Primary Cultures Of Human Cells Research Articles

Benzene affects the response to octreotide treatment of growth hormone secreting pituitary adenoma cells

Growth hormone (GH) secreting pituitary adenomas are the main cause of acromegaly. Somatostatin analogs are the gold standard of medical therapy; however, resistance represents a big drawback in acromegaly management. We recently demonstrated that benzene (BZ) modifies the aggressiveness of GH-secreting rat pituitary adenoma cells (GH3), increasing GH secretion and altering the synthesis of molecules involved in the somatostatin signaling pathway. Based on these pieces of evidence, this study aimed to evaluate the effects of BZ on octreotide (OCT) efficacy in GH-secreting adenoma cells. In GH3 cells, BZ counteracted the anti-proliferative action of OCT. GH gene expression, unmodified by OCT, remained high in BZ-treated cells as well as after treatment with the association of both. GH secretion, reduced by OCT, was increased after treatment with BZ alone or when the pollutant was used with OCT. The combination of BZ and OCT greatly reduced the gene expression of ZAC1 and SSTR2; and this reduction was also present at a protein level. BZ caused an increase in the protein level of the transcription factor STAT3 and in its phosphorylated form. In the presence of BZ, OCT lost the ability to reduce the phosphorylated protein levels. Finally, in primary cultures of human pituitary adenoma cells, BZ caused an increase in GH secretion. OCT decreased GH secretion, but the addition of BZ reversed the OCT effect. In conclusion, our results suggest that BZ may have an important role in the resistance of pituitary adenomas to the pharmacological treatment with somatostatin analogs.

Sodium Acrylate‐Induced Differential Expansion of Cellular Organelles

The physical expansion of biological samples in 3‐D to obtain nanoscale (~70 nm) resolution using light microscopy employing hydration‐competent polymers, an approach termed expansion microscopy (ExM), has not been applied to assess the extent of expansion between different tissues and subcellular organelles, critical to clinical pathological assessments. Ultrastructure of cellular organelles at the nanoscale using electron microscopy, and the distribution of associated antigens, can reflect cell health and is used in the detection and assessment of a wide range of pathologies. Using light microscopy, we performed organelle‐specific fluorescent immunocytochemistry on formalin‐fixed paraffin‐embedded human and rabbit tissue slices, and formalin‐fixed primary human muscle cell cultures, to determine the extent of expansion between different tissues and subcellular organelles. Optical evaluation of subcellular expansion of various organelles in the same and in different tissues demonstrate differential expansion, critical to the rapid and inexpensive optical evaluation, as opposed to electron microscopy. The differential expansion of cellular organelles indicates a growing need to better understand the interactions of the polymers with subcellular structures, the composition of different cellular organelles, and the differences of organelle composition between tissues, before ExM can be applied as a replicable clinical tool.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Regulatory Role of TRAPPC9 in Osteoarthritis

INTRODUCTIONTrafficking Protein Particle Complex 9 (TRAPPC9) is a major subunit of TRAPPII Complex. TRAPPC9 is implicated in endoplasmic to Golgi trafficking pathway and intra Golgi trafficking in yeast cells. TRAPPC9 has been reported to bind IKKb and NIK where it plays a role in NFkB signaling. Hence, plays a role in different cell types differentiation and function. In osteoarthritis, inflammation mediated by the NFkB signaling plays a role in the pathogenesis on the disease. There are two distinct pathways, which can activate the NF‐kB signaling cascades. The first one termed the canonical or the classical pathway, activation of IKKα/IKKβ/IKKβ‐NEMO complex will activate this pathway. On the other hand, the non‐canonical or the alternative pathway, activation relies on NF‐kB‐inducing kinase (NIK) signaling. Our data showed that TRAPPC9 binds IKKβ and NIK in chondrocytes. We believe such interaction could lead to a potential regulatory role of TRAPPC9 during NF‐kB signaling cascades given that the role of TRAPPC9 in OA has not been studied yet.METHODSIn this study, we used human chondrocytes and primary mouse chondrocyte cell culture, immunofluorescence, and retroviral transfection techniques.RESULTSTo explore the role of TRAPPC9 in OA, first, we assessed the expression of TRAPPC9 (mRNA and protein) levels in primary human chondrocytes treated with ILb to resemble chondrocytes within OA knee and found that TRAPPC9 expression is decreased in IL‐1β‐treated chondrocytes compared to controls. Next, we determined whether modulation of TRAPPC9 expression alters the inflammatory markers induced by IL1b that are regulated by NF‐kB signaling pathways. Human chondrocytes were transfected with either TRAPPC9 overexpression plasmid or control plasmid. Gene expression for metalloproteinases (what else a part of MMP) was measured and our data showed that TRAPPC9 overexpression induces inflammation by increasing the gene expression of MMPs‐1,3,9,13, and Interlukin‐6 (IL‐6).CONCLUSIONWe showed that TRAPPC9 binds/interacts with NIK and IKKb in chondrocytes. Also, we were able to show that TRAPPC9 expression is decreased in inflammatory conditions. In addition, our data showed that modulating TRAPPC9 expression prior to IL‐1β induction leads to elevate inflammatory conditions in chondrocytes. Studies are underway to determine the effects of modulating TRAPPC9 using genetic and pharmacologic approaches on osteoarthritis development and identifying the mechanism(s) by which inflammation in chondrocytes mediated by TRAPPC9, NIK, and IKKb interactions. Collectively, these studies are the first to report a novel role for TRAPPC9 in osteoarthritis.SIGNIFICANCE/CLINICAL RELEVANCEUnderstanding the role of TRAPPC9 in osteoarthritis are not reported previously. It is important to investigate whether TRAPPC9, NIK and IKKb interaction mediates inflammation in chondrocytes by modulating the NF‐kB signaling pathways thereby, suggesting new therapeutics for osteoarthritis disease.Support or Funding InformationKent State University, School of Biomedical Sciences. Ohio Physiological Society (Peter K Lauf Travel Award).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

THU-457-DoubleCortin/Like Kinase 1 (DCLK1) expression characterized specific cancer stem cell subpopulations of human cholangiocarcinoma primary cell cultures where its inhibition exerts anti-neoplastic effects

THU-457-DoubleCortin/Like Kinase 1 (DCLK1) expression characterized specific cancer stem cell subpopulations of human cholangiocarcinoma primary cell cultures where its inhibition exerts anti-neoplastic effects

The receptor for advanced glycation end products is a critical mediator of type 2 cytokine signaling in the lungs

Asthma is estimated to effect more than 300 million persons worldwide, leading to nearly 250,000 deaths annually. The majority of patients with mild-to-severe asthma have what is deemed "type-2 high" asthma, which is driven by the prototypical type 2 cytokines IL-4, IL-5, and IL-13. Studies have indicated that the receptor for advanced glycation end products (RAGE) is a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation. More specifically, RAGE expressed on stromal cells, rather than hematopoietic cells, is critical to induction of asthma/allergic airway inflammation by driving type 2 inflammatory responses. However, the role of RAGE in directly mediating type 2 cytokine signaling has never been investigated. The goal of this study was to test the hypothesis that RAGE mediates type 2 cytokine-induced signal transduction, airway inflammation, and mucus metaplasia in the lungs. Wild-type (WT) and RAGE knockout (RAGE-/-) mice, were intranasally administered rIL-5/rIL-13 or rIL-4 alone, and signal transducer and activator of transcription 6 (STAT6) signaling, airway inflammation, and mucus metaplasia were assessed. ARAGE small-molecule antagonist was used to determine the effects of pharmacologically inhibiting RAGE on type 2 cytokine-induced effects. Administration of type 2 cytokines induced pronounced airway inflammation and mucus metaplasia in WT mice, which was nearly completely abrogated in RAGE-/- mice. In addition, treatment with a RAGE-specific antagonist diminished the effects of type 2 cytokines in WT mice and in primary human bronchial epithelial cell cultures. Genetic ablation or pharmacologic inhibition of RAGE blocks the effects of IL-13 and IL-4 by inhibiting sustained STAT6 activation and downstream target gene expression in mice and in human bronchial epithelial cells. This study is the first to indicate that RAGE is a critical component of type 2 cytokine signal transduction mechanisms, which is a driving force behind type 2-high asthma.

Resveratrol inhibits decidualization by accelerating downregulation of the CRABP2-RAR pathway in differentiating human endometrial stromal cells

Pregnancy critically depends on the transformation of the human endometrium into a decidual matrix that controls embryo implantation and placenta formation, a process driven foremost by differentiation and polarization of endometrial stromal cells into mature and senescent decidual cells. Perturbations in the decidual process underpin a spectrum of prevalent reproductive disorders, including implantation failure and early pregnancy loss, emphasizing the need for new therapeutic interventions. Resveratrol is a naturally occurring polyphenol, widely used for its antioxidant and anti-inflammatory properties. Using primary human endometrial stromal cell (HESC) cultures, we demonstrate that resveratrol has anti-deciduogenic properties, repressing not only the induction of the decidual marker genes PRL and IGFBP1 but also abrogating decidual senescence. Knockdown of Sirtuin 1, a histone deacetylase activated by resveratrol, restored the expression of IGFBP1 but not the induction of PRL or senescence markers in decidualizing HESCs, suggesting involvement of other pathways. We demonstrate that resveratrol interferes with the reprogramming of the retinoic acid signaling pathway in decidualizing HESCs by accelerating down-regulation of cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor (RAR). Notably, knockdown of CRABP2 or RAR in HESCs was sufficient to recapitulate the anti-deciduogenic effects of resveratrol. Thus, while resveratrol has been advanced as a potential fertility drug, our results indicate it may have detrimental effects on embryo implantation by interfering with decidual remodeling of the endometrium.

Fluorescence imaging of meningioma cells with somatostatin receptor ligands: an in vitro study.

The use of five-aminolevulinic acid (5-ALA) in the staining of malignant glioma cells has significantly improved intraoperative radicality in the resection of gliomas in the last decade. Currently, there is no comparable selective fluorescent substance available for meningiomas. There is however a demand for intraoperative fluorescent identification of, e.g., invasive skull base meningiomas to help improve safe radical resection. Meningiomas show high expression of the somatostatin receptor type 2, offering the possibility of receptor-targeted imaging. The authors used a somatostatin receptor-labeled fluorescence dye in the identification of meningiomas in vitro. The aim of this study was to evaluate the possibility of selective identification of meningioma cells with fluorescent techniques. Twenty-four primary human meningioma cell cultures were analyzed. The tumor cells were incubated with FAM-TOC (5,6-Carboxyfluoresceine-Tyr3-Octreotide). As a negative control, four human dura tissues were cultured as well as a mixed cell culture in vitro and incubated with the same somatostatin receptor-labeled fluorescence substance. After incubation, fluorescence signal and intensity in all cell cultures were analyzed at three different time points using a fluorescence microscope with 488nm epi-illumination. Sixteen WHO I, six WHO II, two WHO III meningioma primary cell cultures, and four dura cell cultures were analyzed. Fluorescence was detected in all meningioma cell cultures (22 cell culture stained strongly, 2 cell cultures moderately) directly after incubation up until 4h later. There were no differences in the quality and quantity of fluorescence signal between the various meningioma grades. The fluorescence signal persisted unchanged during the analyzed period. In the negative control, dura cell cultures remained unstained. This study demonstrates the use of FAM-TOC in the selective fluorescent identification of meningioma cells in vitro. Further evaluation of the chemical kinetics of the applied somatostatin receptor ligand and fluorescence dye is warranted. As a next step, an experimental animal model is needed to evaluate these promising results in vivo.

PTH Modulation by Aldosterone and Angiotensin II is Blunted in Hyperaldosteronism and Rescued by Adrenalectomy.

Accumulating evidence suggests a link between adrenocortical zona glomerulosa and parathyroid gland through mechanisms that remain unexplored. To test the hypothesis that in vivo angiotensin II blockade affects PTH secretion in patients with hypertension and that aldosterone and angiotensim II directly stimulate PTH secretion ex vivo. We investigated the changes of serum PTH levels induced by oral captopril (50 mg) administration in patients with primary essential hypertension (EH) and with primary aldosteronism (PA) caused by bilateral adrenal hyperplasia (BAH) or aldosterone-producing adenoma (APA), the latter before and after adrenalectomy. We also exposed primary cultures of human parathyroid cells from patients with primary hyperparathyroidism to angiotensin II (10-7 M) and/or aldosterone (10-7 M). Captopril lowered PTH levels (in nanograms per liter) both in patients with EH (n = 63; 25.9 ± 8.3 baseline vs 24.4 ± 8.0 postcaptopril, P < 0.0001) and in patients with APA after adrenalectomy (n = 27; 26.3 ± 11.6 vs 24.0 ± 9.7 P = 0.021). However, it was ineffective in patients with full-blown PA caused by APA and BAH. In primary culture of human parathyroid cells, both aldosterone (P < 0.001) and angiotensin II (P = 0.002) markedly increased PTH secretion from baseline, by acting through mineralocorticoid receptor and angiotensin type 1 receptor, as these effects were abolished by canrenone and irbesartan, respectively. These results collectively suggest an implication of the renin-angiotensin-aldosterone system in PTH regulation in humans, at least in PTH-secreting cells obtained from parathyroid tumors. Moreover, they further support the concept that mild hyperparathyroidism is a feature of human PA that is correctable with adrenalectomy.

Специфичная токсичность кальций-фосфатных бионов для культур венозных и артериальных эндотелиальных клеток человека

Цель исследования - оценка токсичности кальций-фосфатных бионов (КФБ) и магний-фосфатных бионов (МФБ) для культур эндотелиальных клеток. Методика. Эндотелиотоксичность бионов изучена при помощи добавления равных концентраций МФБ или КФБ к: 1) разреженным или конфлюэнтным культурам иммортализованных венозных эндотелиальных клеток человека линии EA.hy 926 с последующим культивированием в течение 24 ч или 4 ч соответственно; 2) конфлюэнтным культурам коммерческих первичных эндотелиальных клеток коронарной и внутренней грудной артерии человека с последующим культивированием в течение 24 ч. Эндотелиотоксические эффекты бионов оценивали при помощи сочетанного окрашивания клеток флюоресцентными красителями Hoechst 33342 и бромистым этидием, а также посредством колориметрического теста. Кроме того, методом проточной цитометрии оценивали пути и стадии гибели клеток вышеуказанных культур. Результаты. В отличие от МФБ, КФБ индуцировали гибель эндотелиальных клеток всех 3 линий путем апоптоза. Устойчивость культур к токсическому действию КФБ определялась степенью их конфлюэнтности (конфлюэнтные культуры более устойчивы чем разреженные) и типом клеточной линии (эндотелиальные клетки внутренней грудной артерии продемонстрировали большую устойчивость в сравнении с эндотелиальными клетками коронарной артерии). Заключение. Токсичность КФБ для культур эндотелиальных клеток специфична, то есть определяется их специфическим минеральным составом, а не общей для всех типов бионов корпускулярной природой. Добавление КФБ к конфлюэнтным культурам первичных артериальных эндотелиальных клеток и к иммортализованным венозным эндотелиальным клеткам вызывало их гибель, при этом экспозиция МФБ не оказывает значимого токсического действия. Эндотелиальные клетки внутренней грудной артерии менее чувствительны к воздействию КФБ в сравнении с эндотелиальными клетками коронарной артерии человека. Aim. To compare toxicity of calcium phosphate bions (CPB) and magnesium phosphate bions (MPB) for endothelial cells. Me-thods. To assess endothelial toxicity of the bions, we first added equal concentrations of either MPB or CPB to: 1) non-confluent or confluent cultures of immortalized human venous endothelial cells EA.hy 926 with the exposure time of 24 h or 4 h, respectively; 2) confluent cultures of commercially available primary human coronary artery and internal thoracic artery endothelial cells, with the exposure time of 24 h. Endothelial toxicity was then evaluated by combined Hoechst 33342 and ethidium bromide staining following fluorescence microscopy and by colorimetric cytotoxicity assay. In addition, we attempted to determine the pathway of bion-induced cell death utilizing flow cytometry. Results. In contrast to the MPB, CPB induced apoptosis of all studied endothelial cell lines. Resistance of endothelial cells to the CPB was defined by their confluence (confluent cultures demonstrated higher resistance), and cell type (internal thoracic artery endothelial cells were more resistant to the CPB as compared to the coronary artery endothelial cells). Conclusions. Endothelial toxicity of the CPB is defined by their specific mineral composition but not by their corpuscular nature, which is common for all nanoparticles. Addition of the CPB to the confluent cultures of primary human arterial cells and to immortalized human venous endothelial cells evoked their death. On the contrary, exposure to the MPB did not cause any toxic effects. Human internal thoracic artery endothelial cells are more resistant to the CPB in comparison with coronary artery endothelial cells.

Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH)

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) β-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) β-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

Formoterol counteracts the inhibitory effect of cigarette smoke on glucocorticoid-induced leucine zipper (GILZ) transactivation in human bronchial smooth muscle cells

Cigarette smokers with asthma and chronic obstructive pulmonary disease (COPD) are less responsive to glucocorticoids (GCs). The anti-inflammatory action of GCs depends also on their ability to transactivate genes such as GC-induced leucine zipper (GILZ). We investigated the effects of aqueous cigarette smoke extract (CSE) on GILZ transactivation evoked by 17-beclomethasone monopropionate (BMP) or fluticasone propionate (FP) in the presence or absence of the long acting β2-adrenoceptor agonist (LABA) bronchodilator formoterol or salmeterol in human primary cultures of human bronchial smooth muscle cells (HBSMC). We monitored GC receptor Ser211 phosphorylation by western blot analysis and GC receptor nuclear translocation by immunostaining followed high-content imaging analysis. BMP, as well as FP, induced GILZ expression in a concentration-dependent manner (EC50 of 0.87 and 0.16 nM respectively). Pre-incubation with CSE inhibited GC-evoked GILZ transactivation (>50%), GC receptor Ser211 phosphorylation and nuclear translocation. Both formoterol and salmeterol counteracted the effect of CSE on GC-induced GILZ expression but not on nuclear translocation or phosphorylation. The effect of formoterol was mimicked by the cAMP-elevating agent forskolin and blocked by ICI 118,551, a selective β2-adrenoceptor antagonist. Pre-incubation with TNF-α also reduced GC-evoked GILZ transactivation but was not counteracted by formoterol undercovering a different responsiveness to LABAs of TNF-α in comparison to CSE.In sum, CSE inhibits GC-evoked transactivation of GILZ and such effect is counteracted by LABAs, through β2-adrenoceptors and a cAMP-dependent mechanism. This study sheds light on a mechanism underlying complementary interactions between LABAs and inhaled GCs that could be relevant in smokers with asthma and COPD.

Effects of high temperature on pandemic and seasonal human influenza viral replication and infection-induced damage in primary human tracheal epithelial cell cultures

High temperature reduces influenza viral replication; however, the treatment of fevers is thought to be necessary to improve patients' conditions. We examined the effects of high temperature on viral replication and infection-induced damage to human tracheal epithelial cells. Cell viability and dome formation were reduced, the number of detached cells was increased and lactate dehydrogenase (LDH) levels tended to be increased from 72 h to 120 h in uninfected cells cultured at 40 °C. Long-term (72 h and/or 120 h) exposure to high temperatures (39 °C and/or 40 °C) decreased RNA levels and/or viral titers of eight influenza virus strains. Cell viability and dome formation were reduced, and the number of detached cells and LDH levels were increased to a similar extent after infection with the A/H1N1 pdm 2009 virus at 37 °C and 40 °C. High temperature increased the endosomal pH, where the viral RNA enters the cytoplasm, in uninfected cells. High temperature reduced the production of IL-6, which mediate viral replication processes, and IL-1β and IL-8 in uninfected and infected cells. Based on these findings, high temperature may cause similar levels of airway cell damage after infection to cells exposed normal temperatures, although high temperature reduces viral replication by affecting the function of acidic endosomes and inhibiting IL-6-mediated processes.

Addition of IL-3 to Stem Cell Factor Dependent Primary Human Mast Cell Cultures Induces a Transient Basophilic Cell Population

Addition of IL-3 to Stem Cell Factor Dependent Primary Human Mast Cell Cultures Induces a Transient Basophilic Cell Population

The FXR agonist obeticholic acid inhibits the cancerogenic potential of human cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive cancer with high resistance to chemotherapeutics. CCA is enriched in cancer stem cells, which correlate with aggressiveness and prognosis. FXR, a member of the metabolic nuclear receptor family, is markedly down-regulated in human CCA. Our aim was to evaluate, in primary cultures of human intrahepatic CCA (iCCA), the effects of the FXR agonist obeticholic acid (OCA), a semisynthetic bile acid derivative, on their cancerogenic potential. Primary human iCCA cell cultures were prepared from surgical specimens of mucinous or mixed iCCA subtypes. Increasing concentrations (0–2.5 μM) of OCA were added to culture media and, after 3–10 days, effects on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC/propidium iodide), cell migration and invasion (wound healing response and Matrigel invasion assay), and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR gene expression was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.05) and in mucinous iCCA vs mixed iCCA cells (p < 0.05) but was upregulated by addition of OCA. OCA significantly (p < 0.05) inhibited proliferation of both mucinous and mixed iCCA cells, starting at a concentration as low as 0.05 μM. Also, CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with its different affinity for FXR. OCA significantly induced apoptosis of both iCCA subtypes and decreased their in vitro cancerogenic potential, as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and Matrigel invasion. In general, these effects were more evident in mixed than mucinous iCCA cells. When tested together with Gemcitabine and Cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics, but mainly in mixed iCCA cells. OCA abolished the capacity of both mucinous and mixed iCCA cells to form colonies when administered together with Gemcitabine and Cisplatin. In subcutaneous xenografts of mixed iCCA cells, OCA alone or combined with Gemcitabine or Cisplatin markedly reduced the tumor size after 5 weeks of treatment by inducing necrosis of tumor mass and inhibiting cell proliferation. In conclusion, FXR is down-regulated in iCCA cells, and its activation by OCA results in anti-cancerogenic effects against mucinous and mixed iCCA cells, both in vitro and in vivo. The effects of OCA predominated in mixed iCCA cells, consistent with the lower aggressiveness and the higher FXR expression in this CCA subtype. These results, showing the FXR-mediated capacity of OCA to inhibit cholangiocarcinogenesis, represent the basis for testing OCA in clinical trials of CCA patients.

Preclinical assessment of MEK1/2 inhibitors for neurofibromatosis type 2-associated schwannomas reveals differences in efficacy and drug resistance development.

Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

Neoplastic Transformation of the Peribiliary Stem Cell Niche in Cholangiocarcinoma Arisen in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory cholangiopathy frequently complicated by cholangiocarcinoma (CCA). Massive proliferation of biliary tree stem/progenitor cells (BTSCs), expansion of peribiliary glands (PBGs), and dysplasia were observed in PSC. The aims of the present study were to evaluate the involvement of PBGs and BTSCs in CCA which emerged in PSC patients. Specimens from normal liver (n = 5), PSC (n = 20), and PSC-associated CCA (n = 20) were included. Samples were processed for histology, immunohistochemistry, and immunofluorescence. In vitro experiments were performed on human BTSCs, human mucinous primary CCA cell cultures, and human cholangiocyte cell lines (H69). Our results indicated that all CCAs emerging in PSC patients were mucin-producing tumors characterized by PBG involvement and a high expression of stem/progenitor cell markers. Ducts with neoplastic lesions showed higher inflammation, wall thickness, and PBG activation compared to nonneoplastic PSC-affected ducts. CCA showed higher microvascular density and higher expression of nuclear factor kappa B, interleukin-6, interleukin-8, transforming growth factor β, and vascular endothelial growth factor-1 compared to nonneoplastic ducts. CCA cells were characterized by a higher expression of epithelial-to-mesenchymal transition (EMT) traits and by the absence of primary cilia compared to bile ducts and PBG cells in controls and patients with PSC. Our in vitro study demonstrated that lipopolysaccharide and oxysterols (PSC-related stressors) induced the expression of EMT traits, the nuclear factor kappa B pathway, autophagy, and the loss of primary cilia in human BTSCs. Conclusion: CCA arising in patients with PSC is characterized by extensive PBG involvement and by activation of the BTSC niche in these patients, the presence of duct lesions at different stages suggests a progressive tumorigenesis.

Heat shock protein 70 is induced by pepsin via MAPK signaling in human nasal epithelial cells.

Recent studies have shown that laryngopharyngeal reflux is associated with chronic rhinosinusitis. Pepsin may be a key factor involved in the injury of nasal mucosal epithelial cells, but the pathogenesis remains unclear. We are to investigate whether a mitogen-activated protein kinase (MAPK) pathway regulates heat shock protein 70 (HSP70) expression in primary cultures of human nasal epithelial cells (HNEpCs) in response to pepsin stimulation. HSP70 protein expression levels in HNEpCs were estimated by Western blot analysis after treatment with pepsin. MAPK pathway activity levels were also evaluated to elucidate the mechanism underlying the effects of pepsin on HSP70 in HNEpCs. Inhibitors of signaling pathways were used to determine the contribution of MAPKs in HSP70 response after pepsin stimulation. Cellular apoptosis and cell viability in HNEpCs after treatment with pepsin were measured. The expression of HSP70 increased after stimulation with pepsin and decreased after the removal of pepsin. Pepsin induced activation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase (JNK) 1/2. Inhibition of JNK1/2 reduced HSP70 expression in HNEpCs. The apoptosis in HNEpCs at 12h after treatment with pepsin at pH 7.0 increased significantly when compared with the control and pH 7.0 groups. Cell viability decreased following exposure to pepsin at pH 7.0. Pepsin, even under neutral pH 7.0, increases the expression of HSP70 in HNEpCs by activating the JNK/MAPK signaling pathway. Increased HSP70 may be the protective mechanism when pepsin presents in the other parts of the body.

Tranexamic acid toxicity in human periarticular tissues.

ObjectivesTranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce perioperative bleeding. Increasingly, topical administration as an intra-articular injection or perioperative wash is being administered during surgery. Adult soft tissues have a poor regenerative capacity and therefore damage to these tissues can be harmful to the patient. This study investigated the effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations.MethodsTendon, synovium, and cartilage obtained from routine orthopaedic surgeries were used for ex vivo and in vitro studies using various concentrations of TXA. The in vitro effect of TXA on primary cultured tenocytes, fibroblast-like synoviocytes, and chondrocytes was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays, fluorescent microscopy, and multi-protein apoptotic arrays for cell death.ResultsThere was a significant (p < 0.01) increase in cell death within all tissue explants treated with 100 mg/ml TXA. MTT assays revealed a significant (p < 0.05) decrease in cell viability in all tissues following treatment with 50 mg/ml or 100 mg/ml of TXA within four hours. There was a significant (p < 0.05) increase in cell apoptosis after one hour of exposure to TXA (100 mg/ml) in all tissues.ConclusionThe current study demonstrates that TXA caused significant periarticular tissue toxicity ex vivo and in vitro at commonly used clinical concentrations.Cite this article: M. McLean, K. McCall, I. D. M. Smith, M. Blyth, S. M. Kitson, L. A. N. Crowe, W. J. Leach, B. P. Rooney, S. J. Spencer, M. Mullen, J. L. Campton, I. B. McInnes, M. Akbar, N. L. Millar. Tranexamic acid toxicity in human periarticular tissues. Bone Joint Res 2019;8:11–18. DOI: 10.1302/2046-3758.81.BJR-2018-0181.R1.

A Study of the Effects of Metformin, a Biguanide Derivative, on Annulus Fibrosus and Nucleus Pulposus Cells.

To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues. Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups’ changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1β (IL-1β) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays. The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrixâ€"like structures. This was statistically significant (p < 0.05). Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.

Triptolide Inhibits Lipopolysaccharide-Induced MUC5AC/5B Expression via Nuclear Factor-Kappa B in Human Airway Epithelial Cells

Background and Objectives The representative mucin genes in the human airway are MUC5AC and MUC5B, which are regulated by several inflammatory and anti-inflammatory substances. Triptolide (TPL), udenafil, betulinic acid, changkil saponin, and glucosteroid are some of the many anti-inflammatory substances that exist. TPL is a diterpenoid compound from the thunder god vine, which is used in traditional Chinese medicine for treatment of immune inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, nephritis and asthma. However, the effects of TPL on mucin expression of human airway epithelial cells have yet to be reported. Hence, this study investigated the effect of TPL on lipopolysaccharide (LPS)-induced MUC5AC and MUC5B expression in human airway epithelial cells. Subjects and Method The NCI-H292 cells and the primary cultures of human nasal epithelial cells were used to investigate the effects of TPL on LPS-induced MUC5AC and MUC5B expression using real-time polymerase chain reaction, enzyme immunoassay, and Western blot. Results TPL significantly decreased the LPS-induced MUC5AC and MUC5B mRNA expression and protein production. TPL also significantly decreased the nuclear factor-kappa B (NF-kB) phosphorylation. Conclusion These results suggest that TPL down regulates MUC5AC and MUC5B expression via inhibition of NF-kB activation in human airway epithelial cells. This study may provide important information about the biological role of triptolide on mucus-secretion in airway inflammatory diseases and the development of novel therapeutic agents for controlling such diseases. Key words: Human airway epithelial cells ㆍ MUC5AC ㆍ MUC5B ㆍ Nuclear factor-kappa B ㆍ Triptolide