials have been studied through experiments conducted on laboratory animals and in vitro. It has been demonstrated that the TiO2@Ag nanocomposite exhibited distinct physicochemical characteristics: it consisted of TiO2 nanoparticles ranging in size from 13 nm to 20 nm and Ag nanoparticles ranging from 35 nm to 40 nm with 4.0 wt% of silver localized on the surface of titanium dioxide. The purity of the modification of synthesized nano-TiO2 and nano-TiO2@Ag has been confirmed. Acute intraperitoneal administration of nanopowders revealed LD50 values of 4783.30 mg/kg for nano-TiO2 and 724.44 mg/kg for nano-TiO2@Ag. A slight accumulation was observed upon repeated (28-fold) intragastric administration of nano-TiO2. The cumulative dose administered, which equated to 15.9 multiples of the LD50 (76040 mg/kg), did not result in animal mortality but led to retardation in body weight gain. TiO2 and TiO2@Ag nanopowders do not irritate the skin, induce mild conjunctival irritation, and may exhibit a weak sensitizing effect. Nano-TiO2 and nano-TiO2@Ag powders accumulate in the tissues of internal organs and cause damage to the liver, kidneys, and lungs of laboratory animals upon intraperitoneal administration. The most characteristic morphological signs of the toxic effect of nano-TiO2 on liver tissue were observed at a level of 67.7% (cytoplasmic vacuolization in hepatocytes), while in the case of nano-TiO2@Ag initial necrotic changes were at a level of 70.0% (hepatocytes with pyknotic nuclei). Immunoassay analysis has demonstrated that TiO2@Ag and TiO2 nanomaterials at concentrations of 30 µg/ml can enhance the functional activity of peripheral blood mononuclear cells in vitro by increasing the production of cytokines IL-1, IL-6, TNF-α, and IL-4 in donors (p<0.05). This indicates the potential for chronic inflammation and allergic reactions among synthesis operators. In the study of the impact of nanomaterials on murine germ cells, it has been established that they affect the activity of mitochondrial enzymes and exert a damaging effect on mitochondrial membranes and overall cell integrity. Estimated approximate safe exposure levels in the workplace air are 0.3 mg/m3 for nano-TiO2 and 0.2 mg/m3 for nano-TiO2@Ag. Nano-TiO2@Ag and nano-TiO2 at a concentration of 100 µg/ml exhibit pronounced extracellular virucidal activity against human adenovirus serotype 2. The TiO2@Ag nanocomposite has a less damaging effect on Нер-2 cells compared to nano-TiO2.

Comparison of lipopolysaccharide-mediated peripheral blood mononuclear cell activation between Brahman and Brahman × Thai native crossbreed cattle.

Lipopolysaccharide (LPS) is a robust endotoxin known to activate the immune system in cattle. The objective of this study was to investigate the effect of LPS on the morphology, cell viability, malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC) of peripheral blood mononuclear cells (PBMCs) in Brahman and Brahman × Thai native crossbreed cattle. PBMCs were isolated from Brahman and Brahman × Thai native crossbreed cattle and treated with 0, 0.1, 1, and 10 μg/mL Escherichia coli LPS, respectively. Morphological changes in PBMCs were assessed at 24 and 48 h. In addition, we measured PBMC cell viability, MDA, NO, and TAC. LPS stimulation caused cell deformation and partial PBMC area enlargement, but there were no differences between Brahman and Brahman × Thai native crossbreed cattle. Stimulation at all levels did not affect the viability of PBMCs (p > 0.05). MDA and NO levels were significantly higher in Brahman cattle than in Brahman Thai native crossbred cattle (p < 0.05). TAC was significantly higher in Brahman × Thai native crossbred cattle than in Brahman cattle (p < 0.05). Immune cells of crossbreed cattle have a higher activation response to LPS than those of purebred cattle, and native crossbreed beef cattle have a higher antioxidant capacity than purebred beef cattle. This result may explain why hybrid cattle of indigenous breeds are more resistant to disease than purebred cattle.

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;

5,6-dimethylxanthenone-4-acetic acid (DMXAA), a partial STING agonist, competes for human STING activation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

Novel protective effect of the FOXO3 longevity genotype on mechanisms of cellular aging in Okinawans

The genetic association of FOXO3 genotypes with human longevity is well established, although the mechanism is not fully understood. We now report on the relationship of the FOXO3 longevity variant rs2802292 with telomere length, telomerase activity, FOXO3 expression, and inflammatory cytokine levels in men and women. In agreement with earlier work, the FOXO3 longevity variant conferred protection against telomere shortening of peripheral blood mononuclear cells from adults aged 55 years and older. This was accompanied by higher levels of telomerase activity in mononuclear cells for carriers of the longevity-associated FOXO3 G-allele of SNP rs2802292 (P = 0.015). FOXO3 mRNA expression increased slightly with age in both young (P = 0.02) and old (P = 0.08) G-allele carriers. Older female G-allele carriers displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with age (P = 0.07). In contrast, older male G-allele carriers displayed an age-dependent increase in levels of anti-inflammatory cytokine IL-10 with age (P = 0.04). Thus, FOXO3 may act through several different pro-longevity mechanisms, which may differ by age and sex.

Ultrasound and microbubble mediated T cell modulation in peripheral blood mononuclear cells

Ultrasound (US)-stimulated microbubbles are emerging as a revolutionary therapeutic technique, notably within the field of cancer immunotherapy. While this approach has been shown to modify cell permeability and to trigger local anti-tumor effects, the interactions between vibrating microbubbles and T-cells is not well understood. Here, we explore the biophysical impact of microbubbles on T-cell permeability and viability, setting the stage for potential advancements in cellular immunotherapy. Following the activation of fresh human peripheral blood mononuclear cells (PBMCs), cells were incubated with FITC-dextran (10kDa)—used here as a surrogate drug– and exposed in the presence of DefinityTM(1:217–cell:bubble ratio) US (1MHz, N = 1000, PRF = 5 ms for 2 minutes) over a range of peak-negative pressures (208-563kPa). Viability was assessed immediately and post-treatment concurrently with FITC macromolecule uptake using flow cytometry. T-cell population was identified using CD3 and CD4 antibodies (60% CD3 + and 20% CD4+). All conditions examined resulted &amp;gt; 95% viability. Results indicated a significant increase in viably permeated PBMCs with higher acoustic pressures, ranging from 0% to 30% (sham-corrected). When analyzed separately both CD4 + and CD4- T-cells exhibited similar permeability rates (0%–37.5%). The 563 kPa condition yielded the highest permeability with minimal viability loss. These findings open avenues for enhancing the efficacy of cellular immunotherapy for solid tumors.

In-Vitro Effect of Ammodaucus Leucotrichus Flower and Fruit Essential Oils on Nitric Oxide Production and Arginase Activity in Human Peripheral Blood Mononuclear Cells (PBMC) from Patients with Behçet Disease

In-Vitro Effect of Ammodaucus Leucotrichus Flower and Fruit Essential Oils on Nitric Oxide Production and Arginase Activity in Human Peripheral Blood Mononuclear Cells (PBMC) from Patients with Behçet Disease

FEATURES OF RELATIONS OF MELATONIN WITH THE STATE OF INTRACELLULAR REGULATORSOF THE FUNCTIONAL ACTIVITY OF WHOLE BLOOD MONONUCLEAR CELLS IN CORONARY HEART DISEASE

Introduction. Coronary heart disease (CHD), leading among the causes of death in adulthood and old age, is an urgent medical and social problem. The pathogenesis of most forms of coronary heart disease is based on stenosing atherosclerosis of the coronary arteries, which develops against the background of dyslipidemia and arterial hypertension and is accompanied by the activation of immunocompetent cells (ICCs) of the vascular wall with the development of a subclinical inflammatory reaction, as well as the production of pro-inflammatory factors such as interleukins, chemokines, growth factors and etc. In turn, ICC activity is determined by the state of their intracellular molecular cascades, which transmit signals into the cell and ensure its reactivity to various external stimuli, such as mitogens, cytokines, pathogen components, etcIt has been shown that the central nervous system plays an important role in the regulation of ICC activity due to the production of neurohumoral molecules, such as melatonin, endorphin, sero-tonin, etc., which ensure the coordination of immune responses and their control by the central nervous system. The aim of this study was to study the relationship between melatonin production and intracellular factors that regulate the pro-inflammatory activi-ty of whole blood mononuclear cells and their metabolism in patients with coronary artery disease. Material and methods. As part of the cohort study, 58 patients of both sexes with coronary artery disease aged 49 to 67 years and 20 practically healthy individuals of both sexes were examined. In accordance with the purpose of the study, the concentration of focal adhesion protein kinase (FAK), 5'AMP-activated protein kinase (AMPK), AKT1 protein kinase, signal transducers and transcription activators (STAT) was determined in nuclear cy-toplasmic lysates of whole blood mononuclear cells: STAT3, STAT5A and STAT6, c-Jun N-terminal protein kinase 1 and 2 isoforms (JNK), mitogen-activated protein kinase p38 (p38), extracellular growth kinase 1 and 2 isoforms (ERK), Janus kinase type 2 (JAK2), nuclear transcription factor NF -kB, caspase-1, cyclooxygenase-2 (COX-2), p70-S6K1 protein kinase, p53, p27, p21 proteins. In addition, the concentration of cyclic adenosine monophos-phate (cAMP) and cyclic guanosine monophosphate (cGMP) was determined in cell supernatants. Melatonin concentration was determined in blood se-rum. The material for the study was venous blood taken from the cubital vein in the morning from 6.00 to 6.15. Results. The analysis showed that in patients with coronary artery disease, in comparison with practically healthy individuals, in MNCs of whole blood, there was an increased level of protein kinases FAK, AKT, JNK, ERK, p70-S6K1, factor STAT6, protein p21, against which there was a decrease in the content of STAT3, STAT5A, JAK2, transcription factor NF-kB and caspase-1. These changes were accompanied by increased levels of cGMP and cAMP. Against this background, a decrease in the content of factors was revealed in the MNC. A high concentration of melatonin in patients with CAD was as-sociated with a decrease in the content of protein kinases AMPK, AKT, Jak2, ERK1, protein p21, caspase-1, and cAMP in MNCs, which was observed against the background of an increase in the level of protein p27 and nuclear factor NF-kB. The results of the correlation analysis indicate a different na-ture of the relationship between the level of melatonin and such factors as caspase-1, protein kinases ERK, JAK2, as well as the transcription factor NF-kB and p21 protein, depending on the characteristics of melatonin production in patients with coronary artery disease. Conclusions. In patients with coronary artery disease, melatonin exhibits a modulating effect on the energy balance of ICCs and their metabolism, helps limit pro-inflammatory activity by limiting the functional activity of MAPK/SAPK signaling pathways in MNCs.

Estimation of Phagocytic activity by normal human peripheral blood mononuclear cells on various oral isolates of Candida species – An in-vitro study

Polymorphonuclear neutrophils (PMN) and mononuclear phagocytes represent an important first line and effector function in control of Candida infections. The aim of the study is to determine the in-vitro phagocytic activity of human peripheral blood mononuclear cells against oral isolates of Candida species and its antifungal susceptibility. The study also evaluates the degree of respiratory burst activity of PBMCs. Phagocytic and lytic indices by PBMCs were determined for Candida spp. The respiratory burst activity was evaluated by nitroblue tetrazolium test. Antifungal disc diffusion susceptibility testing was performed. A total of 100 Candida were isolated belonging to the species C.albicans, C. tropicalis, C.krusei and C.auris. Phagocytic and lytic indices of C.albicans was significant when compared to standard strain of C.albicans. For C.tropicalis and C.krusei phagocytic index was significant while lytic index was not significant when compared to standard strain. The inter species comparison of both the indices was not significant for the clinical isolates of Candida. A significant reduction in phagocytic activity was observed for clinical isolates of Candida spp. but lytic activity was variable when compared to the standard strain of C.albicans.

Abstract B029: Combinations of metformin and BH3-mimetic drugs synergistically induce immunogenic cell death in breast cancer cells

Abstract There is a compelling medical need for novel and safe treatment options for patients with advanced and difficult to treat breast cancer. Our previous findings have demonstrated that a combination of metformin and navitoclax (BCL-2/BCL-XL inhibitor) successfully prevents tumor growth in patient derived xenograft and syngrafted mouse models of aggressive MYC-driven breast cancer. This outcome is due to the synthetic lethal action of these drugs in tumors with elevated MYC expression. Here, we explored the combinations of metformin with all clinically applicable anti-apoptotic BCL-2 family protein inhibitors (BH3-mimetics) in a panel of 15 triple-negative breast cancer cell lines with distinct endogenous MYC levels, as well as inducible MYC models. We used cytotoxicity assays and analyzed the data with SynergyFinder, an interactive analysis and visualization tool, to calculate the degree of combination synergy utilizing Zero interaction potency reference model. Through these cytotoxicity assays, we defined which metformin and BH3-mimetic drug combination induces most synergistic cell death in a MYC-synthetic lethal manner. Additionally, we used DAMP reporter cell lines and peripheral blood mononuclear cell-based dendritic cell activation assays to evaluate the immunogenic potential of combination-induced cell death. We show that targeting MCL-1 or BCL-2/XL with BH3-mimetics MIK-665 and navitoclax, respectively, and combining them with metformin, induces synergistic cell death in MYC-high cancer cells. We also show that cell death caused by these drug combinations promotes maturation of dendritic cells. In conclusion, targeting MYC-high tumors with synthetic lethal strategies, including BH3-mimetics and metformin, may offer new opportunities to combine with immunotherapies for the treatment of breast cancer. Citation Format: Mariel Savelius, Linda Id, Johanna Anttila, Ruixian Liu, Ilida Suleymanova, Jani Saarela, Juha Klefström. Combinations of metformin and BH3-mimetic drugs synergistically induce immunogenic cell death in breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B029.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

Phase I study targeting newly diagnosed grade 4 astrocytoma with bispecific antibody armed T cells (EGFR BATs) in combination with radiation and temozolomide

PurposeThe purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy.MethodsThis phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O6-methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ.ResultsThe highest feasible dose was 80 × 109 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells (p < 0.03) and NK cell activity (p < 0.002) ex vivo, and increased serum concentrations of IFN-γ (p < 0.03), IL-2 (p < 0.007), and GM-CSF (p < 0.009).ConclusionTargeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 109, with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM.ClinicalTrials.gov Identifier: NCT03344250

Dysregulation of Peripheral Blood Mononuclear Cells and Immune-Related Proteins during the Early Post-Operative Immune Response in Ovarian Cancer Patients.

Surgical treatment is a cornerstone of ovarian cancer (OC) therapy and exerts a substantial influence on the immune system. Immune responses also play a pivotal and intricate role in OC progression. The aim of this study was to investigate the dynamics of immune-related protein expression and the activity of peripheral blood mononuclear cells (PBMCs) in OC patients, both before surgery and during the early postoperative phase. The study cohort comprised 23 OC patients and 20 non-cancer controls. A comprehensive analysis of PBMCs revealed significant pre-operative downregulation in the mRNA expression of multiple immune-related proteins, including interleukins, PD-1, PD-L1, and HO-1. This was followed by further dysregulation during the first 5 post-operative days. Although most serum interleukin concentrations showed only minor changes, a distinct increase in IL-6 and HO-1 levels was observed post-operatively. Reduced metabolic and phagocytic activity and increased production of reactive oxygen species (ROS) were observed on day 1 post-surgery. These findings suggest a shift towards immune tolerance during the early post-operative phase of OC, potentially creating a window for treatment. Further research into post-operative PBMC activity could lead to the development of new or improved treatment strategies for OC.

Role of angiotensin II and neuroendocrine factors in immunological regulation in patients with coronary heart disease: prospective cross-sectional study

BACKGROUND: Among chronic noncommunicable diseases, cardiovascular diseases, particularly coronary heart disease (CHD), are the leading cause of death. The rennin-angiotensin-aldosterone system plays an important role in CHD development and progression; however, its role in the regulation of immunoneuroendocrine interactions requires further analysis.&#x0D; OBJECTIVE: To study the relationship between angiotensin II (AT II) and molecular regulators of the activity of whole blood mononuclear cells (MNCs) in patients with angina pectoris.&#x0D; MATERIALS AND METHODS: This cross-sectional study enrolled 65 patients with exertional angina aged 45–67 years, including 19 apparently healthy individuals. The levels of interleukins (ILs), transforming growth factor-β1 (TGF-β1), prostaglandin E2 (PG E2), serotonin, thyroid-stimulating hormone (TSH), and AT II in the blood serum were determined. In MNCs, the concentrations of protein kinases FAK, JNK, p38, and ERK, signal transducers, and activators of transcription (STAT 3, 5A, and 6) were determined.&#x0D; RESULTS: In patients with coronary artery disease, the production of TGF-β1 increased by 7.2% (p=0.00001), AT II by 136.9% (p=0.0001), serotonin by 129.0% (p=0.00001), IL-18 by 92.5% (p=0.00001), TSH by 51.7% (p=0.0012), ERK protein kinase content by 86.4% (p=0.0001), JNK by 56.8% (p=0.0001), and FAK by 55.3% (p=0.00002). The levels of IL-15 also decreased by 38.1% (p=0.0001), PG E2 by 39.5% (p=0.0001), and STAT3 by 52.5% (p=0.0001).&#x0D; CONCLUSION: The nature of the identified relationships among the analyzed factors allows us to consider AT II as a factor that ensures adaptive coupling of immune and neuroendocrine regulatory mechanisms in patients with coronary artery disease, contributing to a change in the balance between macrophages and T-helper types 1 and 2.

C5a stimulation induces caspase-1 activation and mature IL-1β production in human peripheral blood mononuclear cells

The complement component C5a contributes to the recruitment of immune cells to inflamed tissues and local inflammation. The proinflammatory cytokine interleukin (IL)-1β is also related to inflammatory disorders through inflammasome activation. However, the association between inflammasome activation and C5a is unclear. Human peripheral blood mononuclear cells (PBMCs) were stimulated with C5a and measured for IL-1β secretion by enzyme-linked immunosorbent assay (ELISA). The pro-IL-1β expression in cell lysates was also examined by Western blot analysis. Similarly, magnetic bead-isolated CD14+ monocyte-depleted and lymphocyte-depleted PBMCs were stimulated with C5a, and immunoblot analysis was performed using an anti-cleaved-IL-1β (p17) antibody. FACS was performed to detect caspase-1-activated cells. C5a-stimulated PBMCs produced IL-1β in C5a concentration-dependent manner. The protein levels of pro-IL-1β in the cell lysates were significantly increased. Furthermore, the cleaved-IL-1β (p17) was faintly detected in the same lysates. Active caspase-1 was demonstrated in C5a-simulated CD14+ monocytes by FACS. Cleaved-IL-1β (p17) was demonstrated in the supernatant of C5a-stimulated PBMCs. Lymphocyte-depleted PBMCs stimulated with C5a but monocyte-depleted PBMCs produced cleaved-IL-1β (p17). C5a induced the production of mature IL-1β in PBMCs. The IL-1β production is mediated mainly by caspase-1 activation in CD14+ monocytes. These results suggest that C5a alone potentiates mature IL-1β production mainly in monocytes.