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.

"Progressiver Islam" im ausgehenden Zarenreich: Das Plädoyer des St. Petersburger Imams und Regierungsbeamten Ataulla Bajazitov (1846-1911) für die Partizipation der Muslime an der modernen Zivilisation

Ataulla Bajazitov (1846-1911) fulfilled a social double role by serving his Tatar community in St. Petersburg as imam and the Russian state as military Muslim ‘cleric’, translator and teacher. By founding Russia’s first monolingual Tatar newspaper, initiating St. Petersburg’s first Friday mosque and presenting scriptural and rational arguments for the compatibility of Islam and the modern Civilization to a Russian-speaking public as early as 1883, he has been a pioneer among the Muslims in Russia in several respects. In contrast though to similar activities of his Russian contemporary, the Krim Tatar Ismail Gasprinskii (1851-1914), Bajazitov’s endeavours have remained almost unnoticed in Western scholarship. Also in Tatarstan, his books have been only recently reprinted. The present study analyzes Bajazitov’s three monographs written in Russian, namely A Response to Ernest Renan’s lecture “Islam and Science” (1883), The Relationship of Islam towards Science and People of Different Faith (1887) and Islam and Progress (1898). There, he exposes many positions that around that time started to become key arguments of Muslim reformers in the Near East for the progressivness of Islam. The study takes also into account reactions to Bajazitov’s monographs by Russian officers in Tashkent who tried to demonstrate the backwardness of Islam, especially Nikolai Petrovič Ostroumov’s (1846-1930) response in his book entitled Quran and Progress – On the intellectual awakening of today’s Russian Muslims (1901/1903).

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.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Treatment of optic neuritis with erythropoietin (TONE): a randomised, double-blind, placebo-controlled trial-study protocol.

INTRODUCTION Optic neuritis leads to degeneration of retinal ganglion cells whose axons form the optic nerve. The standard treatment is a methylprednisolone pulse therapy. This treatment slightly shortens the time of recovery but does not prevent neurodegeneration and persistent visual impairment. In a phase II trial performed in preparation of this study, we have shown that erythropoietin protects global retinal nerve fibre layer thickness (RNFLT-G) in acute optic neuritis; however, the preparatory trial was not powered to show effects on visual function. METHODS AND ANALYSIS Treatment of Optic Neuritis with Erythropoietin (TONE) is a national, randomised, double-blind, placebo-controlled, multicentre trial with two parallel arms. The primary objective is to determine the efficacy of erythropoietin compared to placebo given add-on to methylprednisolone as assessed by measurements of RNFLT-G and low-contrast visual acuity in the affected eye 6 months after randomisation. Inclusion criteria are a first episode of optic neuritis with decreased visual acuity to ≤0.5 (decimal system) and an onset of symptoms within 10 days prior to inclusion. The most important exclusion criteria are history of optic neuritis or multiple sclerosis or any ocular disease (affected or non-affected eye), significant hyperopia, myopia or astigmatism, elevated blood pressure, thrombotic events or malignancy. After randomisation, patients either receive 33 000 international units human recombinant erythropoietin intravenously for 3 consecutive days or placebo (0.9% saline) administered intravenously. With an estimated power of 80%, the calculated sample size is 100 patients. The trial started in September 2014 with a planned recruitment period of 30 months. ETHICS AND DISSEMINATION TONE has been approved by the Central Ethics Commission in Freiburg (194/14) and the German Federal Institute for Drugs and Medical Devices (61-3910-4039831). It complies with the Declaration of Helsinki, local laws and ICH-GCP. TRIAL REGISTRATION NUMBER NCT01962571.

(Table 2) Geochemistry of ore minerals and magnetization properties at the DSDP Leg 73 Holes

The samples investigated in this study come from DSDP Leg 73 Drill Holes 519A, 522B, and 524, all of which are in the South Atlantic. A general petrographic description of the basalts is given by Carman et al. (1984; doi:10.2973/dsdp.proc.73.120.1984).

Under ice shelf photographs from Drescher Inlet (Riiser Larsen Ice Shelf) taken by seal mounted cameras during expedition DRE2003

While modern sampling techniques, such as autonomous underwater vehicles, are increasing our knowledge of the fauna beneath Antarctic sea ice of only a few meters in depth, greater sampling difficulties mean that little is known about the marine life underneath Antarctic ice shelves over 100 m thick. In this study, we present underwater images showing the underside of an Antarctic ice shelf covered by aggregated invertebrate communities, most likely cnidarians and isopods. These images, taken at an average depth of 145 m, were obtained with a digital still camera system attached to Weddell seals Leptonychotes weddellii foraging just beneath the ice shelf. Our observations indicate that, similar to the sea floor, ice shelves serve as an important habitat for a remarkable amount of marine invertebrate fauna in Antarctica.

Hombre nuevo y Revolución : Indagando en las continuidades y reformulaciones discursivas constitutivas de la identidad colectiva perretista

El objetivo principal de la tesis es dar cuenta, a partir de un análisis intra e interdiscursivo, de las creencias que formaban parte del discurso politico de la organización armada PRT-ERP (Partido Revolucionario de los Trabajadores-Ejército Revolucionario del Pueblo), especialmente, en lo que respecta a su concepto de "hombre nuevo" y de "moral revolucionaria". Para ello, situamos nuestro análisis en diferentes niveles, tanto diacrónicos como sincrónicos. Por un lado, trabajamos aquellos discursos leídos por la militancia de los sesenta y setenta: Así se templó el acero de Nikolai Ostrovski y Reportaje al pie del patíbulo de Julius Fúcik; por el otro, analizamos documentos del PRT-ERP y textos literarios producidos en la misma época: Libro de Manuel de Julio Cortázar y Los pasos previos de Francisco Urondo. A través del análisis y comparación de las distintas interpelaciones subjetivas observamos las características del "hombre nuevo" perretista, en qué medida se parecen a las subjetividades convocadas desde la memoria revolucionaria, y, por otro lado, si las novelas de Urondo y Cortázar, con similares condiciones de producción, producen variaciones, cambios discursivos, o, tomando a Raiter (2003), son discursos opositores dentro del discurso dominante de la izquierda revolucionaria de ese entonces. Así concluimos que la identidad colectiva en cuestión estaba constituida por la creencia en una moral revolucionaria que hacía del sacrificio y la disciplina las virtudes principales del militante. Pero, en contraposición a lo afirmado por Pozzi (2004), estas creencias no son exclusivas de esta organización, ni de la época en que se producen dichas prácticas discursivas sino que forman parte de lo que llamamos "memorias del sacrificio revolucionario". Muchas de estas representaciones, por tanto, son constitutivas de las identidades revolucionarias, más allá de adoptar o no a la lucha armada como táctica política

Hombre nuevo y Revolución : Indagando en las continuidades y reformulaciones discursivas constitutivas de la identidad colectiva perretista

El objetivo principal de la tesis es dar cuenta, a partir de un análisis intra e interdiscursivo, de las creencias que formaban parte del discurso politico de la organización armada PRT-ERP (Partido Revolucionario de los Trabajadores-Ejército Revolucionario del Pueblo), especialmente, en lo que respecta a su concepto de "hombre nuevo" y de "moral revolucionaria". Para ello, situamos nuestro análisis en diferentes niveles, tanto diacrónicos como sincrónicos. Por un lado, trabajamos aquellos discursos leídos por la militancia de los sesenta y setenta: Así se templó el acero de Nikolai Ostrovski y Reportaje al pie del patíbulo de Julius Fúcik; por el otro, analizamos documentos del PRT-ERP y textos literarios producidos en la misma época: Libro de Manuel de Julio Cortázar y Los pasos previos de Francisco Urondo. A través del análisis y comparación de las distintas interpelaciones subjetivas observamos las características del "hombre nuevo" perretista, en qué medida se parecen a las subjetividades convocadas desde la memoria revolucionaria, y, por otro lado, si las novelas de Urondo y Cortázar, con similares condiciones de producción, producen variaciones, cambios discursivos, o, tomando a Raiter (2003), son discursos opositores dentro del discurso dominante de la izquierda revolucionaria de ese entonces. Así concluimos que la identidad colectiva en cuestión estaba constituida por la creencia en una moral revolucionaria que hacía del sacrificio y la disciplina las virtudes principales del militante. Pero, en contraposición a lo afirmado por Pozzi (2004), estas creencias no son exclusivas de esta organización, ni de la época en que se producen dichas prácticas discursivas sino que forman parte de lo que llamamos "memorias del sacrificio revolucionario". Muchas de estas representaciones, por tanto, son constitutivas de las identidades revolucionarias, más allá de adoptar o no a la lucha armada como táctica política