CD8+ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen-presenting cells that induce their proliferation. Here, we show that thapsigargin-induced LAD2 mast cell (MC) line-released products can impair the ability of monocyte-derived DCs to induce CD8+ T-cell proliferation and the generation of Th1 cytokine-producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA-DR. However, thapsLAD2-matured DCs produced no detectable TNFα or IL-12 during the maturation. In addition, although their surface expression of PD-L1 was comparable with the immature or TLR7/8-agonist (R848)-matured DCs, their TIM-3 expression was significantly higher than in immature DCs and even much higher than in R848-matured DCs. In addition, contrary to R848-matured DCs, the thapsLAD2-matured DCs only tended to induce enhanced proliferation of CD4+ T cells than immature DCs. For CD8+ T cells, this tendency was not even detected because thapsLAD2-matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848-matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ- and IFNγ/TNFα-producing T cells. These findings show a novel mechanism of MC-mediated regulation of adaptive immune responses.

• MeSH
• Lymphocyte Activation * drug effects   immunology   MeSH
• Cell Differentiation * drug effects   MeSH
• Cell Line MeSH
• Hepatitis A Virus Cellular Receptor 2 metabolism   MeSH
• CD8-Positive T-Lymphocytes * immunology   drug effects   MeSH
• Cytokines metabolism   MeSH
• Dendritic Cells * immunology   drug effects   metabolism   MeSH
• Imidazoles pharmacology   MeSH
• Humans MeSH
• Mast Cells * immunology   drug effects   metabolism   MeSH
• Monocytes immunology   drug effects   metabolism   MeSH
• Cell Proliferation * drug effects   MeSH
• Thapsigargin * pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

• MeSH
• Aorta MeSH
• Bradykinin * pharmacology   MeSH
• Endothelium, Vascular MeSH
• Rats MeSH
• Lipopolysaccharides pharmacology   MeSH
• Membrane Potentials MeSH
• Rats, Sprague-Dawley MeSH
• Rats, Transgenic MeSH
• Receptor, Bradykinin B1 * genetics   MeSH
• In Vitro Techniques MeSH
• Thapsigargin pharmacology   MeSH
• Toll-Like Receptor 4 MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The preparation of dendritic cells (DCs) for adoptive cellular immunotherapy (ACI) requires the maturation of ex vivo-produced immature(i) DCs. This maturation ensures that the antigen presentation triggers an immune response towards the antigen-expressing cells. Although there is a large number of maturation agents capable of inducing strong DC maturation, there is still only a very limited number of these agents approved for use in the production of DCs for ACI. In seeking novel DC maturation agents, we used differentially activated human mast cell (MC) line LAD2 as a cellular adjuvant to elicit or modulate the maturation of ex vivo-produced monocyte-derived iDCs. We found that co-culture of iDCs with differentially activated LAD2 MCs in serum-containing media significantly modulated polyinosinic:polycytidylic acid (poly I:C)-elicited DC maturation as determined through the surface expression of the maturation markers CD80, CD83, CD86, and human leukocyte antigen(HLA)-DR. Once iDCs were generated in serum-free conditions, they became refractory to the maturation with poly I:C, and the LAD2 MC modulatory potential was minimized. However, the maturation-refractory phenotype of the serum-free generated iDCs was largely overcome by co-culture with thapsigargin-stimulated LAD2 MCs. Our data suggest that differentially stimulated mast cells could be novel and highly potent cellular adjuvants for the maturation of DCs for ACI.

• MeSH
• Adjuvants, Immunologic pharmacology   MeSH
• Cell Differentiation drug effects   MeSH
• Cell Culture Techniques methods   MeSH
• Dendritic Cells cytology   drug effects   immunology   MeSH
• Immunotherapy, Adoptive * MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Mast Cells cytology   drug effects   immunology   MeSH
• Monocytes cytology   drug effects   immunology   MeSH
• Antigen Presentation drug effects   immunology   MeSH
• Thapsigargin pharmacology   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.

• MeSH
• Apiaceae chemistry   metabolism   MeSH
• Butyrates chemistry   isolation & purification   MeSH
• Furans chemistry   isolation & purification   MeSH
• Antineoplastic Agents, Phytogenic chemistry   isolation & purification   MeSH
• Humans MeSH
• Molecular Structure MeSH
• Neoplasms drug therapy   pathology   MeSH
• Fruit chemistry   metabolism   MeSH
• Carbon Dioxide chemistry   MeSH
• Plant Extracts chemistry   MeSH
• Sarcoplasmic Reticulum Calcium-Transporting ATPases antagonists & inhibitors   metabolism   MeSH
• Chromatography, Supercritical Fluid methods   MeSH
• Chemistry Techniques, Synthetic * MeSH
• Thapsigargin analogs & derivatives   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

• MeSH
• Apoptosis MeSH
• Autocrine Communication MeSH
• Biomarkers MeSH
• Epidermal Growth Factor metabolism   MeSH
• Myocytes, Cardiac drug effects   metabolism   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Membrane Glycoproteins metabolism   MeSH
• Mice MeSH
• Disease Susceptibility MeSH
• Neoplasm Proteins metabolism   MeSH
• Paracrine Communication MeSH
• Proteome * MeSH
• Proteomics * methods   MeSH
• Sarcoplasmic Reticulum metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Endoplasmic Reticulum Stress * drug effects   MeSH
• Thapsigargin pharmacology   MeSH
• Calcium metabolism   MeSH
• Calcium Signaling drug effects   MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Sesquiterpene lactones are bioactive natural compounds with anticancer, antiprotozoal, immunomodulatory, antibacterial and antiviral activity which have potential for drug development. The aim of this review article is to provide a brief insight into the field of sesquiterpene lactones: the main mechanisms of their biological actions, as well as particular compounds, are described, some of which have already become a basis of a drug development and are being tested in clinical trials.

• MeSH
• Artemisinins MeSH
• Ethnopharmacology MeSH
• Pharmacognosy MeSH
• Antineoplastic Agents, Phytogenic isolation & purification   MeSH
• Immunomodulation MeSH
• Enzyme Inhibitors MeSH
• Lactones * pharmacokinetics   pharmacology   therapeutic use   MeSH
• Sesquiterpenes, Guaiane pharmacokinetics   pharmacology   therapeutic use   MeSH
• Sesquiterpenes pharmacokinetics   pharmacology   therapeutic use   MeSH
• Tanacetum parthenium chemistry   MeSH
• Thapsigargin MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVE: To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue samples from patients with RA and patients with osteoarthritis (OA) were stained for PTPN2. Synovial fibroblasts were stimulated with tumor necrosis factor (TNF) and interleukin-1β (IL-1β), lipopolysaccharide (LPS), TRAIL, or thapsigargin. The expression of PTPN2 in synovial fibroblasts and peripheral blood mononuclear cells (PBMCs) was analyzed by real-time polymerase chain reaction and Western blotting. Cell death, the release of IL-6 and IL-8, and the induction of autophagy were analyzed after PTPN2 silencing. Methylated DNA immunoprecipitation analysis was used to evaluate DNA methylation-regulated gene expression of PTPN2. RESULTS: PTPN2 was significantly overexpressed in synovial tissue samples from RA patients compared to OA patients. Patients receiving anti-TNF therapy showed significantly reduced staining for PTPN2 compared with patients treated with nonbiologic agents. PTPN2 expression was higher in RA synovial fibroblasts (RASFs) than in OASFs. This differential expression was not regulated by DNA methylation. PTPN2 was further up-regulated after stimulation with TNF, TNF combined with IL-1β, or LPS. There was no significant difference in basal PTPN2 expression in PBMCs from patients with RA, ankylosing spondylitis, or systemic lupus erythematosus or healthy controls. Most interestingly, PTPN2 silencing in RASFs significantly increased the production of the inflammatory cytokine IL-6 but did not affect levels of IL-8. Moreover, functional analysis showed that high PTPN2 levels contributed to the increased apoptosis resistance of RASFs and increased autophagy. CONCLUSION: This is the first study of PTPN2 in RASFs showing that PTPN2 regulates IL-6 production, cell death, and autophagy. Our findings indicate that PTPN2 is linked to the pathogenesis of RA via synovial fibroblasts.

• MeSH
• Apoptosis drug effects   MeSH
• Autophagy drug effects   MeSH
• Biological Products pharmacology   MeSH
• Fibroblasts drug effects   metabolism   pathology   MeSH
• Interleukin-1beta pharmacology   MeSH
• Interleukin-6 metabolism   MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Lipopolysaccharides pharmacology   MeSH
• Osteoarthritis metabolism   pathology   MeSH
• TNF-Related Apoptosis-Inducing Ligand pharmacology   MeSH
• Arthritis, Rheumatoid metabolism   pathology   MeSH
• Aged MeSH
• Synovial Membrane drug effects   metabolism   pathology   MeSH
• Thapsigargin pharmacology   MeSH
• Tumor Necrosis Factor-alpha antagonists & inhibitors   pharmacology   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 2 metabolism   MeSH
• Up-Regulation drug effects   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. METHODS: Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. RESULTS: Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1-24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. CONCLUSIONS: Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

• MeSH
• Cell Differentiation * MeSH
• Endoplasmic Reticulum drug effects   metabolism   MeSH
• Phosphorylation MeSH
• Stress, Physiological * MeSH
• Humans MeSH
• Lipids biosynthesis   MeSH
• Unfolded Protein Response MeSH
• Thapsigargin pharmacology   MeSH
• Adipocytes cytology   MeSH
• Tunicamycin pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.

• MeSH
• Biotin analogs & derivatives   MeSH
• Cell Line MeSH
• Enzyme-Linked Immunosorbent Assay methods   statistics & numerical data   MeSH
• Epitope Mapping MeSH
• Mast Cells drug effects   metabolism   MeSH
• Antibodies, Monoclonal MeSH
• Mice MeSH
• Paclitaxel pharmacology   MeSH
• Polymerase Chain Reaction methods   MeSH
• Protein Isoforms analysis   genetics   immunology   MeSH
• Thapsigargin pharmacology   MeSH
• Tubulin analysis   genetics   immunology   MeSH
• Tyramine analogs & derivatives   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The potential pro-survival role of phosphatidylinositol 3-kinase (PI3K)/Akt during endoplasmic reticulum stress has been well-characterized. However, the detailed mechanisms remain largely unknown. Here, we showed that PI3K/Akt inhibition promoted endoplasmic reticulum stress-induced apoptosis in a glucose-regulated protein 78 (GRP78)-dependent manner. During endoplasmic reticulum stress, high levels of Akt phosphorylation were sustained for at least 18 h in HEK293 cells. Importantly, PI3K/Akt enhanced GRP78 accumulation through increasing its stability following endoplasmic reticulum stress. Furthermore, Akt1, but not Akt2 or Akt3, was involved in GRP78 stability regulation. These results suggest that PI3K/Akt inhibits endoplasmic reticulum stress-induced apoptosis in HEK293 cells, at least in part, by promoting GRP78 protein stability.

• MeSH
• Apoptosis physiology   drug effects   MeSH
• Cell Line MeSH
• NIH 3T3 Cells MeSH
• Dithiothreitol pharmacology   MeSH
• Endoplasmic Reticulum metabolism   drug effects   MeSH
• Phosphatidylinositol 3-Kinases genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Humans MeSH
• Mice MeSH
• Heat-Shock Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-akt genetics   metabolism   MeSH
• Thapsigargin pharmacology   MeSH
• Transcription Factor CHOP metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH