Anti-inflammatory effects of methyl fumarate-derived iron carbonyl complexes (FumET-CORMs) and reactive oxygen species on dendritic cell differentiation

Einer der grundlegenden Schritte einer erfolgreichen Therapie von Th1/Th17-vermittelten Autoimmunerkrankungen ist die Induktion von Typ II dendritischen Zellen (DCs), welche im Folgenden zu einer Immundeviation hin zu einer anti-inflammatorischen Th2 Antwort führen. Im ersten Teil der Arbeit erfolgte deshalb die Charakterisierung neuer Substanzen, welche sowohl Kohlenstoffmonoxid als auch Fumarsäureester freisetzen. Die Behandlung mit diesen FumET-CORMs zeigte sich als sehr effektiv, da es zu einer robusten Induktion von Typ II DCs kam. Die dabei charakteristische Verminderung der STAT1 Phosphorylierung, Induktion von HO-1 und die daraus resultierende Inhibition der Zytokine IL-12p70 und IL-23 erfolgte sogar stärker und bei deutlich niedrigeren Konzentrationen als bei der Kontrollbehandlung mit Dimethylfumarat (DMF). Für eine detaillierte mechanistische Aufklärung der Therapie mit DMF wurde im zweiten Teil der Arbeit ein Cystin/Glutamat Antiporter Knockout Mausmodell herangezogen. In DCs dieser Mäuse wurde damit der Verlust des Radikalfängers Glutathion (GSH) und eine einhergehende Induktion von reaktiven Sauerstoffspezies auf genetischer Ebene erreicht, ohne Nebeneffekte einer pharmakologischen Behandlung. Entgegen der zuvor aufgestellten Hypothese resultierten diese Änderungen der Redox Homöostase nicht in der Differenzierung der DCs hin zu einem Typ II Phänotyp. Stattdessen führte die zusätzliche Behandlung dieser bereits GSH-depletierten Zellen trotzdem zur Modulation des HO-1 und STAT1 Signalweges, sowie der Inhibition der nachgeschalteten Zytokine IL-12p70 und IL-23. Dies zeigt, dass weitere Modifikationen durch das Fumarat für die Vermittlung der Effekte verantwortlich zu machen sind. Des Weiteren konnte auch eine Überkompensation des Antiporterverlustes über den Thioredoxin Signalweg durch den Einsatz von Auranofin ausgeschlossen werden. Die Modulation der Redox Homöostase allein war nicht ausreichend, um DCs in einen anti-inflammatorischen Typ II Phänotyp zu überführen, stattdessen spielen dabei wahrscheinlich noch weitere intrazelluläre Ziele der DMF Behandlung eine entscheidende Rolle.One of the fundamental steps in a successful therapy of Th1/Th17-mediated autoimmune diseases is the induction of type II dendritic cells (DCs), which subsequently cause an immune deviation towards an anti-inflammatory Th2 response. In the first part of the work, the characterization of new substances, which release carbon monoxide as well as fumaric acid esters, was carried out. The treatment with these FumET-CORMs proved to be highly effective, as a robust induction of type II DCs was achieved. The characteristic reduction of STAT1 phosphorylation, induction of HO-1 and the resulting inhibition of the cytokines IL-12p70 and IL-23 occurred even more pronounced and at significantly lower concentrations than in the control treatment with dimethyl fumarate (DMF). For a detailed mechanistic analysis of the DMF therapy, a cystine/glutamate antiporter knockout mouse model was employed in the second part of the study. In DCs of these mice, the loss of the scavenger of reactive oxygen species, glutathione (GSH), and an associated induction of said chemical entities was achieved on the genetic level without additional effects of pharmacological treatment. In contrast to the previously proposed hypothesis, these changes in redox homeostasis did not result in the differentiation of the DCs into a type II phenotype. Instead, the additional treatment of these already GSH-depleted cells still resulted in the HO-1 and STAT1 signaling pathway's modulation and the inhibition of downstream cytokines IL-12p70 and IL-23, indicating that additional targets of the fumarate are responsible for the mediation of these effects. In further experiments, an overcompensation of the antiporter loss via the thioredoxin signaling pathway could be excluded by using the inhibitor auranofin. Accordingly, the modulation of redox homeostasis alone is not sufficient to transform DCs into an anti-inflammatory type II phenotype. Instead, further intracellular targets of DMF treatment probably also play a decisive role in this process

Shear-wave velocity imaging of weathered granite in La Campana (Chile) from Bayesian inversion of micro-tremor H/V spectral ratios

Subsurface imaging of the regolith layer is an important tool for weathering zone characterization. For example, the extent of bedrock modification by weathering processes can be modelled by means of differing seismic velocities. We acquired a 360 m-long seismic profile in central Chile to characterise weathering at a semi-arid site. We used 87 3-component geophones, which continuously recorded ambient seismic noise for three days. The seismic line was centered at an 88 m deep borehole, providing core and downhole logging data for calibration. We extract Horizontal-to-Vertical Spectral Ratio (HVSR) curves along the seismic line to image the subsurface. Temporal analysis of the HVSR curves shows that the ambient noise vibrations recorded during the nighttime provide more stable HVSR curves. The trans-dimensional Bayesian Markov chain Monte Carlo (McMC) approach was used to invert the micro-tremor HVSR curves at each station to reconstruct 1D shear-wave velocity (Vs) models. The resulting individual 1D Vs models were merged to create a 2D Vs model along the linear seismic array in La Campana. The resulting Vs model shows an increase from 0.85 km/s at the surface to ca. 2.5 km/s at 100 m depth. We use the interface probability as a by-product of the Bayesian inversion to apply a more data-driven approach in identifying the different weathering layers. This method identified the boundary between saprolite and fractured bedrock at 42 m depth at the borehole, as evidenced by the interpretation of downhole logging data such as magnetic susceptibility. The resulting 2D Vs model of this site in Mediterranean climate shows a strong correlation between the interpreted weathering front at around 90-m depth and a higher precipitation rate in the study site compared to arid sites. The horizontal alignment of the weathering front indicates a correlation between the weathering front depth with topography and fractures in the bedrock

Cytocompatibility Evaluation of PEG-Methylsulfone Hydrogels

Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide-thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide-thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide-thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future

3-D imaging of the Balmuccia peridotite body (Ivrea–Verbano zone, NW-Italy) using controlled source seismic data

We provide new results from a controlled-source seismic experiment on the deepest part of the Val Sesia crust–mantle section of the Ivrea–Verbano zone (IVZ) in the Italian Alps. The IVZ is a tilted, almost complete section through the continental crust and exposes gabbros and peridotites in the structurally deepest level, coinciding with high-resolution gravity anomalies imaging the Ivrea geophysical body. The seismic experiment SEIZE (SEismic imaging of the Ivrea ZonE) was conducted along two crossing profiles: an NNE-SSW profile of ∼11 km length and an E-W profile of ∼16 km length. 432 vibration points were recorded with 110 receivers resulting in 24 392 traveltime picks. Inversion methods using Markov chain Monte Carlo techniques have been used to derive an isotropic 3-D P -wave velocity model based on first break traveltimes (refracted phases) from controlled source seismic data. Resulting seismic P -wave velocities ( V p ) range from 4.5 to 7.5 km s −1 , with an expected general trend of increasing velocities with depth. A sharp velocity change from low V p in the West to high V p in the East marks the Insubric Zone (ISZ), the Europe–Adria plate boundary. The most prominent feature of the 3-D tomography model is a high-velocity body ( V p increases from 6 to 7.5 km s −1 ) that broadens downwards. Its pointy shape peaks the surface East of Balmuccia at a location coincident with the exposed Balmuccia peridotite. Considering rock physics, high-resolution gravity and other geophysical data, we interpret this high-velocity body as dominantly composed of peridotite. The dimension of this seismically imaged peridotite material is far bigger than interpreted from geological cross-sections and requires a revision of previous models. The interpretation of ultramafic bodies in the IVZ as fragments of mantle peridotites interfingered in the crust during pre-Permian accretion is not supported by the new data. Instead, we re vi ve a model that the contact between the Balmuccia peridotite and the Permian mafic magmas might represent a fossil continental crust–mantle transition zone

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

BACKGROUND AND OBJECTIVES Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis. METHODS In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs. RESULTS Using an in vitro coculture system, we first identified perforin as an essential molecule for killing of BBB-ECs by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. DISCUSSION Therefore, perforin-dependent cytotoxicity plays a key role in the death of BBB-ECs inflicted by autoreactive CD8 T cells in a preclinical model and potentially represents a therapeutic target for CD8 T cell-mediated neuroinflammatory diseases, such as cerebral malaria and Susac syndrome

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (HUN-7293/cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p/Sec61α1 to confer resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and posttranslationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 homolog. We suggest "decatransin" as the name for this novel decadepsipeptide translocation inhibitor

Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial

Background A vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF) in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA). Methods Forty volunteers (35 vaccinees and five placebo recipients) were given two CN54rgp140/GLA-AF immunizations 30-71 weeks after the last HIV-MVA vaccination. These immunizations were delivered intramuscularly four weeks apart. Results The vaccine was safe and well tolerated except for one episode of asymptomatic hypoglycaemia that was classified as severe adverse event. Two weeks after the second HIV-MVA vaccination 34 (97%) of the 35 previously vaccinated developed Env-specific binding antibodies, and 79% and 84% displayed IFN-gamma ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C Env (included in HIV-DNA and protein boost), subtype B Env (included only in HIV-DNA) and CRF01_AE Env (included only in HIV-MVA) were significantly boosted by the CN54rgp140/GLA-AF immunizations. Functional antibodies detected using an infectious molecular clone virus/peripheral blood mononuclear cell neutralization assay, a pseudovirus/TZM-bl neutralization assay or by assays for antibody-dependent cellular cytotoxicity (ADCC) were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-gamma ELISpot responses to Env peptides were significantly enhanced. Four volunteers not primed with HIV-DNA and HIV-MVA before the CN54rgp140/ GLA-AF immunizations mounted an antibody response, while cell-mediated responses were rare. After the two Env subtype C protein immunizations, a trend towards higher median subtype C Env binding antibody titers was found in vaccinees who had received HIV-DNA and HIV-MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07). Conclusion We report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA

Thoracic aortopathy in Turner syndrome and the influence of bicuspid aortic valves and blood pressure: a CMR study

<p>Abstract</p> <p>Background</p> <p/> <p>To investigate aortic dimensions in women with Turner syndrome (TS) in relation to aortic valve morphology, blood pressure, karyotype, and clinical characteristics.</p> <p>Methods and results</p> <p>A cross sectional study of 102 women with TS (mean age 37.7; 18-62 years) examined by cardiovascular magnetic resonance (CMR- successful in 95), echocardiography, and 24-hour ambulatory blood pressure. Aortic diameters were measured by CMR at 8 positions along the thoracic aorta. Twenty-four healthy females were recruited as controls. In TS, aortic dilatation was present at one or more positions in 22 (23%). Aortic diameter in women with TS and bicuspid aortic valve was significantly larger than in TS with tricuspid valves in both the ascending (32.4 ± 6.7 vs. 26.0 ± 4.4 mm; p < 0.001) and descending (21.4 ± 3.5 vs. 18.8 ± 2.4 mm; p < 0.001) aorta. Aortic diameter correlated to age (R = 0.2 - 0.5; p < 0.01), blood pressure (R = 0.4; p < 0.05), a history of coarctation (R = 0.3; p = 0.01) and bicuspid aortic valve (R = 0.2-0.5; p < 0.05). Body surface area only correlated with descending aortic diameter (R = 0.23; p = 0.024).</p> <p>Conclusions</p> <p/> <p>Aortic dilatation was present in 23% of adult TS women, where aortic valve morphology, age and blood pressure were major determinants of the aortic diameter.</p