High Throughput Method to Quantify Anterior-Posterior Polarity of T-Cells and Epithelial Cells

The virologic synapse (VS), which is formed between a virus-infected and uninfected cell, plays a central role in the transmission of certain viruses, such as HIV and HTLV-1. During VS formation, HTLV-1-infected T-cells polarize cellular and viral proteins toward the uninfected T-cell. This polarization resembles anterior-posterior cell polarity induced by immunological synapse (IS) formation, which is more extensively characterized than VS formation and occurs when a T-cell interacts with an antigen-presenting cell. One measure of cell polarity induced by both IS or VS formation is the repositioning of the microtubule organizing center (MTOC) relative to the contact point with the interacting cell. Here we describe an automated, high throughput system to score repositioning of the MTOC and thereby cell polarity establishment. The method rapidly and accurately calculates the angle between the MTOC and the IS for thousands of cells. We also show that the system can be adapted to score anterior-posterior polarity establishment of epithelial cells. This general approach represents a significant advancement over manual cell polarity scoring, which is subject to experimenter bias and requires more time and effort to evaluate large numbers of cells

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

17β-estradiol abrogates apoptosis inhibiting PKCδ, JNK and p66Shc activation in C2C12 cells

17β-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2O2 in skeletal myoblasts. As we have also demonstrated a clear beneficial action of this hormone on skeletal muscle mitochondria, the present work further characterizes the signaling mechanisms modulated by E2 that are involved in mitochondria protection, which ultimately result in antiapoptosis. Here, we report that E2 through estrogen receptors (ERs) inhibited the H2O2-induced PKCδ and JNK activation, which results in the inhibition of phosphorylation and translocation to mitochondria of the adaptor protein p66Shc. In conjunction, the inhibition by the hormone of this H2O2-triggered signaling pathway results in protection of mitochondrial potential membrane. Our results provide basis for a putative mechanism by which E2 exerts beneficial effects on mitochondria, against oxidative stress, in skeletal muscle cells. J. Cell. Biochem. 116: 1454-1465, 2015.Fil: la Colla, Anabela Belén. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentin

Bone resorption predicts for skeletal complications in metastatic bone disease

Relationships between the rate of bone resorption (measured by urinary N-telopeptide (Ntx) excretion) and a range of skeletal complications have been evaluated in patients with metastatic bone disease. A total of 121 patients had monthly measurements of Ntx during treatment with bisphosphonates. All skeletal-related events, plus hospital admissions for bone pain and death during the period of observation, were recorded. Data were available for 121 patients over the first 3-month period of monitoring (0–3 months) and 95 patients over the second 3-month period (4–6 months). N-telopeptide levels were correlated with the number of skeletal-related events and/or death (r=0.62, P<0.001 for 0–3 months and r=0.46, P<0.001 for 4–6 months, respectively). Patients with baseline Ntx values greater than or equal to100 nmol mmol−1 creatinine (representing clearly accelerated bone resorption) were 19.48 times (95% CI 7.55, 50.22) more likely to experience a skeletal-related event/death during the first 3 months than those with Ntx <100 (P<0.001). In a multivariate logistic regression model, Ntx was highly predictive for events/death. This study is the first to indicate a strong correlation between the rate of bone resorption and the frequency of skeletal complications in metastatic bone disease. N-telopeptide appears useful in the prediction of patients most likely to experience skeletal complications and thus benefit from bisphosphonate treatment

NIK signaling in dendritic cells but not in T cells is required for the development of effector T cells and cell-mediated immune responses

NIK expression in thymic dendritic cells is required for the development of effector T cells and their ability to promote EAE

Functional features of gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression

<p>Abstract</p> <p>Background</p> <p>Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in <it>KIT </it>or <it>PDGFRA </it>of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations.</p> <p>Methods</p> <p>Total RNA was isolated from 29 frozen gastric GISTs and processed for hybridization on GENECHIP^®HG-U133 Plus 2.0 microarrays (Affymetrix). <it>KIT </it>and <it>PDGFRA </it>were analyzed by sequencing, while related mRNA levels were analyzed by quantitative RT-PCR.</p> <p>Results</p> <p>Fifteen and eleven tumours possessed mutations in <it>KIT </it>and <it>PDGFRA</it>, respectively; no mutation was found in three tumours. Gene expression analysis identified no discriminative profiles associated with clinical or pathological parameters, even though expression of hundreds of genes differentiated tumour receptor mutation and expression status. Functional features of genes differentially expressed between the two groups of GISTs suggested alterations in angiogenesis and G-protein-related and calcium signalling.</p> <p>Conclusion</p> <p>Our study has identified novel molecular elements likely to be involved in receptor-dependent GIST development and allowed confirmation of previously published results. These elements may be potential therapeutic targets and novel markers of <it>KIT </it>mutation status.</p

Triterpenoid modulation of IL-17 and Nrf-2 expression ameliorates neuroinflammation and promotes remyelination in autoimmune encephalomyelitis

Inflammatory cytokines and endogenous anti-oxidants are variables affecting disease progression in multiple sclerosis (MS). Here we demonstrate the dual capacity of triterpenoids to simultaneously repress production of IL-17 and other pro-inflammatory mediators while exerting neuroprotective effects directly through Nrf2-dependent induction of anti-oxidant genes. Derivatives of the natural triterpene oleanolic acid, namely CDDO-trifluoroethyl-amide (CDDO-TFEA), completely suppressed disease in a murine model of MS, experimental autoimmune encephalomyelitis (EAE), by inhibiting Th1 and Th17 mRNA and cytokine production. Encephalitogenic T cells recovered from treated mice were hypo-responsive to myelin antigen and failed to adoptively transfer the disease. Microarray analyses showed significant suppression of pro-inflammatory transcripts with concomitant induction of anti-inflammatory genes including Ptgds and Hsd11b1. Finally, triterpenoids induced oligodendrocyte maturation in vitro and enhanced myelin repair in an LPC-induced non-inflammatory model of demyelination in vivo. These results demonstrate the unique potential of triterpenoid derivatives for the treatment of neuroinflammatory disorders such as MS

M-CSF Induces Monocyte Survival by Activating NF-κB p65 Phosphorylation at Ser276 via Protein Kinase C

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/β inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes