Phorbol 12-myristate 13-acetate Research Articles

Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current.

To investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VAs). Rabbits hearts were isolated and prepared for Langendorff perfusion technique. The stimuli-extra-stimulus (S1-S2) method and dynamic S1 pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of phorbol-12-myristate-13-acetate (PMA) (100nM) (n=15) greatly steepened the restitution curves (Smax>1) (p<.01) at each site compared to the control group (n=15). Furthermore, treatment with PMA also induced larger spatial dispersions of Smax (p<.05) and decreased the thresholds of the VA and APD alternans (p<.01). However, perfused with the PKC inhibitor, bisindolylmaleimide (BIM) (500nM) (n=10), reversibly flattened the APD restitution curves at each site (Smax<1), decreased the spatial dispersions of Smax, and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca2+ current was significantly increased by addition of PMA compared with control (CTL) group (p<.05). Antagonize this current with verapamil (n=10) can fully inhibited the PMA induced increasing of Smax and inducibility of VA and alternans. PKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca2+ influx.

Production of Peroxymonocarbonate by Steady-State Micromolar H2O2 and Activated Macrophages in the Presence of CO2/HCO3- Evidenced by Boronate Probes.

Peroxymonocarbonate (HCO4-/HOOCO2-) is produced by the reversible reaction of CO2/HCO3- with H2O2 (K = 0.33 M-1, pH 7.0). Although produced in low yields at physiological pHs and H2O2 and CO2/HCO3- concentrations, HCO4- oxidizes most nucleophiles with rate constants 10 to 100 times higher than those of H2O2. Boronate probes are known examples because HCO4- reacts with coumarin-7-boronic acid pinacolate ester (CBE) with a rate constant that is approximately 100 times higher than that of H2O2 and the same holds for fluorescein-boronate (Fl-B) as reported here. Therefore, we tested whether boronate probes could provide evidence for HCO4- formation under biologically relevant conditions. Glucose/glucose oxidase/catalase were adjusted to produce low steady-state H2O2 concentrations (2-18 μM) in Pi buffer at pH 7.4 and 37 °C. Then, CBE (100 μM) was added and fluorescence increase was monitored with time. The results showed that each steady-state H2O2 concentration reacted more rapidly (∼30%) in the presence of CO2/HCO3- (25 mM) than in its absence, and the data permitted the calculation of consistent rate constants. Also, RAW 264.7 macrophages were activated with phorbol 12-myristate 13-acetate (PMA) (1 μg/mL) at pH 7.4 and 37 °C to produce a time-dependent H2O2 concentration (8.0 ± 2.5 μM after 60 min). The media contained 0, 21.6, or 42.2 mM HCO3- equilibrated with 0, 5, or 10% CO2, respectively. In the presence of CBE or Fl-B (30 μM), a time-dependent increase in the fluorescence of the bulk solution was observed, which was higher in the presence of CO2/HCO3- in a concentration-dependent manner. The Fl-B samples were also examined by fluorescence microscopy. Our results demonstrated that mammalian cells produce HCO4- and boronate probes can evidence and distinguish it from H2O2 under biologically relevant concentrations of H2O2 and CO2/HCO3-.

Predicting Organ Dysfunction in Septic and Critically Ill Patients: A Prospective Cohort Study Using Rapid Ex Vivo Immune Profiling.

While cytokine response patterns are pivotal in mediating immune responses, they are also often dysregulated in sepsis and critical illness. We hypothesized that these immunological deficits, quantifiable through ex vivo whole blood stimulation assays, may be indicative of subsequent organ dysfunction. In a prospective observational study, adult septic patients and critically ill but nonseptic controls were identified within 48 hours of critical illness onset. Using a rapid, ex vivo assay based on responses to lipopolysaccharide (LPS), anti-CD3/anti-CD28 antibodies, and phorbol 12-myristate 13-acetate with ionomycin, cytokine responses to immune stimulants were quantified. The primary outcome was the relationship between early cytokine production and subsequent organ dysfunction, as measured by the Sequential Organ Failure Assessment score on day 3 of illness (SOFAd3). Patients were recruited in an academic medical center and data processing and analysis were done in an academic laboratory setting. Ninety-six adult septic and critically ill nonseptic patients were enrolled. None. Elevated levels of tumor necrosis factor and interleukin-6 post-endotoxin challenge were inversely correlated with SOFAd3. Interferon-gamma production per lymphocyte was inversely related to organ dysfunction at day 3 and differed between septic and nonseptic patients. Clustering analysis revealed two distinct immune phenotypes, represented by differential responses to 18 hours of LPS stimulation and 4 hours of anti-CD3/anti-CD28 stimulation. Our rapid immune profiling technique offers a promising tool for early prediction and management of organ dysfunction in critically ill patients. This information could be pivotal for early intervention and for preventing irreversible organ damage during the acute phase of critical illness.

THP-1 Monocytic Cells Are Polarized to More Antitumorigenic Macrophages by Serial Treatment with Phorbol-12-Myristate-13-Acetate and PD98059.

Background and Objectives: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. Materials and Methods: Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11bhigh THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DRlow and CD206low), M1 (HLA-DRhigh and CD206low), and M2 (HLA-DRlow and CD206high), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. Conclusions: We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.

Signal-Sustained Imaging of Mitophagy with an Enzyme-Activatable Metabolic Lipid Labeling Probe

ABSTRACT Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an enzyme-activatable probe covalently attached on mitochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents. Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DBCO, dibenzocyclooctyne; ECAM, enzyme-activated probe covalently attached on mitochondrial inner membrane; GFP, green fluorescent protein; LAMP2, lysosomal associated membrane protein 2; LNPEP/LAP, leucyl and cystinyl aminopeptidase; PMA, phorbol-12-myristate-13-acetate; ΔΨm, mitochondrial transmembrane potential; RFP, red fluorescent protein; TPP, triphenylphosphonium.

Short communication: Goat mastitis and the formation of neutrophil extracellular traps (NETs)

Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis.

Ubiquitination is involved in PKC-mediated degradation of cell surface Kv1.5 channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K+ current (IKur) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

Neutrophil glucose flux as a therapeutic target in antiphospholipid syndrome.

Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that neutrophils from patients with APS used glycolysis more avidly than neutrophils from people in the healthy control group, especially when the neutrophils were from patients with APS with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.

Lysophosphatidic Acid Receptor 3 (LPA3): Signaling and Phosphorylation Sites.

LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.

Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice

AbstractNeutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4–/–) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava–stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4–/– mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)–stimulated PDK2/4–/– neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4–/– neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4–/– neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

HIV-1 latency reversal agent boosting is not limited by opioid use.

The opioid epidemic may impact the HIV-1 reservoir and its reversal from latency in virally suppressed people with HIV (PWH). We studied forty-seven PWH and observed that lowering the concentration of HIV-1 latency reversal agents (LRA), used in combination with small molecules that do not reverse latency, synergistically increases the magnitude of HIV-1 re-activation ex vivo, regardless of opioid use. This LRA boosting, which combines a Smac mimetic or low-dose protein kinase C agonist with histone deacetylase inhibitors, can generate significantly more unspliced HIV-1 transcription than phorbol 12-myristate 13-acetate (PMA) with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation in CD4+ T cells and modulated T cell activation-induced markers and intracellular cytokine production; Smac mimetic-based boosting was less likely to induce immune activation. We found that HIV-1 reservoirs in PWH contain unspliced and polyadenylated (polyA) virus mRNA, the ratios of which are greater in resting than total CD4+ T cells and can correct to 1:1 with PMAi exposure. Latency reversal results in greater fold-change increases to HIV-1 poly(A) mRNA than unspliced message. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent post-transcriptional block. LRA boosting can be leveraged to probe the mechanisms of an effective cellular HIV-1 latency reversal program.

Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Resveratrol and piceid enhance efferocytosis by increasing the secretion of MFG-E8 in human THP-1 macrophages.

The process of apoptotic cell clearance by phagocytes, known as efferocytosis, plays an essential role in maintaining homeostasis. Defects in efferocytosis can lead to inflammatory diseases such as atherosclerosis and autoimmune disorders. Therefore, the maintenance and promotion of efferocytosis are considered crucial for preventing these diseases. In this study, we observed that resveratrol, a representative functional food ingredient, and its glycoside, piceid, promoted efferocytosis in both human THP-1 macrophages differentiated with phorbol 12-myristate 13-acetate and peritoneal macrophages from thioglycolate-elicited mice. Resveratrol and piceid significantly increased mRNA expression and protein secretion of MFG-E8 in THP-1 macrophages. Furthermore, the activation of efferocytosis and the increment in MFG-E8 protein secretion caused by resveratrol or piceid treatment were canceled by MFG-E8 knockdown in THP-1 macrophages. In conclusion, we have demonstrated for the first time that resveratrol and piceid promote efferocytosis through the upregulation of MFG-E8 excretion in human THP-1 macrophages.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

PECULIARITIES OF NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN CHINCHILLA RABBITS

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

First-in-human study of HMPL-295, an ERK1/2 inhibitor, in patients with advanced solid tumors: Dose-escalation results of monotherapy.

e15112 Background: HMPL-295 (’295) is an extracellular signal-regulated kinase 1 and 2 (ERK1/2) inhibitor with potent activity in non-clinical studies. This is a first-in-human, dose escalation study of ’295 in patients (pts) with advanced solid tumors. Here, we report the results of the mono dose escalation stage. Methods: Pts with advanced solid tumors who have failed standard therapy were enrolled to receive ’295 once daily (QD) in 28-day cycles or ’295 QD 2 weeks on/1 week off (2w on/1w off) in 21-day cycles. The mTPI-2 design was used for dose escalation, having explored from 5 to 75 mg QD in 6 cohorts, as well as 75 mg and 100 mg QD 2w on/1w off. The study aims to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), safety, tolerability, pharmacokinetics/pharmacodynamics (PK/PD), and preliminary efficacy (best of response, BOR). Results: As of Dec 11, 2023, 47 pts with advanced solid tumors were enrolled (n=1, 1, 3, 6, 12, 5, 13, 6 in 5, 10, 20, 40, 50, 75 mg QD cohorts, 75, 100 mg QD 2w on/1w off cohorts, respectively), with a median age of 58 years, 23 (48.9%) male pts. During the dose escalation from 5 to 75 mg QD cohort, 1 pt given 40 mg QD experienced a dose-limiting toxicity (DLT) of grade 3 dermatitis acneiform and 2 pts given 75 mg QD experienced DLTs of grade 3 rash and acute kidney injury (AKI). As for intermittent-administration cohorts, 2 pts given 100 mg QD 2w on/1w off experienced DLTs of grade 3 AKI and rash maculo-papular. After multiple-dose oral administration, ’295 plasma concentrations reach peak at 1.0-2.0 hours post dose and the exposure (Cmax and AUC) displayed dose dependent increase. The accumulation ratio (AUC) was 2.1–3.4 after repeat dosing, reaching steady-state around day 15. ’295 inhibited ribosomal S6 kinase (RSK) phosphorylation which is a downstream signaling molecule regulated by ERK1/2 and stimulated by phorbol 12-myristate 13-acetate (PMA). The BOR was partial response each in 1 pt with duodenal adenocarcinoma in 40 mg QD cohort,1 pt with endometrial cancer in 50 mg QD cohort and 1 pt with non-small cell lung cancer (NSCLC) in 75 mg QD 2w on/1w off cohort. Stable disease with tumor shrinkage was also observed in 2 pts with NSCLC on 50 mg QD and 75 mg QD 2w on/1w off cohort respectively, and 1 pt with pancreatic ductal adenocarcinoma in 100 mg QD 2w on/1w off cohort. 25 (53.2%) pts reported grade ≥3 TEAEs. The most common (≥10.0%) ≥ grade 3 TEAEs were rash (10.6%) and anemia (10.6%). Conclusions: Considering safety, tolerability, PK/PD and preliminary efficacy results, RP2D was determined to be 50 mg QD. Clinical trial information: NCT04908046 .

Non-invasive Physical Plasma Modulates Macrophage Polarization: A Potential Strategy for Tumor Microenvironment Remodeling.

Non-invasive physical plasma (NIPP) has shown promise in the treatment of cancer. However, conflicting results have been reported regarding the effect of NIPP on macrophage polarization. As tumor-associated macrophages (TAMs) are essential in the regulation of cancer development, this study aimed to determine the role of NIPP treatment in macrophage polarization and tumor-microenvironment (TME) remodeling. A portable NIPP device, Plasma Care (Terraplasma Medical, Garching, Germany), was employed as the source of NIPP. The human monocytic cell line THP-1 was adopted as the cell model for macrophage differentiation and polarization. The effects of NIPP treatment on temperature, pH value, and oxidative stress induction of the culture medium were examined to validate the feasibility of applying the NIPP device in subsequent cell treatment. The changes in morphology, viability, and proliferation of THP-1 cells after NIPP treatment were determined. The expression of M1/M2 macrophage markers was examined by real-time quantitative polymerase chain reaction. No significant changes were observed in temperature and pH value after NIPP treatment, while the formation of hydrogen peroxide was promoted in a time-dependent manner. Cell morphology, viability, and proliferation were not affected by up to 6 minutes of NIPP treatment. In monocytes, 6 minutes of NIPP treatment significantly increased the expression of M1 markers (TNF-α and IL-6) and suppressed the M2 marker (CD206), findings which were consistent in the monocyte-derived macrophages. Furthermore, NIPP treatment also significantly promoted M1 polarization in the monocyte-derived macrophages induced by phorbol 12-myristate 13-acetate. NIPP is a safe and robust oxidative stress inducer and showed potential in TAM regulation by promoting M1 macrophage polarization.

Ameliorative effects of Wikstroemia trichotoma 95% EtOH extract on a mouse model of DNCB-induced atopic dermatitis

Ethnopharmacological relevanceThe genus Wikstroemia has been extensively utilized in traditional Chinese medicine (TCM) for the management of conditions such as coughs, edema, arthritis, and bronchitis. Studies have indicated that the crude extracts of Wikstroemia exhibit anti-inflammatory, anti-allergy, anti-aging, skin psoriasis, anti-cancer, and antiviral properties. In addition, these extracts are known to contain bioactive substances, including flavonoids, coumarins, and lignans. However, few studies have investigated the anti-inflammatory or anti-allergic activities of Wikstroemia trichotoma (Thunb.) Makino against atopic dermatitis (AD). Aim of the studyThe study aimed to explore the potential of a 95% ethanol extract of W. trichotoma (WTE) on the dysfunction of skin barrier and immune system, which are primary symptoms of AD, in 2,4-dinitrochlorobenzene (DNCB)-induced SKH-1 hairless mice and phorbol 12-myristate 13-acetate (PMA)/ionomycin or immunoglobulin E (IgE) + 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulated rat basophilic leukemia cell line (RBL-2H3). Furthermore, we sought to identify the chemical contents of WTE using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-PDA). Materials and methodsAn in vitro study was conducted using RBL-2H3 cells stimulated with PMA/ionomycin or IgE + DNP-BSA to assess the inhibitory effects of WTE on mast cell degranulation and interleukin-4 (IL-4) mRNA expression levels. For the in vivo study, AD was induced in SKH-1 hairless mice by applying 1% DNCB to the dorsal skin daily for 7 days. Subsequently, 0.1% DNCB solution was applied on alternate days, and mice were orally administered WTE (at 30 or 100 mg/kg/day) dissolved in 0.5% carboxymethyl cellulose (CMC) daily for 2 weeks. Transepidermal water loss (TEWL), skin hydration, skin pH, and total serum IgE levels were measured. ResultsIn DNCB-stimulated SKH-1 hairless mice, WTE administration significantly improved AD symptoms and ameliorated dorsal skin inflammation. Oral administration of WTE led to a significant decrease in skin thickness, infiltration of mast cells, and level of total serum IgE, thus restoring skin barrier function in the DNCB-induced skin lesions. In addition, WTE inhibited β-hexosaminidase release and reduced IL-4 mRNA levels in RBL-2H3 cells. Chemical profile analysis of WTE confirmed the presence of three phenolic compounds, viz. chlorogenic acid, miconioside B, and matteucinol-7-O-β-apiofuranosyl (1 → 6)-β-glucopyranoside. ConclusionsWTE ameliorates AD symptoms by modulating in the skin barrier and immune system dysfunction. This suggests that W. trichotoma extract may offer therapeutic benefits for managing AD.

Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

Decreased atherosclerosis and abdominal aortic aneurysm in mice deficient in polymeric immunoglobulin receptor

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): La Caixa Banking Foundation and Comunidad de Madrid Background Polymeric immunoglobulin receptor (PIGR) is a transmembrane protein involved in the transcytosis of polymeric immunoglobulins that is widely expressed in mucosal epithelial cells. Recent data from our group described increased PIGR levels in plasma and tissue of atherosclerotic subjects. However, the role for PIGR in the mechanisms involved in vascular remodeling has not been addressed. Purpose To analyse the role for PIGR in experimental mouse models of atherosclerosis and abdominal aortic aneurysm (AAA). Methods We analyzed PIGR expression, secretion and distribution in early atherosclerotic plaques and AAA tissues compared to healthy aortic samples, as well as in macrophages derived from the human monocytic THP-1 cell line. Next, we studied the effect of global PIGR deficiency in experimental atherosclerosis (Ldlr-/- Pigr-/- mice fed with atherogenic diet for 10 weeks). In addition, the contribution of hematopoietic PIGR was assessed in both experimental atherosclerosis and AAA (Ldlr-/- mice fed with atherogenic diet and 1 µg/Kg/min angiotensin II infusion for 28 days) by bone marrow transplantation experiments. Finally, bone marrow-derived macrophages (BMDMs) from Pigr-/- and control mice were isolated to assess their inflammatory expression profile and modulation of polarization in response to oxidized LDL (ox-LDL), as well as their ability to promote foam cell formation. Results PIGR expression was increased in the intima of early human atherosclerotic lesions and showed colocalization with CD68+ expression. PIGR expression was also increased during the in vitro differentiation of THP-1 monocytes to macrophages with phorbol myristate acetate. Both global and hematopoietic-specific PIGR deficient mice displayed reduced plaque size in the aortic sinuses as well as a strong decrease in macrophage infiltration (CD68+ cells), while no changes in smooth muscle cells (α-SMA+ cells) and collagen content were observed. Regarding AAA, increased PIGR levels were found in human AAA tissues, associated with infiltrating CD68+ cells. PIGR deficiency in hematopoietic cells also showed significantly reduced AAA dilatation and reduced macrophage infiltration. In vitro experiments with BMDMs obtained from Pigr-/- mice and stimulated with ox-LDL showed reduced proinflammatory gene expression (TNF-a and IL-6) and M1 markers (iNOS and CD11c) as well as diminished foam cell formation. Conclusions PIGR deficiency in hematopoietic cells decreases atherosclerosis and AAA progression by modulating macrophage inflammatory responses, suggesting a novel proinflammatory role for PIGR in vascular pathologies. Future experiments will test the potential therapeutic effect of modulating PIGR to dampen the inflammatory profile in vascular diseases.Results in human and mouse studies