Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.

Inhibition of the kynurenine-NAD+ pathway leads to energy failure and exacerbates apoptosis in pneumococcal meningitis

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1? and -? were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Inhibition of direct and indirect TLR-mediated activation of human NK cells by low molecular weight dextran sulfate

NK cells express toll-like receptors (TLR) that recognize conserved pathogen or damage associated molecular patterns and play a fundamental role in innate immunity. Low molecular weight dextran sulfate (DXS), known to inhibit the complement system, has recently been reported by us to inhibit TLR4-induced maturation of human monocyte-derived dendritic cells (MoDC). In this study, we investigated the capability of DXS to interfere with human NK cell activation triggered directly by TLR2 agonists or indirectly by supernatants of TLR4-activated MoDC. Both TLR2 agonists and supernatants of TLR4-activated MoDC activated NK cells phenotypically, as demonstrated by the analysis of NK cell activation markers (CD56, CD25, CD69, NKp30, NKp44, NKp46, DNAM-1 and NKG2D), and functionally as shown by increased NK cell degranulation (CD107a surface expression) and IFN-gamma secretion. DXS prevented the up-regulation of NK cell activation markers triggered by TLR2 ligands or supernatants of TLR4-activated MoDC and dose-dependently abrogated NK cell degranulation and IFN-gamma secretion. In summary our results suggest that DXS may be a useful reagent to inhibit the direct and indirect TLR-mediated activation of NK cells.

Identification of bracovirus particle proteins and analysis of their transcript levels at the stage of virion formation

http://www.ncbi.nlm.nih.gov/pubmed/20554796

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Biomedical nanoparticles modulate specific CD4+ T cell stimulation by inhibition of antigen processing in dendritic cells

Understanding how nanoparticles may affect immune responses is an essential prerequisite to developing novel clinical applications. To investigate nanoparticle-dependent outcomes on immune responses, dendritic cells (DCs) were treated with model biomedical poly(vinylalcohol)-coated super-paramagnetic iron oxide nanoparticles (PVA-SPIONs). PVA-SPIONs uptake by human monocyte-derived DCs (MDDCs) was analyzed by flow cytometry (FACS) and advanced imaging techniques. Viability, activation, function, and stimulatory capacity of MDDCs were assessed by FACS and an in vitro CD4+ T cell assay. PVA-SPION uptake was dose-dependent, decreased by lipopolysaccharide (LPS)-induced MDDC maturation at higher particle concentrations, and was inhibited by cytochalasin D pre-treatment. PVA-SPIONs did not alter surface marker expression (CD80, CD83, CD86, myeloid/plasmacytoid DC markers) or antigen-uptake, but decreased the capacity of MDDCs to process antigen, stimulate CD4+ T cells, and induce cytokines. The decreased antigen processing and CD4+ T cell stimulation capability of MDDCs following PVA-SPION treatment suggests that MDDCs may revert to a more functionally immature state following particle exposure.

Inhibition of the miR-143/145 cluster attenuated neutrophil differentiation of APL cells

MicroRNAs can influence hematopoietic cell lineage commitment and aberrant expression of hematopoietic miRNAs contributes to AML pathology. We found that miR-143 and miR-145 expression is significantly repressed in primary AML patient samples as compared to neutrophils of healthy donors. Further analysis revealed impaired neutrophil differentiation of APL cells upon inhibition of miR-145 expression. Lastly, we identified p73 as transcriptional regulator of miR-143/145 during neutrophil differentiation of APL cells. Our data suggest that low miR-145 levels in APL, possibly due to aberrant expression of p73 transcription factors, contribute to the differentiation block seen in this disease.

Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

The role of the BMP signaling antagonist noggin in the development of prostate cancer osteolytic bone metastasis

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein

Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release.