Anti-Siglec-1 antibodies block Ebola viral uptake and decrease cytoplasmic viral entry.

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.

CD5L is a pleiotropic player in liver fibrosis controlling damage, fibrosis and immune cell content.

BACKGROUND: Chronic hepatic inflammation leads to liver fibrosis, which may progress to cirrhosis, a condition with high morbidity. Our aim was to assess the as yet unknown role of innate immunity protein CD5L in liver fibrosis. METHODS: CD5L was measured by ELISA in plasma samples from cirrhotic (n = 63) and hepatitis (n = 39) patients, and healthy controls (n = 7), by immunohistochemistry in cirrhotic tissue (n = 12), and by quantitative RT-PCR in mouse liver cell subsets isolated by cell sorting. Recombinant CD5L (rCD5L) was administered into a murine model of CCl4-induced fibrosis, and damage, fibrosis and hepatic immune cell infiltration, including the LyC6hi (pro-fibrotic)-LyC6low (pro-resolutive) monocyte ratio were determined. Moreover, rCD5L was added into primary human hepatic stellate cells to study transforming growth factor ß (TGFß) activation responses. FINDINGS: Cirrhotic patients showed elevated plasma CD5L concentrations as compared to patients with hepatitis and healthy controls (Mann-Whitney test p < 0·0001). Moreover, plasma CD5L correlated with disease progression, FIB4 fibrosis score (r:0·25, p < 0·0001) and tissue expression (r = 0·649; p = 0·022). Accordingly, CCl4-induced damage increased CD5L levels in total liver, particularly in hepatocytes and macrophages. rCD5L administration attenuated CCl4-induced injury and fibrosis as determined by reduced serum transaminase and collagen content. Moreover, rCD5L inhibited immune cell infiltration and promoted a phenotypic shift in monocytes from LyC6hi to LyC6low. Interestingly, rCD5L also had a direct effect on primary human hepatic stellate cells promoting SMAD7 expression, thus repressing TGFß signalling. INTERPRETATION: Our study identifies CD5L as a key pleiotropic inhibitor of chronic liver injury. FUND: Fundació Marató TV3, AGAUR and the ISCIII-EDRF.

Author Correction: Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

The original version of this Article contained an error in the spelling of the author Álvaro Sebastián-Serrano, which was incorrectly given as Álvaro Sebastián Serrano. This has now been corrected in both the PDF and HTML versions of the Article.

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts.

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2(-/-) mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.

Immunogenic characterization and epitope mapping of transmissible gastroenteritis virus RNA dependent RNA polymerase.

Coronavirus RNA synthesis is a sophisticated process performed by a viral multienzymatic replicase complex, together with cellular factors. A key enzyme of this replication complex is the RNA dependent RNA polymerase (RdRp). To study the replication of coronavirus genome, six monoclonal antibodies (mAbs) specific for transmissible gastroenteritis virus (TGEV) RdRp were generated and characterized. His-tagged RdRp was expressed in baculovirus, purified and used as immunogen to produce mAbs. The TGEV RdRp was recognized by these mAbs in the context of virus infection by immunofluorescence analysis and Western blot. Epitope mapping by Pepscan indicated that RdRp mAbs recognized four non-overlapping linear epitopes located in a 62-amino acid region of the N-terminal domain, suggesting that this region may constitute an immunodominant domain. The availability of TGEV RdRp mAbs will be instrumental to study coronavirus replication and to analyze the function of RdRp in pathogenesis.

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis.

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.

Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neurons.

The N-methyl-D-aspartate receptor (NMDAR) is fundamental to normal and pathological functioning of neurons. The receptor subunits are N-glycosylated proteins synthesized in the endoplasmic reticulum (ER) that fold, mature, and oligomerize as they transit through the secretory pathway. Although the early processes of biogenesis are fundamental to NMDAR expression and function, our knowledge of them is nevertheless limited. Additionally, the investigation of NMDAR synthesis is highly relevant, in that ER dysfunction, frequently associated with acute and degenerative brain diseases, might alter this process. We characterize here the effect of ER stress produced by inhibition of N-glycosylation on NMDAR synthesis and function. We use first heterologous systems of NMDAR expression in which NR1 and NR2A subunits are synthesized in nonneuronal cells. The function of these NMDARs as Ca2+ channels is repressed by tunicamycin, because of the inhibition of NR1, but no NR2A, synthesis. The regulation of NR1 is relevant to the central nervous system, in that a dramatic decrease in synthesis of this subunit and assembly of NMDARs is observed in cortical neurons treated with tunicamycin. The inhibition of NR1 synthesis is not due to changes in levels of mRNA but associated with the earliest stages in NMDAR biogenesis. The inhibition of N-glycosylation activates ER-specific stress responses in neurons, which include the ER-associated degradation (ERAD) mechanism responsible for differential and extremely efficient degradation of nonglycosylated NR1 by the proteasome after ubiquitination. Because this is an obligatory NMDAR component, the significant sensitivity of NR1 to ER stress will have important consequences on receptor function.

The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis.

A specific interaction of ASFV p54 protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural protein p54 in apoptosis, transient expression experiments of p54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, p54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of p54. This is the first reported ASFV protein which induces apoptosis.