Microstates in language-related brain potential maps show noun-verb differences

Brain processing of grammatical word class was studied analyzing event-related potential (ERP) brain fields. Normal subjects observed a randomized sequence of single German nouns and verbs on a computer screen, while 20-channel ERP field map series were recorded separately for both word classes. Spatial microstate analysis was applied, based on the observation that series of ERP maps consist of epochs of quasi-stable map landscapes and based on the rationale that different map landscapes must have been generated by different neural generators and thus suggest different brain functions. Space-oriented segmentation of the mean map series identified nine successive, different functional microstates, i.e., steps of brain information processing characterized by quasi-stable map landscapes. In the microstate from 116 to 172 msec, noun-related maps differed significantly from verb-related maps along the left–right axis. The results indicate that different neural populations represent different grammatical word classes in language processing, in agreement with clinical observations. This word class differentiation as revealed by the spatial–temporal organization of neural activity occurred at a time after word input compatible with speed of reading.

Gender-specific modulation of immune system complement gene expression in marine medaka Oryzias melastigma following dietary exposure of BDE-47

BDE-47 is one of the most widely found congeners of PBDEs in marine environments. The potential immunomodulatory effects of BDE-47 on fish complement system were studied using the marine medaka Oryzias melastigma as a model fish. Three-month-old O. melastigma were subjected to short-term (5 days) and long-term (21 days) exposure to two concentrations of BDE-47 (low dose at 290 +/- 172 ng/day; high dose at 580 +/- 344 ng/day) via dietary uptake of BDE-47 encapsulated in Artemia nauplii. Body burdens of BDE-47 and other metabolic products were analyzed in the exposed and control fish. Only a small amount of debrominated product, BDE-28, was detected, while other metabolic products were all under detection limit. Transcriptional expression of six major complement system genes involved in complement activation: C1r/s (classical pathway), MBL-2 (lectin pathway), CFP (alternative pathway), F2 (coagulation pathway), C3 (the central component of complement system), and C9 (cell lysis) were quantified in the liver of marine medaka. Endogenous expression of all six complement system genes was found to be higher in males than in females (p < 0.05). Upon dietary exposure of marine medaka to BDE-47, expression of all six complement genes were downregulated in males at day 5 (or longer), whereas in females, MBl-2, CFP, and F2 mRNAs expression were upregulated, but C3 and C9 remained stable with exposure time and dose. A significant negative relationship was found between BDE-47 body burden and mRNA expression of C1r/s, CFP, and C3 in male fish (r = -0.8576 to -0.9447). The above findings on changes in complement gene expression patterns indicate the complement system may be compromised in male O. melastigma upon dietary exposure to BDE-47. Distinct gender difference in expression of six major complement system genes was evident in marine medaka under resting condition and dietary BDE-47 challenge. The immunomodulatory effects of BDE-47 on transcriptional expression of these complement components in marine medaka were likely induced by the parent compound instead of biotransformed products. Our results clearly demonstrate that future direction for fish immunotoxicology and risk assessment of immunosuppressive chemicals must include parallel evaluation for both genders.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Combined inhibition of complement C5 and CD14 markedly attenuates inflammation, thrombogenicity, and hemodynamic changes in porcine sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

Biactantial agreement in the Gongduk transitive verb in the broader Tibeto-Burman context

The Gongduk language is spoken in an enclave in south central Bhutan comprising several villages and hamlets in the mountains west of the Kurichu. The language occupies a distinct phylogenetic position within the Tibeto-Burman language family. The intransitive verb agrees for person and number with the subject, and the transitive shows biactantial agreement for person and number with both agent and patient. A morphological analysis has identified the individual agreement morphemes, their precise grammatical meaning and their patterns of allomorphy. The cognacy of the greater part of the desinences of the Gongduk verb with morphemes identifiable in the biactantial agreement systems of other Tibeto-Burman languages supports the view that at least a portion of such conjugational morphology must be reconstructed to the common ancestral language.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Different IVIG glycoforms affect in vitro inhibition of anti-ganglioside antibody-mediated complement deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.

Multiple roles of complement MASP-1 at the interface of innate immune response and coagulation

MASP-1 is a versatile serine protease that cleaves a number of substrates in human blood. In recent years it became evident that besides playing a crucial role in complement activation MASP-1 also triggers other cascade systems and even cells to mount a more powerful innate immune response. In this review we summarize the latest discoveries about the diverse functions of this multi-faceted protease. Recent studies revealed that among MBL-associated serine proteases, MASP-1 is the one responsible for triggering the lectin pathway via its ability to rapidly autoactivate then cleave MASP-2, and possibly MASP-3. The crystal structure of MASP-1 explains its more relaxed substrate specificity compared to the related complement enzymes. Due to the relaxed specificity, MASP-1 interacts with the coagulation cascade and the kinin generating system, and it can also activate endothelial cells eliciting pro-inflammatory signaling.

MASP-1 of the complement system promotes clotting via prothrombin activation.

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity, and it has been shown to activate coagulation factors. Here we studied the effects of MASP-1 on clot formation in whole blood (WB) and platelet-poor plasma (PPP) by thrombelastography and further elucidated the underlying mechanism. Cleavage of prothrombin by MASP-1 was investigated by SDS-PAGE and N-terminal sequencing of cleavage products. Addition of MASP-1 or thrombin to WB and PPP shortened the clotting time and clot formation time significantly compared to recalcified-only samples. The combination of MASP-1 and thrombin had additive effects. In a purified system, MASP-1 was able to induce clotting only in presence of prothrombin. Analysis of MASP-1-digested prothrombin confirmed that MASP-1 cleaves prothrombin at three cleavage sites. In conclusion, we have shown that MASP-1 is able to induce and promote clot formation measured in a global setting using the technique of thrombelastography. We further confirmed that MASP-1-induced clotting is dependent on prothrombin. Finally, we have demonstrated that MASP-1 cleaves prothrombin and identified its cleavage sites, suggesting that MASP-1 gives rise to an alternative active form of thrombin by cleaving at the cleavage site R393.

Hypofibrinolysis in type 2 diabetes: the role of the inflammatory pathway and complement C3

AIMS/HYPOTHESIS Plasminogen activator inhibitor-1 (PAI-1) has been regarded as the main antifibrinolytic protein in diabetes, but recent work indicates that complement C3 (C3), an inflammatory protein, directly compromises fibrinolysis in type 1 diabetes. The aim of the current project was to investigate associations between C3 and fibrinolysis in a large cohort of individuals with type 2 diabetes. METHODS Plasma levels of C3, C-reactive protein (CRP), PAI-1 and fibrinogen were analysed by ELISA in 837 patients enrolled in the Edinburgh Type 2 Diabetes Study. Fibrin clot lysis was analysed using a validated turbidimetric assay. RESULTS Clot lysis time correlated with C3 and PAI-1 plasma levels (r = 0.24, p < 0.001 and r = 0.22, p < 0.001, respectively). In a multivariable regression model involving age, sex, BMI, C3, PAI-1, CRP and fibrinogen, and using log-transformed data as appropriate, C3 was associated with clot lysis time (regression coefficient 0.227 [95% CI 0.161, 0.292], p < 0.001), as was PAI-1 (regression coefficient 0.033 [95% CI 0.020, 0.064], p < 0.05) but not fibrinogen (regression coefficient 0.003 [95% CI -0.046, 0.051], p = 0.92) or CRP (regression coefficient 0.024 [95% CI -0.008, 0.056], p = 0.14). No correlation was demonstrated between plasma levels of C3 and PAI-1 (r = -0.03, p = 0.44), consistent with previous observations that the two proteins affect different pathways in the fibrinolytic system. CONCLUSIONS/INTERPRETATION Similarly to PAI-1, C3 plasma levels are independently associated with fibrin clot lysis in individuals with type 2 diabetes. Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fibrinolysis potential in this population.

Ischemia/reperfusion injury: effect of simultaneous inhibition of plasma cascade systems versus specific complement inhibition.

Ischemia/reperfusion injury (IRI) may occur from ischemia due to thrombotic occlusion, trauma or surgical interventions, including transplantation, with subsequent reestablishment of circulation. Time-dependent molecular and structural changes result from the deprivation of blood and oxygen in the affected tissue during ischemia. Upon restoration of blood flow a multifaceted network of plasma cascades is activated, including the complement-, coagulation-, kinin-, and fibrinolytic system, which plays a major role in the reperfusion-triggered inflammatory process. The plasma cascade systems are therefore promising therapeutic targets for attenuation of IRI. Earlier studies showed beneficial effects through inhibition of the complement system using specific complement inhibitors. However, pivotal roles in IRI are also attributed to other cascades. This raises the question, whether drugs, such as C1 esterase inhibitor, which regulate more than one cascade at a time, have a higher therapeutic potential. The present review discusses different therapeutic approaches ranging from specific complement inhibition to simultaneous inhibition of plasma cascade systems for reduction of IRI, gives an overview of the plasma cascade systems in IRI as well as highlights recent findings in this field.