Sophia Koutsogiannaki

Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria

The clinical management of paroxysmal nocturnal hemoglobinuria (PNH), a rare but life-threatening hematologic disease, has fundamentally improved with the introduction of a therapeutic that prevents complement-mediated intravascular hemolysis. However, a considerable fraction of PNH patients show insufficient treatment response and remain transfusion dependent. Because the current treatment only prevents C5-induced lysis but not upstream C3 activation, it has been speculated that ongoing opsonization with C3 fragments leads to recognition and phagocytosis of PNH erythrocytes by immune cells. Here, for the first time, we provide experimental evidence for such extravascular hemolysis and demonstrate that PNH erythrocytes from anti-C5-treated patients are phagocytosed by activated monocytes in vitro. Importantly, we show that this uptake can be mediated by the end-stage opsonin C3dg, which is not traditionally considered a phagocytic marker, via interaction with complement receptor 3 (CR3). Interaction studies confirmed that C3dg itself can act as a ligand for the binding domain of CR3. The degree of C3dg-mediated erythrophagocytosis in samples from different PNH patients correlated well with the individual level of C3dg opsonization. This finding may guide future treatment options for PNH but also has potential implications for the description and management of other complement-mediated diseases.

Effects of cadmium and 17β-estradiol on Mytilus galloprovincialis redox status. Prooxidant–antioxidant balance (PAB) as a novel approach in biomonitoring of marine environments

Cadmium and 17β-estradiol are rapidly accumulated in mussel tissues, making mussels excellent pollution sentinel organisms. The aim of the present study was to compare the oxidative responses of the mussels after 1, 3 and 7 days of exposure to cadmium with those to 17β-estradiol and subsequently, to suggest a multi-parametric approach for biomonitoring studies. Our results showed that environmentally relevant concentrations of either cadmium or 17β-estradiol for 1, 3 and 7 days induced oxidative stress in hemocytes of exposed mussels. The latter was determined by significantly increased ROS levels and apoptosis, by suppression of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) expression levels and subsequent increased prooxidant levels, as measured by prooxidant-antioxidant balance (PAB) assay. To our knowledge this is the first time that prooxidant-antioxidant balance is evaluated in invertebrates as an index of oxidative stress. The simultaneous use of the parameters of prooxidant-antioxidant balance and antioxidant enzymes expression patterns, in combination with ROS production levels and apoptosis, in mussel hemocytes is suggested as an approach that may help to better evaluate the impact of environmental pollution on marine organisms and thereupon ecosystems.

Structural Implications for the Formation and Function of the Complement Effector Protein iC3b

Complement-mediated opsonization, phagocytosis, and immune stimulation are critical processes in host defense and homeostasis, with the complement activation fragment iC3b playing a key effector role. To date, however, there is no high-resolution structure of iC3b, and some aspects of its structure-activity profile remain controversial. Here, we employed hydrogen–deuterium exchange mass spectrometry to describe the structure and dynamics of iC3b at a peptide resolution level in direct comparison with its parent protein C3b. In our hydrogen–deuterium exchange mass spectrometry study, 264 peptides were analyzed for their deuterium content, providing almost complete sequence coverage for this 173-kDa protein. Several peptides in iC3b showed significantly higher deuterium uptake when compared with C3b, revealing more dynamic, solvent-exposed regions. Most of them resided in the CUB domain, which contains the heptadecapeptide C3f that is liberated during the conversion of C3b to iC3b. Our data suggest a highly disordered CUB, which has acquired a state similar to that of intrinsically disordered proteins, resulting in a predominant form of iC3b that features high structural flexibility. The structure was further validated using an anti-iC3b mAb that was shown to target an epitope in the CUB region. The information obtained in this work allows us to elucidate determinants of iC3b specificity and activity and provide functional insights into the protein’s recognition pattern with respect to regulators and receptors of the complement system.

Prolonged exposure to volatile anesthetic isoflurane worsens the outcome of polymicrobial abdominal sepsis

Sepsis continues to result in high morbidity and mortality. General anesthesia is often administered to septic patients, but the impacts of general anesthesia on host defense are not well understood. General anesthesia can be given by volatile and intravenous anesthetics. Our previous in vitro study showed that volatile anesthetic isoflurane directly inhibits leukocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1), critical adhesion molecules on leukocytes. Thus, the role of isoflurane exposure on in vivo LFA-1 and Mac-1 function was examined using polymicrobial abdominal sepsis model in mice. As a comparison, intravenous anesthetic propofol was given to a group of mice. Wild type, LFA-1, Mac-1, and adhesion molecule-1 knockout mice were used. Following the induction of polymicrobial abdominal sepsis by cecal ligation and puncture, groups of mice were exposed to isoflurane for either 2 or 6 h, or to propofol for 6 h, and their outcomes were examined. Bacterial loads in tissues and blood, neutrophil recruitment to the peritoneal cavity and phagocytosis were studied. Six hours of isoflurane exposure worsened the outcome of abdominal sepsis (P < .0001) with higher bacterial loads in tissues, but 2 h of isoflurane or 6 h of propofol exposure did not. Isoflurane impaired neutrophil recruitment to the abdominal cavity by inhibiting LFA-1 function. Isoflurane also impaired bacterial phagocytosis via complement receptors including Mac-1. In conclusion, prolonged isoflurane exposure worsened the outcome of experimental polymicrobial abdominal sepsis and was associated with impaired neutrophil recruitment and bacterial phagocytosis via reduced LFA-1 and Mac-1 function.

Complement C3 inhibitor Cp40 attenuates xenoreactions in pig hearts perfused with human blood

The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3.

The effect of different anesthetics on tumor cytotoxicity by natural killer cells.

A number of retrospective studies have suggested that choice of anesthetic drugs during surgical tumor resection might affect tumor recurrence/metastasis, or outcome of patients. The recent study showed that volatile anesthetics-based general anesthesia was associated with the worse outcomes than intravenous anesthetics-based general anesthesia. However, the underlying mechanism is yet to be determined. Because natural killer (NK) cells are implicated as important immune cells for tumor recurrence/metastasis in the perioperative period, we examined the effect of different anesthetics on NK cell-mediated tumor cytotoxicity. Because adhesion molecule leukocyte function-associated antigen-1 (LFA-1) is functionally important in NK cells and is inhibited by commonly used volatile anesthetics isoflurane and sevoflurane, we hypothesized that these anesthetics would attenuate NK cell-mediated cytotoxicity. Using human NK cell line NK92-MI cells and tumor cell line K562 cells as a model system, we performed cytotoxicity, proliferation, conjugation and degranulation assays. Lytic granule polarization was also assessed. We showed that isoflurane, sevoflurane and LFA-1 inhibitor BIRT377 attenuated cytotoxicity, and reduced conjugation and polarization, but not degranulation of NK cells. Our data suggest that isoflurane and sevoflurane attenuated NK cell-mediated cytotoxicity at least partly by their LFA-1 inhibition in vitro. Whether or not isoflurane and sevoflurane attenuate NK cell-mediated tumor cytotoxicity in patients needs to be determined in the future.

Unanticipated hospital admission in pediatric patients with congenital heart disease undergoing ambulatory noncardiac surgical procedures

An increasing number of surgical and nonsurgical procedures are being performed on an ambulatory basis in children. Analysis of a large group of pediatric patients with congenital heart disease undergoing ambulatory procedures has not been undertaken.

Safety profile after prolonged C3 inhibition

The central component of the complement cascade, C3, is involved in various biological functions, including opsonization of foreign bodies, clearance of waste material, activation of immune cells, and triggering of pathways controlling development. Given its broad role in immune responses, particularly in phagocytosis and the clearance of microbes, a deficiency in complement C3 in humans is often associated with multiple bacterial infections. Interestingly, an increased susceptibility to infections appears to occur mainly in the first two years of life and then wanes throughout adulthood. In view of the well-established connection between C3 deficiency and infections, therapeutic inhibition of complement at the level of C3 is often considered with caution or disregarded. We therefore set out to investigate the immune and biochemical profile of non-human primates under prolonged treatment with the C3 inhibitor compstatin (Cp40 analog). Cynomolgus monkeys were dosed subcutaneously with Cp40, resulting in systemic inhibition of C3, for 1 week, 2 weeks, or 3 months. Plasma concentrations of both C3 and Cp40 were measured periodically and complete saturation of plasma C3 was confirmed. No differences in hematological, biochemical, or immunological parameters were identified in the blood or tissues of animals treated with Cp40 when compared to those injected with vehicle alone. Further, skin wounds showed no signs of infection in those treated with Cp40. In fact, Cp40 treatment was associated with a trend toward accelerated wound healing when compared with the control group. In addition, a biodistribution study in a rhesus monkey indicated that the distribution of Cp40 in the body is associated with the presence of C3, concentrating in organs that accumulate blood and produce C3. Overall, our data suggest that systemic C3 inhibition in healthy adult non-human primates is not associated with a weakened immune system or susceptibility to infections.

Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production

Propofol is an intravenous anesthetic that produces its anesthetic effect, largely via the GABAA receptor in the CNS, and also reduces the N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐induced neutrophil respiratory burst. Because fMLP‐stimulated neutrophils produce leukotriene (LT)B4, we examined the effect of propofol on LTB4 production in vivo and in vitro. Cecal ligation and puncture surgery was performed in mice, with or without exposure to propofol. Propofol attenuated the production of 5‐lipoxygenase (5‐LOX)‐related arachidonic acid (AA) derivatives in the peritoneal fluid. Also, in the in vitro experiments on fMLP‐stimulated neutrophils and 5‐LOX‐transfected human embryonic kidney cells, propofol attenuated the production of 5‐LOX‐related AA derivatives. Based on these results, we hypothesized that propofol would directly affect 5‐LOX function. Using meta‐azi‐propofol (AziPm), we photolabeled stable 5‐LOX protein, which had been used to solve the X‐ray crystallographic structure of 5‐LOX, and examined the binding site(s) of propofol on 5‐LOX. Two propofol binding pockets were identified near the active site of 5‐LOX. Alanine scanning mutagenesis was performed for the residues of 5‐LOX in the vicinity of propofol, and we evaluated the functional role of these pockets in LTB4 production. We demonstrated that these pockets were functionally important for 5‐LOX activity. These two pockets can be used to explore a novel 5‐LOX inhibitor in the future. —Okuno, T., Koutsogiannaki, S., Ohba, M., Chamberlain, M., Bu, W., Lin, F.‐Y., Eckenhoff, R. G., Yokomizo T., Yuki, K. Intravenous anesthetic propofol binds to 5‐lipoxygenase and attenuates leukotriene B4 production. FASEB J. 31, 1584–1594 (2017) www.fasebj.org

The Differential Effects of Anesthetics on Bacterial Behaviors.

Volatile anesthetics have been in clinical use for a long period of time and are considered to be promiscuous by presumably interacting with several ion channels in the central nervous system to produce anesthesia. Because ion channels and their existing evolutionary analogues, ion transporters, are very important in various organisms, it is possible that volatile anesthetics may affect some bacteria. In this study, we hypothesized that volatile anesthetics could affect bacterial behaviors. We evaluated the impact of anesthetics on bacterial growth, motility (swimming and gliding) and biofilm formation of four common bacterial pathogens in vitro. We found that commonly used volatile anesthetics isoflurane and sevoflurane affected bacterial motility and biofilm formation without any effect on growth of the common bacterial pathogens studied here. Using available Escherichia coli gene deletion mutants of ion transporters and in silico molecular docking, we suggested that these altered behaviors might be at least partly via the interaction of volatile anesthetics with ion transporters.