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Objectives: Apoptosis plays an important role in the progression of the ischemic penumbra after reperfusion. Estrogen and progesterone have neuroprotective effects against ischemic brain damage, however the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In this study, we investigated the possible regulatory effects of a combined steroid treatment on extrinsic and intrinsic apoptotic signaling pathways after cerebral ischemia. Methods: Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (tMCAO) using an intraluminal filament technique for 1 h followed by 23 h reperfusion. Estrogen and progesterone were immediately injected after tMCAO subcutaneously. Sensorimotor functional tests and the infarct volume were evaluated 24 h after ischemia. Protein expression of calpain-1 and Fas receptor (FasR), key members of intrinsic and extrinsic apoptosis, were determined in the penumbra region of the ischemic brain using western blot analysis, immunohistochemistry, and TUNEL staining. Results: Neurological deficits and infarct volume were significantly reduced following hormone therapy. Calpain-1 up-regulation and caspase-3 activation were apparent 24 h after ischemia in the peri-infarct area of the cerebral cortex. Steroid hormone treatment reduced infarct pathology and attenuated the induction of both proteases. FasR protein levels were not affected by ischemia and hormone application. Conclusion: We conclude that a combined steroid treatment inhibits ischemia-induced neuronal apoptosis through the regulation of intrinsic pathways. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that oxidative injury is involved in the pathogenesis of many age-related neurodegenerative disorders, including Alzheimer's disease (AD). Identifying the protein targets of oxidative stress is critical to determine which proteins may be responsible for the neuronal impairments and subsequent cell death that occurs in AD. In this study, we have applied a high-throughput shotgun proteomic approach to identify the targets of protein carbonylation in both aged and PS1+AβPP transgenic mice. However, because of the inherent difficulties associated with proteomic database searching algorithms, several newly developed bioinformatic tools were implemented to ascertain a probability-based discernment between correct protein assignments and false identifications to improve the accuracy of protein identification. Assigning a probability to each identified peptide/protein allows one to objectively monitor the expression and relative abundance of particular proteins from diverse samples, including tissue from transgenic mice of mixed genetic backgrounds. This robust bioinformatic approach also permits the comparison of proteomic data generated by different laboratories since it is instrument- and database-independent. Applying these statistical models to our initial studies, we detected a total of 117 oxidatively modified (carbonylated) proteins, 59 of which were specifically associated with PS1+AβPP mice. Pathways and network component analyses suggest that there are three major protein networks that could be potentially altered in PS1+AβPP mice as a result of oxidative modifications. These pathways are 1) iNOS-integrin signaling pathway, 2) CRE/CBP transcription regulation and 3) rab-lyst vesicular trafficking. We believe the results of these studies will help establish an initial AD database of oxidatively modified proteins and provide a foundation for the design of future hypothesis driven research in the areas of aging and neurodegeneration. [ABSTRACT FROM AUTHOR]

Solamargine (SM), isolated from Solanum incanum herb, displayed a superior cytotoxicity in four human lung cancer cell lines. The half-inhibitory concentrations (IC50), of the cell viability assay for H441, H520, H661 and H69 cells were 3, 6.7, 7.2 and 5.8 μM, respectively. SM-induced apoptosis of these cells by PS externalization in a dose-dependent manner and increased sub-G1 fraction were observed. Quenching of the expression of tumor necrosis factor receptors (TNFRs) during the progress of human lung carcinogenesis has been previously reported. SM may induce cell apoptosis via modulating the expression of TNFRs and their subsequent TRADD/FADD signal cascades. Subsequently, SM treatment increased the binding activities of TNF-α and TNF-β to the lung cancers, and the intrinsic TNFs-resistant cancer cells became susceptible to TNF-α and -β. In addition, SM caused release of cytochrome c, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, increase of caspase-3 activity, and DNA fragmentation. Thus, SM could modulate the expressions of TNFRs and Bcl-2, and might be a potential anticancer agent for TNFs and Bcl-2 related resistance of human lung cancer cells. [Copyright &y& Elsevier]

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological and clinical similarities to the major human demyelinating disease multiple sclerosis (MS). Multiple lines of evidence in recent years implicate the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) in the pathogenesis of both EAE and MS. TNF-α cellular responses are mediated by signaling through receptors, which are expressed in two functional forms, designated according to molecular weight p55/60 and p75/80. We report a treatment trial using the extracellular domain of the p80 TNFR in a bivalent fusion construct designated soluble tumor necrosis factor receptor (sTNFR):Fc to treat EAE. sTNFR:Fc/p80, given after the onset of clinical signs, reduced the clinical deficit of the first attack of relapsing-remitting EAE ( RR-EAE ) and the exacerbation rate for subsequent attacks. The effect was reversible as mice treated with sTNFR:Fc/p80 reverted to an exacerbation rate and disease severity typical of placebo-treated animals after treatment was discontinued. Treatment of RR-EAE with sTNFR:Fc/p80 decreased expression of chemokines MIP-1α (Monocyte Inflammatory Protein)/CCL3, MIP-1β/CCL4 and MIP-2/CXCL1-2 in the central nervous system. This treatment trial reveals an important function of TNF in the pathogenesis of RR-EAE and propose the mechanism of beneficial action of sTNFR:Fc/p80 in this disease. [ABSTRACT FROM AUTHOR]

Using a signal sequence trap for selection of differentially expressed secretory and membrane proteins, we identified a novel member of the adhesion family of G-protein coupled receptors (GPCRs), termed vascular inducible GPCR (VIGR). VIGR contains C1r-C1s, Uegf and Bmp1 (CUB) and pentraxin (PTX)-like modules and a mucin-like spacer, followed by seven transmembrane domains. By surface biotinylation as well as by immunofluorescence analysis we demonstrate that endogenous, highly glycosylated VIGR is expressed on the cell surface of endothelial cells (ECs) upon LPS or thrombin treatment, and inducible expression is mediated by MAP kinases, but not NF-κB. We show that VIGR is selectively expressed in ECs derived from larger vessels, but not from microvessels. In summary, VIGR represents a novel GPCR of the adhesion family, which is unique in its long extra-cellular domain comprising CUB and PTX-like modules and in its inducibility by LPS and thrombin in a subset of ECs, suggesting an important function in cell-adhesion and potentially links inflammation and coagulation. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) α-induced adipose-related protein (TIARP) is a novel TNFα-stimulated protein in adipocytes. Besides TNFα, interleukin (IL)-6 has recently been shown to be another adipocytokine implicated in insulin resistance. Therefore, the impact of IL-6 on TIARP gene expression in 3T3-L1 adipocytes was determined by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, TIARP mRNA expression was stimulated up to 3.8-fold by IL-6 in a dose-dependent fashion with significant stimulation detectable at effector concentrations as low as 3 ng/ml and maximal effects seen at 100 ng/ml IL-6. Induction of TIARP mRNA by IL-6 was time-dependent with significant upregulation occurring as early as 2 h after effector addition and maximal effects observed at 4 h. In parallel, TIARP protein synthesis was upregulated with maximal effects seen after 8 h of IL-6 treatment. Furthermore, the Janus kinase 2 inhibitor AG490 decreased TIARP mRNA expression. The increase of TIARP mRNA could be reversed by withdrawal of IL-6 for 24 h. Furthermore, TIARP mRNA induction by IL-6 was also seen in brown adipocytes but not in muscle and liver cells. Taken together, these results show that TIARP is acutely regulated in adipose tissue not only by TNFα but also by IL-6 which has been shown to be another important cytokine implicated in the pathogenesis of insulin resistance. [Copyright &y& Elsevier]

Activation of nuclear factor kappa B (NF-κB) signaling in response to cell stimulation by wear debris may be critical in the pathogenesis of aseptic loosening. Erythromycin (EM), a macrolide antibiotic, has been shown to effectively suppress some types of inflammatory reactions. In this study, we examined the effect of EM on wear debris-induced osteoclastic bone resorption in vitro. EM inhibited Ca+ release from neonatal calvaria co-cultured with conditioned medium from mouse RAW264.7 macrophages activated by wear debris. Inhibition of Ca+ release was associated with a decreased number of tartrate-resistant acid phosphatase (TRAP)-positive cells in cultured bones. To investigate the mechanism whereby EM inhibits bone-resorption, RAW cells were incubated with wear debris in the presence EM. Real time RT-CR analysis revealed that EM (5 μg/ml) significantly inhibited mRNA expression of NF-κB, cathepsin K (CPK), IL-1β and TNFα, but not RANK in RAW cells stimulated with wear debris. Furthermore, electrophoretic mobility-shift assay showed that EM (0.2 μg–5 μg/ml) could reduce DNA-binding activity of NF-κB in RAW cells stimulated with wear debris. The inhibition of inflammatory osteoclastogenesis by EM treatment was further confirmed by an osteoclast (OC) formation assay using primary cultures of mouse bone marrow progenitor cells stimulated with macrophage colony-stimulating factor and RANK ligand (RANKL). EM treatment (5 μg/ml) resulted in more than 70% reduction in multinucleated OC formation and 50% reduction of TRAP+ cells by bone marrow progenitor cells. Our findings support that EM suppresses wear debris-induced osteoclastic bone resorption by, at least, down-regulation of NF-κB signaling pathway. It appears that EM represents a potential therapeutic candidate for the treatment and prevention of aseptic loosening. [Copyright &y& Elsevier]

We previously showed that a metalloprotease and a serine protease mediate shedding of the TNF-R75 (75-kDa tumor necrosis factor receptor) in neutrophils. Here we show that elastase is the TNF-R75 solubilizing serine protease. Release of the TNF-R75 by resting cells was almost totally inhibited by the serine protease inhibitor diisopropylfluorophosphate (DFP), by two synthetic, chemically unrelated, elastase-specific inhibitors and by α1-protease inhibitor. Release after TNF or FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) stimulation was blocked by DFP and a metalloprotease inhibitor used in combination. Supernatants from resting neutrophils contained a 28-kDa fragment of the receptor, compatible with that generated by elastase, whose appearance was inhibited by DFP. Upon FMLP stimulation, the release of 28-kDa and 40-kDa fragments was observed, which was inhibited by DFP and a metalloprotease inhibitor, respectively. We conclude that elastase is the TNF-R75 sheddase of resting neutrophils and that it contributes to shedding of this receptor in stimulated cells. [Copyright &y& Elsevier]

Synthetic lipopeptides derived from the bacterial cell wall component lipoprotein activate B-lymphocytes and macrophages/monocytes in vitro. In vivo they constitute potent immunoadjuvants for a broad range of different antigens and species comparable or superior to Freund’s adjuvant. Here, we demonstrate that P3CSK4, representing a highly active lipopentapeptide derivative in vitro, significantly enhances and accelerates the humoral immune response to tetanus toxoid. P3CSK4 could substitute for up to 90% of the antigen without any decrease in the specific IgG level, and the presence of the lipopeptide resulted in a prolonged production of specific IgG in time. Investigations using P3CSK4 as an adjuvant in genetic immunization confirmed earlier data demonstrating that lipopeptides constitute adjuvants for low-immunogenic DNA constructs and/or for application routes resulting in weak immune responses. We monitored a lipopeptide-dependent shift from a Th1-type to Th2-type response, when DNA immunization was followed by i.p. administration of the lipopeptide adjuvant. [Copyright &y& Elsevier]

The effects of apoptosis induction on glucose and phospholipid metabolite levels in cancer were studied using human colon adenocarcinoma cells (HT-29). Apoptosis was induced by co-incubation with 200 U/ml tumor necrosis factor (TNF)-α for 4, 8 or 15 h, after sensitization with 500 U/ml interferon (IFN)-γ for 7 h. Perchloric acid extracts were analyzed by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Significantly increased lactate and NTP (all nucleoside 5’-triphosphates) signals were detected 4 h after apoptosis-inducing IFN-γ/TNF-α treatment, but not in cells which were TNF-α-treated without IFN-γ preincubation. Simultaneous lactate and NTP changes, if confirmed in vivo, may serve as early, non-invasive markers of treatment response in some tumors. [Copyright &y& Elsevier]

4-1BB(CD137) is a member of the tumor necrosis factor receptor superfamily and provides a costimulatory signal by interaction with 4-1BB ligand expressed on antigen-presenting cells. The expression of 4-1BB is known to be activation-dependent. Here, we investigated the transcriptional machinery required for T cell receptor (TCR) activation-dependent induction of 4-1BB expression in CD3-CEM cells treated with phorbol myristate acetate and ionomycin. Using 5′-deletion constructs of 4-1BB promoter in luciferase reporter assays, we demonstrated that the transcriptional elements mediating 4-1BB upregulation were located in the region between ∼0.9 and ∼1.1 kb from the translational start site. Characterization of these sites by electrophoretic mobility shift assay and site-directed mutagenesis revealed that nuclear factor κB (NF-κB) and activating protein-1 (AP-1) are involved. MEK and c-Jun N-terminal kinase-1 activity was required for activation-dependent 4-1BB upregulation. Thus, NF-κB and AP-1 are involved in the TCR stimulation-dependent transcriptional regulation of the 4-1BB promoter. [Copyright &y& Elsevier]

Interleukin-6 (IL-6) production is up-regulated by several stimuli through the activation of transcription factors. We have previously demonstrated that CCAAT/enhancer binding protein homologous protein (CHOP) positively regulates IL-6 production at the transcriptional level in the human melanoma cell line A375. In this study, we provide evidence that CHOP up-regulates the IL-6 transcription without binding to the IL-6 promoter. CHOP dimerized more preferentially with an inhibitory isoform of nuclear factor for IL-6 expression (LIP (liver-enriched inhibitory protein)) than with a positively acting isoform (LAP, liver-enriched activator protein). These results indicate that CHOP plays an important role in IL-6 production without binding to its promoter, probably by trapping protein(s) such as LIP, which would otherwise inhibit IL-6 transcription. [Copyright &y& Elsevier]

A tridecapeptide (MITLAIPVNKPGR) that stimulates phagocytosis of human neutrophils was isolated from a trypsin digest of soybean proteins. This peptide is derived from the soybean β-conglycinin α′ subunit and was named soymetide-13. The N-terminal methionine residue of soymetide-13 is essential for its activity, and removal of C-terminal residues revealed that soymetide-4 (MITL) is the minimal structure required for phagocytosis stimulation. Although they are not formylated at their N-termini, soymetides have a weak affinity for the N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor and their phagocytosis-stimulating activity is inhibited by the fMLP antagonist Boc-MLP. Interestingly, soymetide-4 promotes tumor necrosis factor α production at a higher level than soymetide-13 following oral administration in mice. [Copyright &y& Elsevier]

To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [3H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [3H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria. [Copyright &y& Elsevier]

Nuclear factor κB (NF-κB)-dependent gene expression plays an important role in numerous cellular processes including stress responses, inflammation and cell proliferation. Therefore, the activity of this transcription factor needs to be tightly regulated. Among others, the NF-κB-dependent zinc finger protein A20 is involved in the negative feedback regulation of NF-κB activation in response to tumor necrosis factor (TNF). We previously demonstrated that A20 can interact with A20-binding inhibitors of NF-κB activation (ABINs), which have the potential to inhibit TNF-induced activation of NF-κB upon overexpression. The ABIN proteins were therefore proposed to mediate the NF-κB inhibiting function of A20. Here we demonstrate the presence of a short homologous region in ABINs and IκB kinase γ, the regulatory subunit of the IκB kinase complex. Site-specific mutagenesis of this region abolished the NF-κB inhibiting function of ABIN-1, without affecting the interaction with A20. Furthermore, coexpression of these ABIN-1 mutants interfered in a dominant negative manner with the NF-κB inhibiting function of ABIN-1, whereas the A20-mediated inhibition was unaffected. These results suggest that A20 and ABIN-1 probably act independently of their mutual interaction. [Copyright &y& Elsevier]

TRAIL-R3 is a decoy receptor for TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a member of the tumor necrosis factor ligand family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and preventing its binding to TRAIL pro-apoptotic receptors. Here we report the cloning of the promoter region of human TRAIL-R3 and the mapping of the transcriptional start sites. This gene contains a consensus TATA box and the minimal promoter lies within the first 33 nucleotides upstream of the transcription start site. Transient transfection assays of luciferase reporter plasmids demonstrate that human TRAIL-R3 promoter can be induced in doxorubicin-treated MCF-7 cells in a p53-independent manner. [Copyright &y& Elsevier]

A major part of the proinflammatory activity of tumor necrosis factor (TNF) is brought about by cytosolic phospholipase A2 (cPLA2) that generates arachidonic acid, the precursor for the production of leukotrienes and prostaglandins. The activation of cPLA2 and induction of proinflammatory lipid mediators is in striking contrast to the teleologic meaning of apoptosis, which is to avoid an inflammatory reaction. In this review we highlight the evidence for a caspase-mediated cleavage and inactivation of cPLA2, which seems to be an important mechanism by which TNF downregulates cPLA2 activity in cells undergoing apoptosis. [Copyright &y& Elsevier]

The cellular redox state regulates nuclear factor-κB (NF-κB) signaling systems. We investigated the effects of H2O2 on inhibitor of NF-κB (IκB) kinases (IKKα and IKKβ), which phosphorylate IκB leading to its degradation and NF-κB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H2O2 induced a slight activation of IKK within 30 min. Furthermore, co-stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H2O2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H2O2 enhanced TNF-induced NF-κB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKα and Ser181 of IKKβ, in the activation loops was essential for the H2O2-mediated activation of IKK. H2O2-induced activation of IKKα and IKKβ was reduced by IKKβ and IKKα kinase-negative mutants, respectively, indicating that IKKα and IKKβ were stimulated by H2O2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-κB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops. [Copyright &y& Elsevier]

The CD95/Fas/Apo-1 ligand (CD95L, CD178) induces apoptosis through the death receptor CD95. CD95L was also described as a co-stimulatory receptor for T-cell activation in mice in vivo. The molecular basis for the bidirectional signaling capacity and directed expression of CD95L is unknown. In the present study we identify proteins that precipitate from T-cell lysates with constructs containing fragments of the CD95L cytosolic tail. The determined peptide mass fingerprints correspond to Grb2, actin, β-tubulin, formin binding protein 17 (FBP17) and PACSIN2. Grb2 had been identified as a putative mediator of T-cell receptor-to-CD95L signaling before. FBP17 and PACSIN2 may be associated with expression and trafficking of CD95L. When overexpressed, CD95L co-precipitates with FBP17 and PACSIN. Protein–protein interactions are mediated via Src homology 3 (SH3) domain binding to the polyproline region of CD95L and can be abolished by mutation or deletion of the respective SH3 domain. [Copyright &y& Elsevier]

The Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors have recently been found to function in preventing apoptosis. In this study, we demonstrated that overexpression of transforming growth factor α (TGFα) has a dramatic protective effect on Fas-mediated hepatic apoptosis at the biochemical and histological levels. Moreover, 85.7% (six out of seven) of TGFα transgenic mice survived the lethal liver damage, whereas all wild-type mice died. Expression of Bcl-xL, an anti-apoptotic protein, was greatly increased in the transgenic mice. Taken together, our findings suggest that TGFα protects against Fas-mediated liver apoptosis in vivo and up-regulation of Bcl-xL may participate in protective effect of TGFα. [Copyright &y& Elsevier]

The adenovirus E3-14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF-α and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3-14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five β-strands and one α-helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution. [ABSTRACT FROM AUTHOR]

To determine the apoptotic signaling pathway which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen-activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L-induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L-induced apoptosis is mediated by ROS-activated p38 MAP kinase followed by caspase activation in HeLa cells. [Copyright &y& Elsevier]

It has been reported that prostate apoptosis response-4 (PAR-4) binds to and inhibits protein kinase Cζ (PKCζ) which phosphorylates IκB kinase β (IKKβ) for nuclear factor κB (NFκB) activation, while p62 binds to and recruits PKCζ to the NFκB signaling complex. Thus, a mechanism to coordinate the two binding proteins for the regulation of PKCζ is expected to exist. The present data show that p62 and PAR-4 do not compete for PKCζ binding but directly interact each other and form a ternary complex with PKCζ. Furthermore, p62 not only enhances the catalytic activity of PKCζ but also reactivates catalytically inactive PAR-4-bound PKCζ. As the result, over-expression of p62 protects cells from PAR-4-mediated inactivation of NFκB and apoptotic death. Thus, the regulatory role of p62 for free and PAR-4-bound PKCζ is important in activation of NFκB. [ABSTRACT FROM AUTHOR]

Gamma delta (γδ) T cells are the all-rounders of our immune-system with their major histocompatibility complex-unrestricted cytotoxicity, capacity to secrete immunosti-mulatory cytokines and ability to promote the generation of tumor antigen-specific CD8+and CD4+T cell responses. Dendritic cell (DC)-based vaccine therapy has the prospective to harness these unique features of the γδ T cells in the fight against cancer. In this review, we will discuss our current knowledge on DC-mediated γδ T cell activation and related opportunities for tumor immunologists. [ABSTRACT FROM PUBLISHER]

The CD8+T-cell response comprises terminally differentiated effector cells and antigen-experienced memory T cells. The latter encompass central (TCM) and effector (TEM) memory cells. TCMcells are superior in their protection against viral and bacterial challenges and mediation of antitumor immunity due to their higher proliferative ability upon antigen re-encounter. Defining a mechanism to enhance TCMcells and delay terminal differentiation of CD8+T cells is crucial for cancer immune therapy, as it can promote a better tumor immune response. The differentiation of CD8+memory T cells is thought to be coordinated by the phosphoinositide 3-kinase (PI3K)/Akt pathway. We, therefore, investigated the role of Akt isoforms in the differentiation and proliferation of memory CD8+T cells. We found that Akt1 and Akt2, but not Akt3, drive the terminal differentiation of CD8+T cells, and their inhibition enhances the therapeutically superior TCMphenotype. Furthermore, the inhibition of Akt1 and Akt2, but not Akt 3, delays CD8+T-cell exhaustion and preserves naïve and TCMCD8+T cells, thus enhancing their proliferative ability and survival and prolonging their cytokine and Granzyme B production ability. Here, we define a mechanism in which proliferative potential, function, and survival of CD8+T cells are enhanced by maintaining a reservoir of TCMand naïve cells using only Akt1 and Akt2 inhibition. Therefore, our findings strongly suggest the utility of using Akt1 and Akt2 inhibitors to modulate CD8+T cells, both for adoptive cell transfer and vaccine-based cancer immune therapies. [ABSTRACT FROM AUTHOR]