Constitutive Release Research Articles

Stromal Endothelial Cells Establish a Bidirectional Crosstalk with Chronic Lymphocytic Leukemia Cells through the TNF-Related Factors BAFF, APRIL, and CD40L

Chronic lymphocytic leukemia (CLL) is a clonal B cell disorder of unknown origin. Accessory signals from the microenvironment are critical for the survival, expansion, and progression of malignant B cells. We found that the CLL stroma included microvascular endothelial cells (MVECs) expressing BAFF and APRIL, two TNF family members related to the T cell-associated B cell-stimulating molecule CD40L. Constitutive release of soluble BAFF and APRIL increased upon engagement of CD40 on MVECs by CD40L aberrantly expressed on CLL cells. In addition to enhancing MVEC expression of CD40, leukemic CD40L induced cleavases that elicited intracellular processing of pro-BAFF and pro-APRIL proteins in MVECs. The resulting soluble BAFF and APRIL proteins delivered survival, activation, Ig gene remodeling, and differentiation signals by stimulating CLL cells through TACI, BAFF-R, and BCMA receptors. BAFF and APRIL further amplified CLL cell survival by upregulating the expression of leukemic CD40L. Inhibition of TACI, BCMA, and BAFF-R expression on CLL cells; abrogation of CD40 expression in MVECs; or suppression of BAFF and APRIL cleavases in MVECs reduced the survival and diversification of malignant B cells. These data indicate that BAFF, APRIL, and CD40L form a CLL-enhancing bidirectional signaling network linking neoplastic B cells with the microvascular stroma.

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

BackgroundATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices.ResultsUnder control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone.ConclusionsThe constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.

Enhanced renin release and content in juxtaglomerular (JG) cells from VAMP8 knockout mice

Renin is essential for angiotensin II production and blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells. We showed that vesicle associated membrane protein 2 (VAMP2), a member of SNARE family, mediates stimulated renin exocytosis in JG cells. However, the VAMP isoform required for constitutive renin release is unknown. In other cells VAMP8 mediates granule maturation and constitutive exocytosis. We hypothesized that VAMP8 mediates constitutive renin release in JG cells. We used primary cultures of JG cells from wild type (WT) and VAMP8 knockout (V8‐KO) mice and measured VAMP8 localization and renin released to the medium during 1 hr. In wild type, VAMP8 was expressed in JG cell granules. Baseline renin release was 5‐fold higher in V8‐KO than WT (WT=0.06±0.01; KO=0.3±0.05 μgAngI/hr/mg prot.; p<0.05). Total renin content was 5‐fold higher in V8‐KO than WT (WT=2.9±0.2; KO=16.7±0.6 μgAngI/hr/mg prot.; p<0.05). The fraction of total renin released to the media was similar in V8‐KO and WT JG cells (WT: 1.6±0.3; KO: 1.7±0.5% of total renin) indicating higher number of granules in V8‐KO, or higher renin content/granule. Immunolabeling of renin and confocal imaging showed enlarged granules and vacuoles in JG cells from V8‐KO. We concluded that: 1) VAMP8 does not mediate constitutive renin exocytosis; and 2) VAMP8 may play an inhibitory role in transcription, translation or processing of renin. 5F32HL096346

Multidrug resistant proteins mediate extracellular ATP release in Caco2 cell monolayers

Epithelial ATP release is an essential component of extracellular purinergic secretory regulation; ATP transporting channels, pannexin (Panx) hemichannels and multidrug resistant proteins (MDR/MRP) have been implicated. The robust intestinal ecto‐ATPase activity present in Caco‐2 cells impairs measurements of medium ATP. We thus transfected c‐Myc‐tagged GPI‐anchored plasma membrane luciferase (pmeLuc) to Caco2 cell monolayer to monitor ATP release at the cell surface. Confluent Caco‐2 cell monolayers were transfected with pmeLuc, confirmed by immunolocalization. Although transfection efficacy into Caco2 cell is ~ 10 %, much less than in HEK293 ~ 80 %, luminescence was measurable over the background (~ 3000 in Caco‐2, ~ 30,000 in HEK293). Luminescence was stable over 40 min, suggesting constitutive ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific MDR/MRP inhibitor dose‐dependently inhibited ATP release. Stilbene anion transport inhibitors or Panx inhibitors had no effect. Vesicles expressing P‐glycoprotein (Mdr1) and MRP4, but not MRP2 accumulated ATP inside. MDR/MRP mediates ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport. Regulation of ATP release may have important implications for the physiological functioning of intestinal and other secretory epithelia. NIH‐NIDDK R01 DK54221 & R21 DK081199

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection.

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.

Disease‐stabilizing treatment with all‐trans retinoic acid and valproic acid in acute myeloid leukemia: Serum hsp70 and hsp90 levels and serum cytokine profiles are determined by the disease, patient age, and anti‐leukemic treatment

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study, we have investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile when compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA + valproic acid + theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra), and hepatocyte growth factor (HGF) levels. Furthermore, disease-stabilizing therapy altered the serum-cytokine profile, but the correlations between HSP70/HSP90/IL-1ra/HGF were maintained only when ATRA + valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML.

CAR T cells transform to trucks: chimeric antigen receptor-redirected T cells engineered to deliver inducible IL-12 modulate the tumour stroma to combat cancer.

Adoptive T cell therapy recently achieved impressive efficacy in early-phase clinical trials; this significantly raises the profile of immunotherapy in the fight against cancer. A broad variety of tumour cells can specifically be targeted by patients' T cells, which are redirected in an antibody-defined, major histocompatibility complex-unrestricted fashion by endowing them with a chimeric antigen receptor (CAR). Despite promising results for some haematologic malignancies, the stroma of large, established tumours, the broad plethora of infiltrating repressor cells, and cancer cell variants that had lost the target antigen limit their therapeutic efficacy in the long term. This article reviews a newly described strategy for overcoming some of these shortcomings by engineering CAR T cells with inducible or constitutive release of IL-12. Once redirected, these T cells are activated, and released IL-12 accumulates in the tumour lesion where it promotes tumour destruction by at least two mechanisms: (1) induction of an innate immune cell response towards those cancer cells which are invisible to redirected T cells and (2) triggering programmatic changes in immune-suppressive cells. Given the enormous complexity of both tumour progression and immune attack, the upcoming strategies using CAR-redirected T cells for local delivery of immune-modulating payloads exhibited remarkable efficacy in pre-clinical models, suggesting their evaluation in clinical trials.

Activation of Peroxisome Proliferator-Activated Receptor-Gamma by Glitazones Reduces the Expression and Release of Monocyte Chemoattractant Protein-1 in Human Mesothelial Cells

Human peritoneal mesothelial cells (MC) play an important role in inflammatory processes of the peritoneal cavity by producing various cytokines and chemokines, such as monocyte chemoattractant protein-1 (MCP-1). The present study was designed to assess the effect of the peroxisome proliferator-activated receptor-gamma- (PPARγ-) activator rosiglitazone on the mesothelial MCP-1 expression and release. Primary cultures of MC were obtained from omental tissue. MCP-1 antigen concentrations were measured in the cell supernatant by ELISA and MCP-1 mRNA levels by real-time polymerase chain reaction. The presence of PPARγ on MC was assayed in a Western Blot analysis. MC constitutively express PPARγ. Activation of this receptor via rosiglitazone (0,1–10 μmol/L) resulted in significantly reduced amounts of mesothelial MCP-1 release as well as MCP-1 mRNA. The use of the PPARγ inhibitor GW-9662 could completely prevent the rosiglitazone effects. Rosiglitazone was also effective in reducing TNFα-induced enhanced secretion of MCP-1. Our findings indicate that glitazones are effective in reducing constitutive and TNFα-stimulated mesothelial MCP-1 mRNA expression and release.

Expression profile of heat shock proteins in acute myeloid leukaemia patients reveals a distinct signature strongly associated with FLT3 mutation status – consequences and potentials for pharmacological intervention

Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSPs are regarded as possible therapeutic targets in acute myeloid leukaemia (AML). We used bioinformatical approaches to characterize the HSP profile in AML cells from 75 consecutive patients, in addition to the effect of the HSP90 inhibitor 17-DMAG. Patients harbouring a FLT3-internal tandem duplication (FLT3-ITD) were extensively overrepresented in the cluster with high HSP levels, indicating a strong dependence of HSPs in stabilizing FLT3-ITD encoded oncoproteins. FLT3 ligation further increased the levels of HSP90 and its co-chaperone HSP70. HSP90 inhibition had a stronger pro-apoptotic effect for AML cells with FLT3-ITD than for cells with wild-type FLT3, whereas the anti-proliferative effect of HSP90 inhibition was similar for the two patient subsets. HSP90 inhibition altered the constitutive cytokine release profile in an anti-angiogenic direction independent of FLT3 mutational status: (i) pro-angiogenic CXCL8, MMP-2 and MMP-9 showed a stronger decrease than anti-angiogenic CXCL9-11, (ii) the Tie-2 agonist Ang-1 showed a stronger decrease than the potentially antagonistic Ang-2, and (iii) VEGF and HGF levels were decreased. Finally, HSP90 inhibition counteracted the leukaemia-stimulating effect of endothelial cells. Our studies demonstrate that HSP90 inhibition mediates anti-leukaemic effects through both direct and indirect activity.

Serum Heat Shock Protein Levels and Cytokine Profiles Are Altered In AML Patients Before Treatment and Following Disease Stabilizing Therapy,

Abstract 3599Heat shock proteins (HSPs) maintain cellular homeostasis and function as molecular chaperones assisting protein folding and translocation. Their intracellular levels are up-regulated in response to a wide variety of insults allowing the cell to survive otherwise lethal conditions. Heat shock proteins are also released to the extracellular space and lead to a more efficient cross-presentation to specific T cells and may also initiate release of proinflammatory cytokines, stimulate NK cells and facilitate DC maturation. Extracellular Heat shock protein (HSP) 70 and HSP90 are characteristics of immunogenic apoptosis, and primary human acute myeloid leukemia (AML) cells release these mediators during spontaneous in vitro apoptosis. Primary AML cells also show constitutive release of several cytokines; these heterogeneous mediators interact through local cytokine networks that regulate growth and differentiation of normal as well as malignant hematopoietic cells. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving disease-stabilizing treatment based on all-trans retinoic acid (ATRA) plus valproic acid.Serum samples from 82 untreated AML patients, including 42 patients receiving AML-stabilizing therapy, and 20 healthy controls were analysed. HSP and cytokine levels were determined by ELISA and multiplex analyses.Patients with untreated AML showed significantly increased HSP90 levels compared to healthy controls (Mann Whitney U-test, p<0.0001). The serum levels of HSP70 were significantly lower than the HSP90 levels both for the patients and for the healthy controls (p<0.0001), but the HSP70 serum levels did not differ significantly between patients and controls (Fig. 1A). HSP70 and HSP90 serum levels were significantly correlated for the AML patients (Fig. 1B, Spearman, p<0.01, R=0.425). HSP70 levels were not altered during AML-stabilizing palliative treatment, whereas HSP90 levels decreased during treatment with ATRA, valproic acid and low-dose cytarabine. High age and low differentiation of AML cells according to the FAB classification predicted lower HSP90 levels. [Display omitted] AML patients showed an altered serum cytokine profile compared to healthy controls with significantly altered levels of several chemokines, interleukins, growth factors and immunomodulatory cytokines (Tab.1). These cytokine levels were usually increased compared to the healthy controls. Hierarchical cluster analysis of the untreated patients showed a close association between HSP70, HSP90 and Hepatocyte growth factor (HGF) levels, these mediators also showed a significant correlation (Spearman, p<0.01). Furthermore, disease-stabilizing therapy altered the serum cytokine profile, but the profiles differed when ATRA + valproic acid was combined with either theophyllin or low-dose cytarabine. The close association between HSP70, HSP90 and HGF levels was then maintained only in the cytarabine group.Tab.1CytokinePatientsControlsP-valueIMMUNOMODULATORY CYTOKINESTNF-α↑8.4 (<1.5–68.5)4.5 (2.3–13.4)0.016INTERLEUKINSIL-1α↓0.5 (<0.4–7.0)2.0 (2.0–2.9)<0.0001IL-4↓<4.5 (<4.5–53.9)11.8 (5–17.5)<0.0001IL-5↑<0.7 (<0.7–39.1)0.9 (<0.7–0.9)0.0001IL-6↑5.8 (<1.1–204.6)<1.1 (<1.1–7.7)<0.0001IL-10↑1.3 (<0.3–238.0)0.3 (<0.3–0.8)0.0005CHEMOKINESCXCL5↓128.4 (<4.1–3252)1283.5 (341–5307)<0.0001CXCL8/IL-8↑30.5 (<2.0–1103)10.7 (2.2–22.8)0.0003CXCL10↑56.7 (0.5–1958)15.25 (8.5–58.4)<0.0001GROWTH FACTORSHGF↑797.3 (1–9703)262.4 (117.3–729.3)<0.0001GM-CSF↑0.5 (<2.0–17.2)0.9 (0.9–3.6)0.0096We conclude that both HSP levels and serum cytokine profiles are altered in untreated AML, and the modulation of these serum levels by AML-stabilizing chemotherapy may reflect a possible mechanism for the clinical effect of this treatment strategy.The table shows serum levels for 82 AML patients and 20 healthy controls where significant differences were found (Mann-Whitney U-test, p<0.02). The results are presented as the cytokine analyzed, the alteration in the patients (↑ increased, ↓ decreased), levels in patients and healthy controls (median and range). All cytokine concentrations are presented as pg/ml, for cytokine levels not detected the values are set to < the detection limit. Disclosures:No relevant conflicts of interest to declare.

Future perspectives: should Th17 cells be considered as a possible therapeutic target in acute myeloid leukemia patients receiving allogeneic stem cell transplantation?

Th17 cells seem to promote proinflammatory effects, and their development seems to depend on intracellular signaling initiated by IL1β, supported by IL6 and IL23 and mediated by STAT3 and RORC2. Even though primary human AML cells may affect Th17 development through their constitutive cytokine release, the levels of circulating Th17 cells in older patients with untreated AML do not differ from healthy controls and show only minor variations during and following conventional intensive chemotherapy. IL17-A is the signature cytokine of Th17 cells, but in vitro studies have failed to demonstrate a direct antileukemic effect of IL17 on primary human AML cells for most patient samples. However, several observations suggest that Th17 cells mediate antileukemic effects through other mechanisms and are important in allogeneic stem cell transplantation. Firstly, genetic variants in IL23/Th17 pathway have a prognostic impact with regard to both development of GVHD and posttransplant infections. Secondly, circulating IL17-secreting cells are detected during early posttransplant pancytopenia, and their ability to release IL17 is associated with later GVHD. Thirdly, a high number of Th17 cells in allogeneic stem cell grafts are associated with later acute GVHD, levels of circulating Th17 cells are increased at the onset of acute GVHD, and these levels normalize during treatment. In the present article, we review previous studies of Th17 cells in AML and in the development of GVHD, possible therapeutic strategies and available therapeutic tools for targeting of Th17 cells.

EGCG downregulates IL-1RI expression and suppresses IL-1-induced tumorigenic factors in human pancreatic adenocarcinoma cells

Human pancreatic cancer is currently one of the fifth-leading causes of cancer-related mortality with a 5-year survival rate of less than 5%. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative therapeutic strategies. Increasing evidence suggests an association of carcinogenesis and chronic inflammation. Because IL-1 plays a crucial role in inflammation-associated carcinogenesis, we analyzed the biological effects of IL-1 and its modulation by the chemopreventive green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) in the human pancreatic adenocarcinoma cell line Colo357. Proinflammatory IL-6 and PGHS-2 as well as proangiogenic IL-8 and VEGF were induced by IL-1, whereas the secretion of invasion-promoting MMP-2 remained unaffected. IL-1 responsiveness and constitutive MMP-2 release in Colo357 were downregulated by EGCG in a dose- and time-dependent manner. Moreover, EGCG reduced cell viability via induction of apoptosis in Colo357. Since EGCG effects on cytokine production precede reduction in cell viability, we hypothesize that these findings are not only a result of cell death but also depend on alterations in the IL-1 signaling cascade. In this context, we found for the first time an EGCG-induced downregulation of the IL-1RI expression possibly being caused by NF-κB inhibition and causative for its inhibitory action on the production of tumorigenic factors. Thus, our data might have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in high-risk patients with human pancreatic carcinoma.

Cell coupling regulates Ins1, Pdx-1 and MafA to promote insulin secretion in mouse pancreatic beta cells

The intracellular contact between pancreatic β-cells plays an important role in functional insulin secretion. We propose cell coupling may regulate glucose-stimulated insulin gene expression. MIN-6, a murine β-cell line, was cultured on the Lipidure-coat dish for cell clusters formation. The responses to glucose-stimulation between monolayers and clusters were compared. The coupling of β-cells was evaluated by immunohistochemical (IHC) staining. After the establishment of cell coupling was confirmed, the transcription factors of insulin gene expression were assessed by RT-PCR. Results showed that in addition to secrete insulin in a glucose-regulated manner, MIN-6 clusters had biphasic insulin secretion. Relative to monolayer, clusters have higher stimulation-index and lower constitutive insulin release. IHC staining showed the connexin 36 highly expressed in clusters. Compared with monolayers, the mRNA expression of insulin 1 (Ins1) and homeodomain transcription factor pancreatic/duodenal homebox-1 (Pdx-1) of clusters were downregulated in low glucose level. On the contrary, these gene expressions were upregulated when clusters cultured in high glucose environment. The V-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA) was relative highly expressed in clusters under high glucose condition. This study reveals that cell coupling regulates glucose-stimulated insulin gene expression to promote insulin secretion in mouse β-cells.

Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells

Mesenchymal stromal cells (MSC) derived from Wharton's jelly (WJ) of the umbilical cord are increasingly gaining prominence as substitutes for bone marrow (BM) MSC. While MSC isolated from different tissue sources may share common mesenchymal properties, the difference in their plasticity to individual lineages is ill-defined. Thus the focus of this study was to estimate the neuronal plasticity of WJ MSC to the dopaminergic (DA) cell type in comparison with BM MSC. For neuronal differentiation, MSC were exposed to developmentally relevant cues for midbrain DA neurons: sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8), along with basic fibroblast growth factor (bFGF). Naive MSC from both sources constitutively expressed neuronal markers. Flow cytometry data revealed that the control WJ MSC shared a signature similar to BM MSC for early neuronal markers (nestin, musashi12 and A2B5) and DA-specific markers [tyrosine hydroxylase (TH) and Nuclear Receptor related protein 1 (Nurr1) but differed for mature neuronal proteins [β-tubulin III and microtubule-associated protein 2 (Map2ab)]. Similar populations of cells in both sources of MSC were positive for the SHH receptors [patched (PTCH) and smoothened (SMO)]. In induced BM and WJ MSC, real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis showed similar levels of DA-related transcription factors Nurr1 and Engrailed (En) 1. Immunocytochemical and flow cytometry analysis showed an increase in mature neuronal marker Map2ab. Kv4.2, a K(+) channel marker, was observed only in the induced MSC. Induced MSC also expressed several DA-specific markers, TH, dopamine and cyclic AMP regulated phosphoprotein (DARPP) 32, paired-like homeodomain transcription factor (PitX) 3 and vesicular monoamine transporter (VMAT) 2, in comparable levels between the two sources. The efficiency (c. 65%) of transdifferentiation of WJ MSC to TH-positive cells was similar to that of induced BM MSC. Constitutive and inducible release of dopamine was found to be similar between induced BM and WJ MSC, as measured by dopamine enzyme-linked immunosorbent assay (ELISA). Interestingly, an adenosine triphosphate (ATP)-stimulated change in intracellular Ca(2+) was observed in both control and induced MSC, but only the induced MSC was capable of releasing dopamine. Our data demonstrate that MSC from the two different sources respond similarly to inductive cues to differentiate terminally to a DA cell type, and the neuronal plasticity of human WJ MSC is comparable with that of BM MSC.

Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release

Purkinje cell protein 4 (PCP4), also called brain-specific polypeptide 19 (PEP19), is a neurospecific, small calmodulin-binding protein that binds both calcium-free and calcium-binding calmodulin to regulate the calmodulin-mediated signal. The expression level of this molecule is decreased in the brain in Alzheimer's disease, Huntington's disease, and alcoholism. However, little is known of the function of PCP4 regarding neuronal or neuroendocrine cell differentiation and neurotransmitter release. To address this, we established a PCP4 tetracycline-inducible rat chromaffin cell line, PC12. When PCP4 expression was induced with doxcycline, neurite outgrowth was significantly advanced in the presence of nerve growth factor (NGF) and dibutyryl cAMP, which was inhibited by W-7, a calmodulin inhibitor, and PD98059, an ERK inhibitor. In addition, size of the cell body also was increased by treatment with NGF in the PCP4-induced PC12 cells. Constitutive and potassium-evoked release of acetylcholine and dopamine was increased and apoptosis induced by hydrogen peroxide (H(2)O(2)) was inhibited in PCP4-induced PC12 cells. On the other hand, knockdown of PCP4 by siRNA transfection decreased neurite outgrowth and dopamine release and increased H(2)O(2)-induced apoptosis in PC12 cells. These results indicate that PCP4 promotes neuroendocrine cell differentiation and neurotransmitter release by activating calmodulin function.

MHV-68 producing mIFNα1 is severely attenuated in vivo and effectively protects mice against challenge with wt MHV-68

Human gammaherpesviruses such as Epstein–Barr virus (EBV) cause lifelong infections and associated diseases, by virtue of their ability to establish latent infection. Many studies performed in the past years in murine herpesvirus 68 (MHV-68) model of infection suggested that the limited immunity generated against isolated viral components by subunit vaccines cannot counteract the multiple immune evasion strategies operated by gammaherpesviruses. Indeed, a significant inhibition of long-term latency establishment could be observed in mice vaccinated with strains of genetically modified MHV-68 defective in reactivation or establishment of latency. In this study, we focused on the effects of interferon-α (IFN-α) on both the lytic and latent phase of MHV-68 infection, as exerted by the constitutive release of IFN-α1 by a clone of MHV-68 genetically modified to produce this cytokine (MHV-68mIFNα1). Although the MHV-68mIFNα1 recombinant virus exhibited in vitro replication features indistinguishable from those of the wild type MHV-68, its pathological properties were severely attenuated in vivo in immunocompetent mice and not in mice rendered genetically unresponsive to type I IFN, suggesting that a stronger immune response was primed in the presence of the cytokine. Notably, MHV-68mIFNα1 attenuation did not result in a reduced level of long-term spleen latency establishment. These results prompted us to evaluate the efficacy of MHV-68mIFNα1 in a prophylactic vaccination regimen aimed at inhibiting the symptoms of acute virus infection and the establishment of long-term latency after MHV-68 challenge. Our results show that mice vaccinated with MHV-68mIFNα1, administered as a live-attenuated or partially inactivated (by Psoralen and UV treatment) vaccine, were protected against the challenge with wt MHV-68 from all phases of infection. The ability of MHV-68mIFNα1 to produce IFN-α at the site of the infection, thus efficiently stimulating the immune system in case of virus reactivation from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the previously reported latency-deficient clones.

Peptidoglycan Induces Necrosis and Regulates Cytokine Production in Murine Trophoblast Stem Cells

Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1β secretion while inhibiting the constitutive release of RANTES. Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1β may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications.

Double-Stranded RNA Induces Shedding of the 34-kDa Soluble TNFR1 from Human Airway Epithelial Cells via TLR3–TRIF–RIP1-Dependent Signaling: Roles for Dual Oxidase 2- and Caspase-Dependent Pathways

TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (NCI-H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ectodomains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experiments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated.

Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes

Summary 1. Indirect plant defences are well documented for the above‐ground constituents of plants. Although less investigated to date, below‐ground defences that mediate multitrophic interactions are equally important. Entomopathogenic nematodes (Steinernema diaprepesi) are attracted to herbivore‐induced volatiles from Swingle var. (Citrus paradisi×Poncirus trifoliata) when fed upon by root weevil, Diaprepes abbreviatus. 2. We examined the extent to which below‐ground volatiles modify behaviour of nematode species representing various foraging strategies (cruisers versus ambushers) and trophic levels (plant parasites versus entomopathogens). We compared attraction to volatiles of weevil‐infested and non‐infested roots from Swingle citrus rootstock and a parent line of the Swingle hybrid, Poncirus trifoliata (Pt). 3. Swingle weevil‐infested roots attracted more nematodes than non‐infested roots irrespective of nematode foraging strategy and trophic status. The parental line, Pt, attracted all nematode species irrespective of insect herbivory. 4. Dynamic in situ collection and GC–MS analysis of volatiles from soil revealed that Pt roots release attracting cues constitutively. A different non‐hybrid citrus species (sour orange, Citrus aurantium) released nematode attracting cues only in response to larval feeding, similar to responses found in Swingle. Volatile collections from above‐ and below‐ground portions of citrus plants revealed that above‐ground feeding by weevils does not induce production of nematode attracting cues analogous to that induced by root damage, nor does damage by larvae below‐ground induce a similar volatile above ground. 5. Synthesis. Our results suggest that release of nematode attractants by citrus roots occurs broadly and can be constant or herbivore‐induced. The major constituent of this indirect defence is produced by roots and not shoots and in response to below‐ground, but not above‐ground herbivory. Our findings suggest that this cue acts on nematode species broadly, attracting entomopathogenic nematodes that exhibit various foraging strategies. Unexpectedly, we also found that this cue attracts a plant parasitic nematode species. It appears, thus, that release of nematode attracting cues by citrus plants can cause ecological costs. The plants, however, appear to counteract against these costs, because constitutive release was found only in a cultivar that is resistant to phytopathogenic nematodes, while herbivore‐induced release occurred in lines susceptible to pathogenic nematode species.

The Crosstalk Between the Matrix Metalloprotease System and the Chemokine Network in Acute Myeloid Leukemia

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.