TNF-alpha Secretion Research Articles

Abstract 042: Cd74-dysregulation of Macrophage-trophoblastic Interactions in the Preeclamptic Placenta

Objectives: Preeclamptic pregnancies feature placental anomalies. Villous trophoblast differentiation during placental development is regulated by feto-placental macrophages and disturbance of this well-balanced regulation can lead to pathological pregnancies. We hypothesized that Cluster of differentiation 74 (CD74) dysregulation of placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. Methods and Results: We performed microarray analysis of placental tissue. CD74 was one of the most down-regulated (-2.5 fold) genes in placentas from preeclamptic women. We confirmed this finding in early onset (<34 gestational week, n=26) and late onset (≥34 gestational week, n=24) samples from preeclamptic women, compared to healthy pregnant controls (n=28) by real-time RT-PCR and on protein level by Western blot and flow cytometry. We localized CD74 expression in placental macrophages by immunofluorescence, flow cytometry, and real-time RT-PCR. Number and mean fluorescence intensity (MFI) of CD74-positive macrophages were significantly lower in preeclamptic placentas (7692 MFI ± 4402), as compared to controls (16283 MFI ± 3047). In CD74-silenced macrophages, expression of adhesion molecules ALCAM (-2 fold), ICAM4 (-2.1 fold), and Syndecan-2 (-1.9 fold) was lower compared to control. Macrophage adhesion to a trophoblast layer were diminished (-1.3 fold). Naïve and activated macrophages lacking CD74 showed a shift towards a pro-inflammatory signature with an increased secretion of TNFalpha(21.8 pg/ml ± 13.2 vs. 8 pg/ml ± 4.3), CCL5 (1.9 ng/ml ± 0.4 vs. 0.8 ng/ml ± 0.2) and MCP-1 (3 ng/ml ± 2.6 vs. 1 ng/ml ± 0.5), when co-cultured with trophoblasts compared to control macrophages. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone (1.6 mm2 ± 0.3 vs. 2.3 mm2 ± 0.5) and smaller placentas (0.09 g ± 0.01 vs. 0.11 g ± 0.02) with fetal growth restriction (0.7 g ± 0.1 vs. 0.9 g ± 0.2) when compared to WT mice. Conclusions: We found that CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation led to altered macrophage activation towards a pro-inflammatory signature, a disturbed crosstalk with trophoblasts and an abnormal placental morphology.

Quantitative defects in invariant NKT cells and TLR responses in patients with hyper-IgE syndrome

BackgroundAutosomal dominant hyper-IgE syndrome (AD-HIES) is a primary immunodeficiency mainly caused by mutations in STAT3, a signalling molecule implicated in the development of appropriate immune responses. We aimed to characterise the innate immune response in AD-HIES. MethodsThe frequency of innate immune cells in peripheral blood (PB) from seven AD-HIES patients and healthy controls were determined. CD80/CD86 surface expression and cytokine levels in supernatants from PBMC after stimulation with TLR-2, -4 and -9 agonists were also measured by flow cytometry. In addition, several SNPs within these TLR genes in genomic DNA samples from patients and controls were examined. ResultsA significantly reduced number of PB iNKT cells was observed in the AD-HIES group. CpG-stimulated pDC and mDC from patients exhibited a lower increase in the expression of the costimulatory molecule CD80. We also observed an increase in the secretion of IL-12p70, TNF-alpha and IL-10 in PBMC from HIES patients after LTA or LPS stimuli. No association was found between the different SNPs detected and the HIES phenotype. ConclusionsThese findings demonstrate that important mediators of the innate immunity responses are affected in AD-HIES. More studies are necessary to investigate how the STAT3 function interferes with development of iNKT cells and TLR-mediated responses.

Outbreak MDR Strains of M. tuberculosis (Mtb) Induce Differential TNF-alpha Secretion by Macrophages Altering CD54 Expression on Bronchial Epithelial Cells

Outbreak MDR Strains of M. tuberculosis (Mtb) Induce Differential TNF-alpha Secretion by Macrophages Altering CD54 Expression on Bronchial Epithelial Cells

P-199 Lipocalin2 Affecting Bacterial Clearance in Activated Macrophages Contributes the Onset of Intestinal Inflammation

Lipocalin2 (Lcn2) is an extracellular transport protein expressed in neutrophil granules, activated macrophages, and epithelial cells of the respiratory and gastrointestinal tract in response to inflammatory signals. It is well known that Lcn2 exerts bacteriostatic effect by sequestrating iron-containing siderophores because iron is essential for the growth and virulence of most bacterial species. In this regards, Lcn2 is considered to be involved in innate immune response. However, little is known about the exact role of Lcn2 in the intestinal inflammation. The aim of this study is to investigate the role of Lcn2 in the intestinal inflammation using IL-10 knock out (KO) mice. (1) Time-dependent expression of Lcn2 in the colonic tissues of IL-10 KO mice was compared with that of C57BL/6 wild type (WT) mice by quantitative PCR (qPCR) and immunohistochemistry (IHC). (2) We compared intestinal inflammation between IL-10 KO and Lcn2/IL-10 double knock out (DKO) mice. Inflammation was quantified by histologic scores, TNF-alpha and IL-12p40 secretion from cultured colonic explants, and IFN-gamma and IL-17A secretion from unseparated mesenteric lymph node (MLN) cells stimulated with cecal bacterial lysate. (3) Thioglycollate-elicited peritoneal macrophages were obtained from IL-10 KO (IL-10 KO-derived TEPM) and Lcn2/IL-10 DKO mice (DKO-derived TEPM), and infected with Escherichia coli. We examined the bacteria-killing function of macrophages by gentamicin protection assay. Also, TNF-alpha secretion from infected macrophages was determined by enzyme-linked immune-sorbent assay (ELISA). (1) qPCR and IHC demonstrated that Lcn2 expression in the colonic tissues of IL-10 KO mice was apparently up-regulated compared to that of WT mice from early 4 weeks after birth, and further increased over time. (2) Histologic scores in DKO mice were significantly higher than those in IL-10 KO mice at 4 weeks after birth (DKO versus IL-10 KO mice, 15.0 ± 0.0 versus 0.6 ± 0.5). Gene expressions of IL-1beta, TNF-alpha, IFN-gamma, and IL-17A in the colonic tissues of DKO mice were significantly up-regulated compared to those in IL-10 KO mice. TNF-alpha and IL-12p40 secretion from colonic explants of DKO mice were significantly higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, TNF-alpha; 6.5 ± 0.7 versus 3.6 ± 1.2 ng/mL, IL-12p40; 30.6 ± 9.2 versus 5.3 ± 1.9 ng/mL). IFN-gamma and IL-17A production from MLN cells of DKO mice were higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, IFN-gamma; 28.6 ± 33.7 versus 21.3 ± 32.7, P = 0.29, IL-17A; 2.5 ± 3.8 versus 0.6 ± 0.8 ng/mL, P < 0.05). (3) Uptake of bacteria was significantly enhanced in DKO-derived TEPM compared to that in IL-10 KO-derived TEPM (colony forming unit (CFU) at 2 hours after infection; DKO versus IL-10 KO-derived TEPM, 1461.7 ± 201.0 versus 868.3 ± 300.0 cfu/mL). In addition, intracellular survival of phagocytosed bacteria in DKO-derived TEPM persisted compared to that in IL-10 KO-derived TEPM (CFU at 8 hours after infection; DKO versus IL-10 KO-derived TEPM, 30.0 ± 17.9 versus 8.3 ± 13.3 cfu/mL, P < 0.05). Consequently, TNF-alpha production from DKO-derived TEPM significantly increased compared to IL-10 KO-derived TEPM (DKO versus IL-10 KO-derived TEPM, 14.6 ± 1.2 versus 4.0 ± 0.9 pg/mL at 16 hours after infection). Lcn2 affecting bacterial clearance in activated macrophages contributes to the onset of intestinal inflammation.

Monocyte expression of MICA enhances the Natural Killer Cell response to antibody-coated tumor targets

Natural Killer (NK) cells are large granular lymphocytes that are uniquely equipped to promote the anti-tumor response via communication with other immune cell types in the tumor microenvironment. We have shown that NK cells secrete a distinct profile of immune stimulatory factors e.g., interferon-gamma (IFN-γ) with potent anti-tumor activity in response to dual stimulation with antibody (Ab)-coated tumor cells and cytokines, such as interleukin (IL)-12 and that this response is significantly enhanced 10 fold in the presence of autologous monocytes (p < 0.05). Similar results were obtained when NK cell secretion of TNF-alpha (a) and MIP1-a were examined. This enhancement of the NK cell cytokine response by monocytes held true for every known stimulator of NK cell IFN-γ production. Further, autologous monocytes significantly increased the ability of IL-12 activated NK cells to lyse Trastuzumab-coated breast cancer cells in an ADCC assay (p < 0.05). Monocyte enhancement of NK cell activity was shown to be dependent on direct cell-cell contact as determined by a transwell assay. We hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of the stimulatory ligand MICA on monocytes to the NKG2D receptor on NK cells. Activation of monocytes with bacterial components (e.g., LPS) or IFN-a led to increased surface expression of the NKG2D ligand MICA and further enhanced the ability of monocytes to act as stimulators of NK cell cytokine secretion. The stimulatory effects of MICA-positive monocytes were duplicated via use of a MICA over-expressing cell line (C1R-MICA) (inducing a 4 fold increase in IFN-γ production) but not the parental MICA-negative cell line. Down-modulation of MICA via siRNA or incubation with a MICA neutralizing FAb fragment prior to co-culture of monocytes with NK cells led to a significant reduction in NK cell IFN-γ secretion (p < 0.05). Blockade of NKG2D on NK cells with a neutralizing Ab also reduced IFN-γ production in this co-culture system. In vivo, depletion of monocytes from Balb/c mice resulted in decreased IFN-γ production by murine NK cells upon exposure to Trastuzumab-coated tumor cells and IL-12. Extended depletion of monocytes in vivo abrogated the immune response to combination therapy with Trastuzumab and IL-12, resulting in significantly larger tumors in monocyte-depleted mice in comparison to mock-depleted controls (p < 0.02). These data suggest that direct cell-cell interaction between NK cells and monocytes may constitute an important mechanism for enhancement of the NK cell anti-tumor response in the setting of monoclonal Ab therapy for cancer.

Enhanced spontaneous and induced secretion of the proinflammatory cytokine TNF-alpha by monocytes-macrophages from the blood of the patients presenting with type 2 diabetes mellitus

Type 2 diabetes mellitus (DM2) is known to be associated with the accelerated development of atherosclerosis. The current concepts of atherosclerosis take into consideration the possible role of inflammation in its pathogenesis which theoretically implies the modification of macrophages in accordance with the proinflammatory phenotype and the production of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-α) by these cells. In connection with this, we undertook a study of spontaneous and induced secretion of proinflammatory cytokine TNF-α by monocytes-macrophages from the blood of 20 patients presenting with newly diagnosed type 2 diabetes mellitus (HbA1c - 8,9%) and the healthy volunteers showing up no disturbances of carbohydrate metabolism. It was shown that blood monocytes-macrophages of the patients presenting with DM2 are characterized by the enhanced ability (compared with the cells from the healthy subjects) to synthesize TNF-α in both native and interferon-y stimulated states up to 750 versus 270 pg/ml culture medium and to 1653 pg/ml versus 378 pg/ml culture respectively. This difference was statistically significant (p &lt;0.05). It is concluded that the results of the study help to explain the elevated blood TNF-α level in the patients with type 2 diabetes mellitus and provide an insight into the mechanism underlying the development of the systemic inflammatory reaction accelerating the progression of atherosclerosis associated with diabetes mellitus.

Abstract 3195: Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1

Abstract Bryostatin 1 (bryo 1) is a natural compound tested in several clinical trials as an anticancer agent and in preclinical studies for treatment of Alzheimer disease. Despite the clinical interest, the mechanisms responsible for its unique behavior are not fully understood. Bryo 1 is a protein kinase C activator that paradoxically antagonizes many but not all phorbol ester responses. To obtain a broader view of the differences in response to bryo 1 and to the phorbol ester PMA, we have compared their effects on global RNA expression as well as on the levels of important regulatory proteins in two cellular systems (LNCaP and U937 cell lines) where bryo 1 has different biology than PMA. In LNCaP cells, where bryo 1 does not induce secretion of TNF alpha or cause apoptosis, bryo 1 fails to induce translocation of PKC alpha, delta, epsilon and pPKD1 to the nuclear enriched fraction or the phosphorylation of PKCdelta at Y311. For many other early responses detected at 60 min (activation of MEK/ERK pathway and of several transcription factors including NFKB, AP1, EGR1) bryo 1 acts similarly to PMA. In contrast, bryo 1 fails to induce the late responses detected at 6 hrs (phosphorylation of p65, translocation of cRel and RelB) and induces early termination of the induced responses both at the protein and RNA levels as detected for a selected panel of genes. The early termination of the induced responses by bryo 1 can be detected as well in U937 cells, where bryo 1 fails to induce differentiation and apoptosis, unlike PMA. Microarray analysis of LNCaP and U937 cells treated for 6 and 8 hrs, respectively, revealed significant, extensive changes in gene expression compared to control but did not identify genes uniquely responsive to bryo 1. Typically, the effect of bryo 1 was smaller than that of PMA. qPCR analysis of about 3 dozen genes spanning the breadth of the microarray response patterns indicated a single mechanism - genes were similarly activated by PMA and bryo 1 early on but the effect of bryo 1 was transient. Further microarray analysis was performed on LNCaP and U937 samples treated for 60 min and 6 hrs to address the following questions: 1) Are there genes that are induced selectively by bryo 1 at 1 hr? 2) Does transient response to bryo 1, which was only shown for selected genes by qPCR but was inferred to explain the extensive reduced expression at 6 hr in response to bryo 1 compared to PMA, actually explain the reduced expression? While some genes are selectively induced by bryo 1 treatment, the altered levels of expression are limited, suggesting that it does not reflect a unique site of action. Looking at genes showing a reduced level of expression at 6 hr in response to bryo 1 compared to PMA, we confirm that transiency of action is the primary mechanism. These insights provide a guide for evaluation of second generation bryostatin analogs and a focus on the biochemical basis for the transiency of bryo 1 effect. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Peter M. Blumberg. Transient duration of action is the primary mechanism responsible for the unique biology of bryostatin 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3195. doi:10.1158/1538-7445.AM2014-3195

Abstract 5315: Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation

Abstract Bryostatin 1 (bryo 1) is an anticancer agent currently in multiple clinical trials with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. In LNCaP prostate cancer cells the typical phorbol ester PMA induces TNFalpha secretion and apoptosis while bryo 1 fails to do so and antagonizes the PMA effect when used in combination. The mechanisms responsible for the unique effect of bryo 1 are still unknown. We have shown previously, using transcription factor analysis of microarray data from LNCaP cells, that multiple transcription factor families are involved in the dramatic transcriptional response to PMA and bryo 1 (3131 genes for PMA and 1569 genes for bryo 1). Further, analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. To further investigate the altered pathways, the novel Simple Western capillary immunoassay system (ProteinSimple) was used to analyze protein level changes following PKC activation. Simple Western system performs automated western analysis in capillaries using nanograms of lysate and provides digital quantitation with good sensitivity and excellent reproducibility. The system runs at a through-put of 96 sample / target combinations in a single overnight run. We analyzed 35 signals in total lysates (for detection of changes in protein levels and phosphorylation) and in nuclear extracts (for detection of translocation) from LNCaP cells treated for 60 min and 6 hrs. Changes downstream of PKC activation were measured, including PKC/PKD1 phosphorylation, activation of multiple members of the MEK/ERK pathway and levels of transcription factors of the NFKB, AP1, Stat and EGR families. Simple Western results correlated very well with conventional western analysis. The analysis validated the changes described previously using other methods on activation of MEK/ERK pathway and the changes in PKC phosphorylation, translocation and degradation. Bryo1 is different from PMA in failing to induce phosphorylation of PKCdelta at Tyr311 and the translocation of PKCalpha, delta, epsilon, and PKD1 to the nuclear fraction. Bryo1 is similar to PMA in inducing many of the early responses detected at 60 min (pPKCdeltaSer299, pMEK, pERK1/2, pPKD1 in total lysates, decrease in STAT3 levels etc.) but it fails to sustain the induced responses or to induce the late responses detected at 6 hours (pNFκBp65, increase in Fra1 and FosB levels, degradation of cMyc, translocation of cRel and RelB etc.). Using Simple Western system, we developed an approach for precise and accurate measurement of differential PMA and bryo 1 signaling in PKC activation. Further investigation needs to be performed to identify the mechanism/s responsible for the early termination of bryo 1 induced responses. Citation Format: Jin-Qiu Chen, Noemi Kedei, Michelle A. Herrmann, Peter M. Blumberg. Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2014-5315

The anti-inflammatory effect of human amnion-derived mesenchymal stem cells

s / Placenta 35 (2014) A1eA23 A23 allele whose GC-rich promoter is epigenetically regulated. In this study, we compared themethylation status of the upstream CpG-related promoter of C19MC between chorionic villous trophoblast (VT) and extravillous trophoblast (EVT). Methods: First-trimester placental tissues from patients who gave informed consent were aseptically obtained after legal abortions (at 8-10 weeks of gestation, n1⁄45). Chorionic villous tips including VT were defined to be VT; EVT growing from explanted human chorionic villi was isolated. We employed bisulfite genome sequencing for analysis of the methylation status of 31 CpG sites in the C19MC promoter. The rate of methylation change was calculated using the following formula; [(extent of methylation in EVT cells/extent of methylation in VT) -1] X 100. Results: In some patients, the levels of methylation at the 31 CpG sites in EVT were higher than those in VT. However, the other patients did not show remarkable differences in methylation between EVT and VT. The levels of methylation across the 31 CpG sites was greater by approximately 30% (the average rate of methylation change of 5 samples) in EVT compared to VT. Conclusion: These findings suggest that the observed difference in epigenetic profile reflects a disparity in C19MCmiRNA expression between EVT and VT cells. The detailed epigenetic regulatory mechanisms underlying C19MC miRNA expression in trophoblast cells require further investigation. O-108. THE ANTI-INFLAMMATORY EFFECT OF HUMAN AMNION-DERIVED MESENCHYMAL STEM CELLS Reizo Onishi , Shunsuke Ohnishi , Ryosuke Higashi , Kenichi Yamahara , Jun Yoshimatsu , Takehiko Katsurada , Naoto Okubo , Koji Nakagawa , Hiroshi Takeda , Naoya Sakamoto . Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Japan; b Laboratory of Pathophysiology and Therapeutics, Hokkaido University Graduate School of Pharmacy, Japan; Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Japan; Division of Maternal and Fetal Medicine and Gynecology, National Cerebral and Cardiovascular Center, Japan Aims: Several studies have reported that mesenchymal stem cells (MSCs) have the potential to improve damaged tissues by the secretion of antiinflammatory molecules. Recent investigations have demonstrated that amnion-derived MSCs can be easily isolated from human fetal membrane, and a large amount of cells can be obtained. We examined whether human amnion-derivedMSCs (AMSCs) have the anti-inflammatory effects in vitro. Methods: Mouse macrophage RAW264.7 cells were stimulated with lipopolysaccharide (LPS) for 24h under co-incubationwith AMSCs, or were cultured with the conditioned medium (CM) obtained from AMSCs. The expression levels and production of proinflammatory cytokines were measured by quantitative RT-PCR and the enzyme-linked immunosorbent assay (ELISA). HEK293 cells transfected with NF-kB reporter plasmid were treated with TNF-a or LPS for 6h under incubation with CM, and reporter gene assay was performed. RAW264.7 cells were stimulated by LPS after co-incubation for 3h with CM. Nuclear translocation of NF-kB was investigated with immunofluorescent staining. Phosphorylation of IkB was examined by Western blot analysis. Results: Coculture with AMSCs or incubation with CM obtained from AMSCs decreased the expression of proinflammatory cytokines, and CM down-regulated the secretion of TNF-alpha. AMSC coculture inhibited the NF-kB activation induced by TNF-a or LPS. Nuclear translocation of NF-kB by LPS was significantly inhibited by CM. However, Western blotting analysis demonstrated that the phosphorylation of IkB induced by TNF-a or LPS was not inhibited by the conditioned medium. Conclusion: AMSC coculture and incubation with CM downregulated the inflammatory response in cultured macrophages. CM may have suppressed the inflammatory signal of NF-kB at the step of nuclear translocation. O-109. WNT5A DECREASES SFLT1 EXPRESSION IN TROPHOBLAST CELLS Mari Ujita, Eiji Kondoh, Kaoru Kawasaki, Yoshitsugu Chigusa, Hiroshi Takai, Hikaru Kiyokawa, Fumitomo Nishimura, Haruta Mogami, Ikuo Konishi. Department of Obstetrics and Gynecology, Kyoto University, Japan Objective: Wnt signaling is implicated in mice placentation, and human trophoblast migration and invasion. However, the role of Wnt signaling in preeclampsia(PE) has not been clarified yet. We previously reported the expression of Wnt5a, which is involved in non-beta-catenin dependent Wnt pathway, was significantly decreased in PE placenta. The aim of the present study was to elucidate the influence of Wnt5a on vascular growth factor related genes. Methods: HTR-8/SVneo trophoblast cells were cultured with or without recombinant Wnt5a(50ng, 100ng, 200ng/ml)for 24 hours. The mRNA expressions of sFlt-1, PlGF, TGF-b1, TGF-b2 and TGF-b3 were measured by quantitative RT-PCR. Moreover, we inhibited Wnt5a expression of HTR-8/ SVneo trophoblast cells using shRNA vector. Results: sFlt1 was significantly decreased by recombinant Wnt5a in a dose-dependentmanner. The expression levels of PlGF, TGF-b1 and TGF-b3 were significantly increased by Wnt5a in a concentration-dependent manner. TGF-b3 was significantly decreased in HTR-8/SV neo cells via knockout of Wnt5a, while no alteration was observed in TGF-b1 and TGFb2 expression. Conclusion: Wnt5a may be involved in the regulation of vascular growth factor related genes in trophoblast cells.

Intervention effect of quercetin on inflammatory secretion of cardiac fibroblasts

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).

Tea nanoparticles for immunostimulation and chemo-drug delivery in cancer treatment.

Many health benefits have been associated with tea consumption. In an effort to elucidate the source of these health benefits, numerous phytochemicals have been extracted from tea infusions, some of which have demonstrated promise as clinical therapeutics for cancer therapy. Considering the advantageous properties of organic nanoparticles, the purpose of this study is to develop a method for isolating nanoparticles from tea leaves, and explore potential biomedical applications for these nanoparticles. First, an infusion-dialysis procedure for isolating tea nanoparticles (TNPs) from green tea infusions is developed. Second, atomic force microscopy and scanning electron microscopy reveal that the TNPs are spherical with diameters of 100-300 nm. Third, electrophoretic light scattering is used to determine that the TNPs have a zeta potential of -26.52 mV at pH 7.0. Finally, chemical analysis demonstrates that (-) Epigallocatechin gallate, caffeine, and theobromine are not found in the TNPs. Interestingly, the TNPs do enhance the in vitro secretion of cytokines IL-6, TNF-alpha, and G-CSF, as well as the chemokines RANTES, IP-10, MDC from mouse macrophages RAW264.7, indicating an immunostimulatory effect. As a nanocarrier, the TNPs are able to form complexes with doxorubicin (DOX) and have the potential for applications in drug delivery. Further the DOX-loaded TNPs increase the cellular DOX uptake, compared to free DOX, leading to higher cytotoxicity in the A549 human lung cancer and MCF-7 breast cancer cells. More importantly, the DOX-loaded TNPs significantly increase the DOX uptake and cytotoxicity in MCF-7/ADR multidrug resistant breast cancer cells. In this work, an infusion-dialysis procedure is developed for isolation of the TNPs from green tea, and the potential of these nanoparticles as a multifunctional nanocarrier for cancer therapy in vitro is explored.

Exposure to low doses of formaldehyde during pregnancy suppresses the development of allergic lung inflammation in offspring

Formaldehyde (FA) is an environmental and occupational pollutant, and its toxic effects on the immune system have been shown. Nevertheless, no data are available regarding the programming mechanisms after FA exposure and its repercussions for the immune systems of offspring. In this study, our objective was to investigate the effects of low-dose exposure of FA on pregnant rats and its repercussion for the development of allergic lung inflammation in offspring.Pregnant Wistar rats were assigned in 3 groups: P (rats exposed to FA (0.75ppm, 1h/day, 5days/week, for 21days)), C (rats exposed to vehicle of FA (distillated water)) and B (rats non-manipulated). After 30days of age, the offspring was sensitised with ovalbumin (OVA)-alum and challenged with aerosolized OVA (1%, 15min, 3days). After 24h the OVA challenge the parameters were evaluated.Our data showed that low-dose exposure to FA during pregnancy induced low birth weight and suppressed the development of allergic lung inflammation and tracheal hyperresponsiveness in offspring by mechanisms mediated by reduced anaphylactic antibodies synthesis, IL-6 and TNF-alpha secretion. Elevated levels of IL-10 were found. Any systemic alteration was detected in the exposed pregnant rats, although oxidative stress in the uterine environment was evident at the moment of the delivery based on elevated COX-1 expression and reduced cNOS and SOD-2 in the uterus. Therefore, we show the putative programming mechanisms induced by FA on the immune system for the first time and the mechanisms involved may be related to oxidative stress in the foetal microenvironment.

Proliferative and secretory activity of human umbilical endothelial cells cultivated under various hypoxia conditions

In this study, we examined the impact of 3-day hypoxia of different degrees on the viability, proliferation, and secretory activity of endothelial cells from human umbilical vein (HUVEC). A gas mixture of three components was used (%): 1) 10 O2, 5 CO2, and 85 Ar; 2) 5 O2, 5 CO2, and 80 Ar; and 3) 1 O2, 5 CO2, and 94 Ar. Cells cultivated in a CO2 incubator in atmospheric oxygen (21% O2) served as control. It was found that 3-day HUVEC cultivation at 1% O2 increased NO synthesis; enhanced secretion of endothelin-1, IL-6, IL-8, TNF-alpha, sVCAM-1, sE-cadherin, sE-selectin, VEGF-A, and bFGF; and inhibited proliferation. HUVEC cultivated under 10% O2 and 5% O2 exhibited the lowest level of basal secretion of these substances and increased proliferative activity. These cells cultivated under conditions of atmospheric oxygen for 3 days displayed activated secretion of NO, IL-6, IL-8, and von Willebrand factor; the activation was higher than at 10% O2 and 5% CO2. Thus, the gaseous medium with reduced oxygen content (5%) is a more physiological condition for HUVEC cultivation. An increase in the amount of oxygen up to the atmospheric level causes endotheliocyte activation; the cells exhibit the features of endothelial dysfunction. Oxygen content reduced to 1% induces endothelial dysfunction and reduced proliferative potential.

The production of nitric oxide, IL-6, and TNF-alpha in palmitate-stimulated PBMNCs is enhanced through hyperglycemia in diabetes.

We examined nitric oxide (NO), IL-6, and TNF-α secretion from cultured palmitate-stimulated PBMNCs or in the plasma from type 2 diabetes mellitus (T2MD) patients or nondiabetic (ND) controls. Free fatty acids (FFA) have been suggested to induce chronic low-grade inflammation, activate the innate immune system, and cause deleterious effects on vascular cells and other tissues through inflammatory processes. The levels of NO, IL-6, TNF-α, and MDA were higher in supernatant of palmitate stimulated blood cells (PBMNC) or from plasma from patients. The results obtained in the present study demonstrated that hyperglycemia in diabetes exacerbates in vitro inflammatory responses in PBMNCs stimulated with high levels of SFA (palmitate). These results suggest that hyperglycemia primes PBMNCs for NO, IL-6, and TNF-alpha secretion under in vitro FFA stimulation are associated with the secretion of inflammatory biomarkers in diabetes. A combined therapy targeting signaling pathways activated by hyperglycemia in conjunction with simultaneous control of hyperglycemia and hypertriglyceridemia would be suggested for controlling the progress of diabetic complications.

The Histone Deacetylase Inhibitor LBH589 (Panobinostat) Modulates the Crosstalk of Lymphocytes with Hodgkin Lymphoma Cell Lines

Epigenetic changes have been implicated in the malignant phenotype of Hodgkin Reed Sternberg (HRS) cells in Hodgkin lymphoma (HL), where HRS survival and proliferation depends on the microenvironment. The histone-deacetylase (HDAC) inhibitor LBH589 (panobinostat) showed clinical efficacy but its impact on the HRS microenvironment is unclear. Hence, we analysed the effects of LBH589 on lymphocytes and also potential combination therapies. In lymphocyte-target cell killing assays, LBH589-treatment triggered an enhanced lymphocyte-dependent lysis of HL cells despite of mild lymphocytopenic effects. In co-culture experiments of lymphocytes with HL cells, LBH589 suppressed the IFNgamma-release but increased the TNFalpha secretion. Recombinant TNFalpha boosted the lymphocyte-dependent lysis of HL target cells. In HL cell lines, LBH589 induced cell death, autophagy, and an increase of MICA/B that are ligands to natural killer cell receptors. The combination of LBH589 with Brentuximab Vedotin was inefficient due to down-regulation of CD30 as a target. Combination with gemcitabine revealed highly significant effects, suggesting a potential combination for future therapy. Based on these data we suggest that LBH589 favourably modulates the cytokine network and lymphocyte activity in the HL microenvironment.

Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Abstract Intensified treatment of pediatric acute lymphoblastic leukemia (ALL) has lead to increased survival rates of about 80%, however therapy fails in the remaining patients leading to relapse of the disease associated with inferior prognosis. Because treatment failure is, at least in part, due to defects in apoptosis programs, novel therapeutic strategies that counter apoptosis resistance are needed. “Inhibitor of Apoptosis” (IAP) proteins block the apoptosis machinery at a central point and are highly expressed in acute leukemias, thereby providing a target structure for therapeutic intervention. Molecules antagonizing these apoptosis inhibitors, so called SMAC-mimetics, therefore provide a promising strategy to overcome apoptosis deficiency and effectively treat high risk ALL. In this study, we investigated the effects of the small molecule SMAC-mimetic BV6 (kindly provided by Genentech) in B cell precursor- (BCP-) ALL. BV6 showed a clear induction of cell death at nanomolar concentrations in ALL cell lines. ALL cells sensitive for SMAC-mimetic induced cell death showed rapid cIAP degradation, NFkB activation and secretion of TNF-alpha (TNF-a). Interestingly, mitochondrial perturbation and caspase activation could be inhibited by the soluble TNF-a receptor Etanercept indicating the induction of a TNF-a dependent feed forward loop by the SMAC-mimetic BV6. In addition to cell lines, we investigated the effects of BV6 on a series of 42 primary ALL samples isolated from ALL bearing mice of established patient derived NOD/SCID/huALL xenograft leukemias. Intriguingly, upon treatment with the small molecule SMAC mimetic BV6, induction of cell death was observed in a majority of 70% of all individual patient-derived leukemias and BV6 induced cell death was inhibited by Etanercept demonstrating TNF-a dependency also in primary ALL. We previously described that rapid engraftment of ALL cells transplanted onto NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in the ALL cells and indicative for early patient relapse. Importantly, primary xenograft ALL samples with a TTLshort/early relapse phenotype showed increased cell death upon treatment with SMAC-mimetic BV6 and activation of the constitutive deficient apoptosis signaling pathway, demonstrating that SMAC-mimetics induce intact apoptosis signaling in former apoptosis resistant primary ALL cells. Based on theses findings, we further evaluated the in vivo effectivity of the SMAC-mimetic BV6 on high risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. ALL bearing recipients were treated with either BV6 or solvent for a given time of two weeks and further investigated for the presence of leukemia. Most interestingly, a significant delay of post-treatment leukemia reoccurrence was observed upon BV6 in vivo treatment along with a profound reduction of tumor load in the recipients compared to solvent treated animals. In a clinical setting, high-risk disease is unlikely to be treated by one compound alone. Therefore, we combined BV6 with multidrug chemotherapy resembling ALL induction treatment and observed a significant delay of ALL reoccurrence and prolonged survival of animals treated with the combination of the SMAC-mimetic and chemotherapy in contrast to chemotherapy alone. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death sensitizing effect of BV6, both in single treatment and in combination with chemotherapy. This indicates that BV6 induced apoptosis sensitization involves signaling via TNF-a and thereby provides a potential biomarker for the identification of patients who would benefit from SMAC-mimetic treatment. Taken together, we show that the small molecule SMAC-mimetic BV6 induces cell death via a TNF-a loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL. Thus, induction of cell death by new generation small molecule SMAC-mimetics provides a promising novel strategy for targeted therapy of high-risk acute lymphoblastic leukemia and involvement of TNF-a signaling in BV6-sensitive patients points to its potential use as biomarker indicating effective cell death sensitization. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma

BackgroundGliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function.Methods and ResultsTo better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs.ConclusionsRestoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs.

Hepatic SREBP-2 and cholesterol biosynthesis are regulated by FoxO3 and Sirt6

Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.

ERK pathway is activated in bare-FeNPs-induced autophagy

Iron oxide nanoparticles (FeNPs) are known to be one of the most biocompatible and safe nanoparticles. However, their long-term persistence remains a problem, and macrophages play as an important mediator in continuous stimulation of the immune system due to biopersistence of nanoparticles. In the present study, we identified the mechanisms underlying the uptake and toxicity of bare-FeNPs using RAW264.7 cells, a mouse peritoneal macrophage cell line. The bare-FeNPs penetrated the cell membrane through electrostatic interactions together with the general phagocytic pathway. At 24 h after exposure, they distributed freely in the cytosol or within autophagosome-like vacuoles. Bare-FeNPs induced decrease in the cell viability along with the cell cycle arrest in G1 phase. In addition, they increased the generation of ROS and the secretion of NO and TNF alpha as well as the expression of SOD-1 and SOD-2 proteins, which are an antioxidant. While the mitochondrial calcium level, the intensity of labeled mitochondria, and ATP production decreased, the levels of autophagy-related proteins such as p62, beclin 1, ATG5, and LC3B increased in a dose-dependent manner together with the levels of ATF 3, p-EGFR, and p-ERK proteins. However, the level of p-JNK protein clearly decreased. TEM images also showed that damaged organelle exist within autophagosome-like vacuoles with bare-FeNPs. On the basis of these results, we suggest that bare-FeNPs induce autophagy by initiating oxidative stress in RAW264.7 cells. Furthermore, ERK, but not JNK, pathway is activated in bare-FeNPs-induced autophagy.

The Influence of Toll-like Receptor (TLR-) Agonists on Lysozyme Activity and TNFα-Secretion in THP-1 cells

TLRs (Toll-like receptors) are part of the innate immune system and play a major role in the recognition of pathogens. They participate in the stimulation of monocytes and macrophages by activation of NF-κB and C/EBPβ, two transcription factors that are implicated in lysozyme and TNF-alpha expression. Aim of this study was to investigate the influence of TLR-agonists and other natural compounds on cytokine secretion and lysozyme activity in a comparative assay. To explore the importance of differentiation THP-1 cells were pre-treated with PMA and IFNy. Stimulation of undifferentiated cells with natural products including various bacterial antigens, diterpenes and alkamides had almost no effect on TNF-alpha secretion and lysozyme activity whereas secretion of IL-6 and IL-10 as well as intercellular adhesion was clearly affected. Differentiated THP-1 cells were much more sensitive to further stimulation yet revealed an opposing effect on lysozyme activity and TNF-alpha secretion. The results suggest a coordinated activation of intercellular adhesion and cytokine production in the differentiation process from monocytes to macrophages and highlights the role of natural products as putative immunmodulatory agents.