Sex-dependent expression levels of VAV1 and P2X7 in PBMC of multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system and the leading cause of progressive neurological disability in young adults. It decreases the patient's lifespan by about 10 years and affects women more than men. No medication entirely restricts or reverses neurological degradation. However, early diagnosis and treatment increase the possibility of a better outcome. To identify new MS biomarkers, we tested the expression of six potential markers (P2X4, P2X7, CXCR4, RGS1, RGS16 and VAV1) using qPCR in peripheral blood mononuclear cells (PBMC) of MS patients treated with interferon ß (IFNß), with glatiramer acetate (GA) or untreated. We showed that P2X7 and VAV1 are significantly induced in MS patients. In contrast, the expression of P2X4, CXCR4, RGS1 and RGS16 was not significantly modified by MS in PBMC. P2X7 and VAV1 are essentially induced in female patients, suggesting these markers are connected to sex-specific mechanisms. Strikingly, VAV1 expression is higher in healthy women than healthy men and IFNß treatment of MS reduced VAV1 expression in female MS patients while it up-regulated VAV1 in male MS patients. Our data point to the differential, sex-dependent value of MS markers and treatment effects. Although rgs16 expression in PBMC was not a valid MS marker in patients, the strong upregulation of P2X4 and P2X7 induced in the spinal cord of WT mice by EAE was abrogated in rgs16KO mice suggesting that rgs16 is required for P2X4 and P2X7 induction by neurological diseases.

Dual ELISA using SARS-CoV-2 nucleocapsid protein produced in E. coli and CHO cells reveals epitope masking by N-glycosylation.

Spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-SP, SARS-CoV-2-NP) are the main immunogenic targets for antibodies. We herein demonstrate that the glycosylation of SARS-CoV-2-NP masks some of its antibody epitopes. In many cases, this can lead to false-negative serological tests. Deglycosylation of SARS-CoV-2-NP significantly increased the number of positive tests. The glycosylation pattern analysis of this protein revealed that the putative N-linked glycosylation sites, at the amino acid positions 48 and 270, co-located with two of the main immunodominant B cell epitopes.

Evolutionary Origin of the P2X7 C-ter Region: Capture of an Ancient Ballast Domain by a P2X4-Like Gene in Ancient Jawed Vertebrates.

P2X purinergic receptors are extracellular ATP-gated ion channel receptors present on the cell plasma membrane. P2X receptors have been found in Metazoa, fungi, amoebas, and in plants. In mammals, P2X7 is expressed by a large number of cell types and is involved in inflammation and immunity. Remarkably, P2X7 does not desensitize as other P2X do, a feature linked to a "C-cysteine anchor" intra-cytoplasmic motif encoded by exon 11. Another specific feature of P2X7 is its C-terminal cytoplasmic ballast domain (exon 13) which contains a zinc (Zn) coordinating cysteine motif and a GDP-binding region. To determine the origin of P2X7, we analyzed and compared sequences and protein motifs of the C-terminal intra-cytoplasmic region across all main groups of Metazoa. We identified proteins with typical ballast domains, sharing a remarkably conserved Zn-coordinating cysteine motif. Apart from vertebrates, these ballast domains were not associated with a typical P2X architecture. These results strongly suggest that P2X7 resulted from the fusion of a P2X gene, highly similar to P2X4, with an exon encoding a ballast domain. Our work brings new evidence on the origin of the P2X7 purinergic receptor and identifies the Zn-coordinating cysteine domain as the fundamental feature of the ancient ballast fold.

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

Porcine circovirus type 2 ORF3 protein induces apoptosis in melanoma cells.

BACKGROUND: The current treatment of malignant melanoma is limited by the lack of effective therapeutic approaches, and alternative treatments are needed. Proliferative diseases such as melanoma and other cancers may be treatable by virally-encoded apoptotic proteins that are targeted to rapidly multiplying cells. Caspase-dependent apoptosis, that is frequently used in chemotherapy, can boost the cell proliferation that caspase-independent cell death does not. METHODS: In the current study, the porcine circovirus type 2 (PCV2), proapoptotic protein ORF3 was expressed in mouse and human cancer cell lines, and its apoptotic activity was assessed. RESULTS: Quantitative assessment of the apoptotic cells by flow cytometry showed that apoptotic cell death was significantly increased in ORF3-expressing malignant cells, compared to ORF3 non-expressing cells. Our data show that PCV2 ORF3 induces apoptosis in a caspase-3 and -8 independent manner. ORF3 expression seems to cause an increase in abnormal mitosis in B16F10 melanoma cells by interacting with centrosomes and thereby disrupting the formation of the mitotic spindle. In addition, we show that ORF3 of PCV2 also exhibits significant anti-tumor effects in vivo. Although the expression of Regulator of G protein Signaling (RGS)-16 by recipient mice inhibited the development of grafted melanoma in vivo, it was not required for the antitumoral activity of ORF3. CONCLUSION: PCV2 ORF3 causes abnormal mitosis in rapidly dividing cells and increases the apoptosis of cancer cells. Apoptin might, therefore, be considered to develop future antitumoral strategies.