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Antimicrobial Agents and Chemotherapy Nov 2023A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir...
A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.
Topics: Humans; CD4-Positive T-Lymphocytes; HIV Infections; Proviruses; Virus Activation; Virus Latency; Virus Replication; tat Gene Products, Human Immunodeficiency Virus; HIV-1
PubMed: 37874295
DOI: 10.1128/aac.00417-23 -
BioRxiv : the Preprint Server For... Oct 2023Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their...
Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.
PubMed: 37873256
DOI: 10.1101/2023.10.11.561954 -
Parasites & Vectors Oct 2023Upon encountering exogenous pathogens, polymorphonucleocytes (PMNs) engage in various processes to destroy them, including releasing neutrophil extracellular traps...
Upon encountering exogenous pathogens, polymorphonucleocytes (PMNs) engage in various processes to destroy them, including releasing neutrophil extracellular traps (NETs) that trap pathogens and induce phagocytosis and cytokine production. Parasites have unique strategies with which to evade the host's immune response. However, the strategy employed by Trichinella spiralis in response to the reaction of PMNs has yet to be elucidated. This study explored the effect of excretory/secretory products (ESP) on three major functions: NETs, phagocytosis, and cytokine production. Specifically, PMNs were pre-treated with the ESP of 3-day-old adults and then stimulated with phorbol 12-myristate 13-acetate (PMA). We found that in PMNs pretreated with ESP, PMA-induced NET generation was suppressed by ESP. ROS production is a hallmark of PMA-induced NETosis. The LDH assay results showed that ESP inhibits NETs by suppressing ROS rather than promoting PMN death. Furthermore, ESP enhanced Escherichia coli engulfment by PMNs, improving overall phagocytic function. Finally, cytokine analysis revealed an increase in pro-inflammatory cytokine IL-1β, and other cytokines (IL-10, TNF-α), while IL-4 displayed a significant reduction. In conclusion, this study has unraveled T. spiralis' evasion and regulation mechanisms against innate immune cells, providing insights into parasite strategies to manipulate host immunity, potentially informing new treatments for NET-related autoimmune diseases.
Topics: Animals; Cytokines; Neutrophils; Trichinella spiralis; Reactive Oxygen Species; Gene Expression; Extracellular Traps
PubMed: 37864246
DOI: 10.1186/s13071-023-05979-8 -
PloS One 2023Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic...
Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.
Topics: Animals; Humans; Endothelial Cells; Induced Pluripotent Stem Cells; Brain; Microvessels; Lab-On-A-Chip Devices; Spheroids, Cellular
PubMed: 37856438
DOI: 10.1371/journal.pone.0288025 -
International Journal of Molecular... Sep 2023The neuroinflammatory response after intracerebral hemorrhage (ICH) causes a large amount of neuronal loss, and inhibiting the inflammatory response can improve the...
The neuroinflammatory response after intracerebral hemorrhage (ICH) causes a large amount of neuronal loss, and inhibiting the inflammatory response can improve the prognosis. In previous laboratory studies and clinical trials, ursolic acid (UA) inhibited the inflammatory response, but whether it can be administered to inhibit the neuroinflammatory response after cerebral hemorrhage is unknown. The aim of this study was to investigate the effects of ursolic acid after cerebral hemorrhage. Online databases were used to obtain potential therapeutic targets of ursolic acid for the treatment of cerebral hemorrhage, and possible mechanisms were analyzed by KEGG, GO, and molecular docking. A rat model of cerebral hemorrhage was established using collagenase, and an in vitro cerebral hemorrhage model was constructed by adding hemin to BV2 cell culture medium. Enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), immunofluorescence, TUNEL staining, and calcein/PI staining were used to investigate the degree of microglial M1 polarization, changes in the levels of inflammatory factors, activation of the NF-κB pathway, and changes in the indicators of cellular death after ursolic acid treatment. In addition, phorbol 12-myristate 13-acetate (PMA) was used to activate the NF-κB pathway to verify that ursolic acid exerts its anti-neuroinflammatory effects by regulating the NF-κB/NLRP3/GSDMD pathway. Network pharmacology and bioinformatics analyses revealed that ursolic acid may exert its therapeutic effects on cerebral hemorrhage through multiple pathways. Together, in vivo and in vitro experiments showed that ursolic acid inhibited microglial M1 polarization and significantly reduced the levels of p-NF-κB, GSDMD-N, cleaved caspase-1, TNF-α, IL-6, and IL-1β, which were significantly inhibited by the use of PMA. Ursolic acid inhibits microglial pyroptosis via the NF-κB/NLRP3/GSDMD pathway to alleviate neuroinflammatory responses after cerebral hemorrhage.
Topics: Rats; Animals; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Signal Transduction; Neuroinflammatory Diseases; Microglia; Molecular Docking Simulation; Rats, Sprague-Dawley; Cerebral Hemorrhage; Inflammation; Ursolic Acid
PubMed: 37834220
DOI: 10.3390/ijms241914771 -
Intensive Care Medicine Experimental Oct 2023Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these...
BACKGROUND
Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7-10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality.
RESULTS
Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation.
CONCLUSIONS
Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.
PubMed: 37831231
DOI: 10.1186/s40635-023-00556-w -
Heliyon Sep 2023Methyl lucidone (ML), a methyl derivative of lucidone, has anti-inflammatory properties. However, the molecular mechanisms that reduce the inflammatory effect of ML in...
BACKGROUND
Methyl lucidone (ML), a methyl derivative of lucidone, has anti-inflammatory properties. However, the molecular mechanisms that reduce the inflammatory effect of ML in human lung epithelial cells remain unkown. This study aimed to elucidate the molecular mechanisms underlying the anti-inflammatory effect of ML.
METHODS
Four compounds (ML, methyl linderone, kanakugiol, and linderone) from Makino were evaluated for their ability to reduce MUC5AC secretion levels in phorbol-12-myristate-13-acetate (PMA)-stimulated NCI-H292 cells using ELISA. The expression and secretion levels of inflammatory response-related proteins were analyzed using quantitative reverse transcription-PCR, ELISA, and western blotting. To determine whether ML directly regulates TGF-β-activated kinase 1 (TAK1), we performed an kinase assay.
RESULTS
ML treatment effectively reduced the levels of inflammatory cytokines, including interleukin-1β and TNF-α, increased by stimulation. Furthermore, ML downregulated the pathway cascade of both IκB kinase (IKK)/NF-κB and p38 mitogen-activated protein (MAP) kinase/CREB by inhibiting the upstream kinase TAK1. An kinase analysis confirmed that ML treatment significantly reduced the kinase activity of TAK1.
CONCLUSION
ML pretreatment repressed the PMA-stimulated inflammation reaction by reducing the TAK1-mediated IKK/NF-κB and p38 MAP kinase/CREB signaling. These findings suggest that ML may improve respiratory health and can be used as a dietary supplement or functional food to prevent inflammatory lung diseases.
PubMed: 37809903
DOI: 10.1016/j.heliyon.2023.e20154 -
Frontiers in Immunology 2023The relationship between the tumor microenvironment and the network of key signaling pathways in cancer plays a key role in the occurrence and development of tumors....
BACKGROUND AND OBJECTIVES
The relationship between the tumor microenvironment and the network of key signaling pathways in cancer plays a key role in the occurrence and development of tumors. Tumor-associated macrophages (TAMs) are important inflammatory cells in the tumor microenvironment and play an important role in tumorigenesis and progression. Macrophages in malignant tumors, mainly the M2 subtype, promote tumor progression by producing cytokines and down-regulating anti-inflammatory immune responses. Several articles have investigated the effect of macrophages on the sensitivity of cancer chemotherapeutic agents, but few such articles have been reported in cholangiocarcinoma, so we investigated the effect of M2 macrophage on the sensitivity of cholangiocarcinoma cells to Lenvatinib compared to M1.
METHODS
THP-1 monocytes were polarized to M0 macrophage by phorbol 12-myristate 13-acetate (PMA) and then induced to differentiate into M1 and M2 macrophages by LPS, IFN-γ and IL-4 and IL-13, respectively. Macrophages and cholangiocarcinoma cells were co-cultured prior to 24 hours of Lenvatinib administration, cancer cell apoptosis was detected by western-blot, FACS analysis of Annexin V and PI staining. Furthermore, we use xCELLigence RTCA SP Instrument (ACEA Bio-sciences) to monitor cell viability of Lenvatinib administration in co-culture of cholangiocarcinoma cells and macrophages. After tumorigenesis in immunodeficient mice, Lenvatinib was administered, and the effects of M2 on biological characteristics of cholangiocarcinoma cells were investigated by immuno-histochemistry.
RESULTS
mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1 derived macrophages, which provided a successful and efficient model of monocyte polarization to TAMs. Lenvatinib-induced apoptosis of cholangiocarcinoma cells was significantly reduced when co-cultured with M2 macrophage, whereas apoptosis of cholangiocarcinoma cells co-cultured with M1 macrophage was increased. In the CDX model, Lenvatinib-induced cancer cell apoptosis was markedly reduced, and proliferative cells increased in the presence of M2 macrophages. Angiogenesis related factors was significantly increased in cholangiocarcinoma cells co-cultured with M2.
CONCLUSION
Compared with M1, M2 macrophages can inhibit the anti-tumor effect of Lenvatinib on cholangiocarcinoma through immune regulation, which may be related to the tumor angiogenesis factor effect of M2 macrophage.
Topics: Animals; Mice; Macrophages; Cholangiocarcinoma; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Carcinogenesis; Tumor Microenvironment
PubMed: 37809069
DOI: 10.3389/fimmu.2023.1251648 -
Pflugers Archiv : European Journal of... Jan 2024Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal...
Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis.
Topics: Humans; Protein Kinase C; Glucuronidase; Fibroblast Growth Factors; Protein Isoforms; Gene Expression; Kidney Diseases
PubMed: 37773536
DOI: 10.1007/s00424-023-02863-3 -
Journal of Pharmacological Sciences Nov 2023We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to...
Pharmacological study on the enhancing effects of U46619 on guinea pig urinary bladder smooth muscle contraction induced by acetylcholine and α,β-methylene ATP and the possible involvement of protein kinase C.
We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to acetylcholine (ACh) and an ATP analog (α,β-methylene ATP (αβ-MeATP)) through stimulation of the UBSM TP receptor and whether protein kinase C (PKC) is involved. U46619 (10 M) markedly enhanced UBSM contractions induced by electrical field stimulation and ACh/αβ-MeATP (3 × 10 M each), the potentiation of which was completely suppressed by SQ 29,548 (a TP receptor antagonist, 6 × 10 M). PKC inhibitors did not attenuate the ACh-induced contractions enhanced by U46619 although they partly suppressed the U46619-enhanced, αβ-MeATP-induced contractions. While phorbol 12-myristate 13-acetate (PMA, a PKC activator, 10 M) did not enhance ACh-induced contractions, it enhanced αβ-MeATP-induced contractions, an effect that was completely suppressed by PKC inhibitors. αβ-MeATP-induced contractions, both with and without U46619 enhancement, were strongly inhibited by diltiazem. U46619/PMA enhanced 50 mM KCl-induced contractions, the potentiation of which was partly/completely attenuated by PKC inhibitors. These findings suggest that U46619 potentiates parasympathetic nerve-associated UBSM contractions by stimulating UBSM TP receptors. PKC-increased Ca influx through voltage-dependent Ca channels may partially play a role in purinergic receptor-mediated UBSM contractions enhanced by TP receptor stimulation.
Topics: Guinea Pigs; Animals; Acetylcholine; Urinary Bladder; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Muscle Contraction; Receptors, Thromboxane
PubMed: 37770153
DOI: 10.1016/j.jphs.2023.08.007