Sofia de Oliveira

An overview about erythrocyte membrane

In the sixties and seventies, erythrocytes or red blood cells (RBCs) were extensively studied. Much has been learnt particularly concerning their metabolism and gas transporter function.In the past decade, the use of new approaches and methodologies, such as proteomic analysis, has contributed for a renewed interest on the erythrocyte. Recent studies have provided us with a more detailed and comprehensive picture on the composition and organization of its cellular membrane that will be the main subject of this minireview. Unexpectedly, it has been recognized that this cell expresses several adhesion molecules on its surface, like other cellular types such blood circulating cells or endothelial cells. Taking into consideration the cellular functions of the erythrocyte, the clarification of the role of those adhesion molecules may in the future open new horizons for the biological significance of this cellular player.

Modulation of erythrocyte deformability by PKC activity

The interactions between membrane, peripheral and cytoskeleton proteins are responsible for the maintenance of erythrocyte deformability (EEI) and some of these interactions are modulated by PKC activity. Protein band 3 of the erythrocyte membrane is phosphorylated by phosphotyrosine kinases (PTK) and dephosphorylated by phosphotyrosine phosphatase (PTP). It was previously described by us a signal transduction mechanism that describes a possible pathway connecting an erythrocyte external membrane protein, acetylcholinesterase (AChE), with protein band 3. So how does PKC activity modulate EEI when protein band 3 is phosphorylated or dephosphorylated in absence or presence of AChE effectors?To answer this we used phorbol 12-myristate 13-acetate (PMA) as an activator and chelerythrine chloride as inhibitor of PKC and also band 3 modulators of band 3 phosphorylation degree, in presence and absence of AChE effectors in order to measure in whole blood samples EEI. Our results showed that erythrocyte deformability was significantly (i) decreased by inhibition of PKC, in absence and presence of AChE inhibitor velnacrine (ii) increased with PMA in absence and presence of ACh and (iii) decreased in presence of calpeptin in absence and presence of either chelerythrine or PMA. These results establish dependence between cytoskeleton proteins, PKC activity, band 3 phosphorylation degrees and EEI. Better understanding of those proteins interactions on transduction mechanisms might trigger possible targets for drug action that would modulate EEI.

ATP Modulates Acute Inflammation In Vivo through Dual Oxidase 1–Derived H2O2 Production and NF-κB Activation

Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H2O2 gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H2O2 release from DUOX1 and whether the H2O2 gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H2O2 release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H2O2 is able to trigger the NF-κB inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca2+ signaling pathway and the production of H2O2, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-κB signaling pathway.

Erythrocyte as a biological sensor

The erythrocytes ability of sensing the local oxygen gradient through the hemoglobin conformation, along with changes in nitric oxide mobilization and vasomotor repercussions at the microcirculation, were reviewed in detail in this article. Different approachs trying to explain the erythrocyte death were additionally documented. Also, the influence of several types of molecules (vasoactive, oxidant/reductor) on the erythrocyte roles as sensor of (i) oxygen tissue needs, (ii) blood viscosity and myogenic environment, (iii) and inflammatory conditions were mentioned in order to highlight its physiologycal function and substitute the erroneous idea of the erythrocyte being simply a hemoglobin sac content.

Cxcl8-l1 and Cxcl8-l2 are required in the zebrafish defense against Salmonella Typhimurium.

In recent years zebrafish has emerged as an excellent model for studying the Cxcl8 signaling pathway in inflammation elicited upon tissue damage or infection. Zebrafish has two true homologs of mammalian CXCL8, named Cxcl8-l1 and Cxcl8-l2. Previously, we have shown that in wound-associated inflammation, these chemokines are up-regulated and are relevant for neutrophil recruitment. In infections, no such knowledge is available as most studies performed on this subject in zebrafish have mainly focused on Cxcl8-l1 even though Cxcl8-l2 shares higher homology with human CXCL8. In this study, we aimed to address the biological function of both zfCxcl8s in infection to improve our understanding of their respective roles under different inflammatory conditions. Gene expression analysis first confirmed that both Cxcl8-l1 and l2 are induced upon infection or in PAMP-elicited inflammatory processes. In addition, we also found that cxcl8-deficient larvae show higher susceptibility to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, reduced neutrophil recruitment to the infection site assayed in the line Tg(mpx:gfp), and decreased bacterial clearance. These data indicate that both zebrafish Cxcl8s play important roles in neutrophil recruitment and in the inflammatory response elicited upon infection or tissue damage, suggesting that even though the divergence of lower vertebrates and humans from a common ancestor occurred about 450 millions years ago, the basic principles of neutrophil recruitment are apparently conserved in all vertebrates.

Neurologic Manifestations of Gastrointestinal and Liver Diseases

Hepatic and gastrointestinal disorders can produce a wide spectrum of neurologic complications both affecting the central nervous system (CNS) and the peripheral nervous system. These manifestations range in severity from coma in acute liver failure and acute pancreatitis, to minor cognitive changes in chronic portosystemic encephalopathy and hepatitis C. Cerebrovascular diseases can complicate hepatitis C infection and inflammatory bowel disease. Demyelinating disorders may co-exist with inflammatory bowel disease. Anti-tumor necrosis factor alpha drugs may induce demyelination. Ataxia may occur in malabsorption syndromes and in gluten related disorders. Characteristic movement disorders are key features of acquired hepatocerebral degeneration and of Whipple disease. Multiple types of neuropathy can be found in association with hepatitis, inflammatory bowel disease and gluten related disorders.

Duox1-Derived H2O2 Modulates Cxcl8 Expression and Neutrophil Recruitment via JNK/c-JUN/AP-1 Signaling and Chromatin Modifications

DUOX1-derived hydrogen peroxide (H2O2) and CXCL8 are two key neutrophil chemoattractants. H2O2 is critical at the early phase, whereas CXCL8 plays a key role in the late phases of recruitment, but the crosstalks between the two phases in vivo remain unknown. In this study using zebrafish, we report that H2O2 also contributes to neutrophil recruitment to injuries at the late phase as it induces Cxcl8 expression in vivo through a JNK/c-JUN/AP-1 signaling pathway. However, Erk and NF-κB signaling were not involved in this crosstalk. Strikingly, H2O2 also promotes cxcl8 expression through modulation of histone 3 lysine 4 trimethylation, histone 3 lysine 9 acetylation, and histone 3 lysine 9 trimethylation levels at its promoter. These results explain how early H2O2 signal regulates neutrophil recruitment at all phases, directly via Lyn oxidation or indirectly by modulating cxcl8 gene expression, via the activation of redox-sensitive signaling pathways, and further point out H2O2/DUOX1 as a key drug target for anti-inflammatory therapies.

Interleukin 8 mediates bcl-xL-induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model

The protein bcl‐xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl‐xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild‐type embryos, bcl‐xL‐overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino‐mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl‐xL‐overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA‐seq public databases of human melanoma biopsies revealed that bcl‐xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co‐expression of bcl‐xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl‐xL‐induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl‐xL‐expressing melanoma.

Correction: Cutting Edge: TRPV4-Mediated Detection of Hyposmotic Stress by Skin Keratinocytes Activates Developmental Immunity.

Galindo-Villegas, J., A. Montalban-Arques, S. Liarte, S. de Oliveira, C. Pardo-Pastor, F. Rubio-Moscardo, J. Meseguer, M. A. Valverde, and V. Mulero. 2016. TRPV4-mediated detection of hyposmotic stress by skin keratinocytes activates developmental immunity. J . Immunol . 196: [738–749][1]. A

Cerebral Venous Thrombosis: Genetic Aspects

Cerebral venous thrombosis (CVT), an unusual form of stroke affecting the venous circulation rather than the more common arterial circulation, is being diagnosed with higher frequency due to increased awareness and ready access to MR. Multiple conditions have been reported in association with CVT. Risk factors and associated conditions for CVT can be categorized as permanent or transient. The most frequent permanent risk factors are genetic prothrombotic disorders. More than half of CVT patients have more than one associated condition or risk factor, in general a combination of a permanent (e.g. genetic prothrombotic disorder) with a transient risk factor (e.g. oral contraceptives, infection). Several publications from different regions of the world, systematic reviews and meta-analysis demonstrated that inherited thrombophilia increases the risk of CVT, especially in children. The most common genetic thrombophilic disorders which are important risk factors for CVT are Factor V Leiden and prothrombin G20210A mutations and protein C, protein S and antithrombin deficiencies. Routine screening of thrombophilia in CVT patients is not recommended. Thrombophilia screening may be performed in patients with high pre-test probability to carry severe thrombophilia (i.e., a personal and/or family history of venous thrombosis, young age at CVT, CVT without a transient or a permanent risk factor) to prevent recurrent venous thrombotic events.