FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.     

Impact of Hormone-Associated Resistance to Activated Protein C on the Thrombotic Potential of Oral Contraceptives: A Prospective Observational Study

Introduction

The increased thrombotic risk of oral contraceptives (OC) has been attributed to various alterations of the hemostatic system, including acquired resistance to activated protein C (APC). To evaluate to what extent OC-associated APC resistance induces a prothrombotic state we monitored plasma levels of thrombin and molecular markers specific for thrombin formation in women starting OC use. Elevated plasma levels of thrombin have been reported to characterize situations of high thrombotic risk such as trauma-induced hypercoagulability, but have not yet been studied during OC use.

Patients and Methods

Blood samples were collected prospectively from healthy women (n = 21) before and during three menstruation cycles after start of OC. APC resistance was evaluated using a thrombin generation-based assay. Plasma levels of thrombin and APC were directly measured using highly sensitive oligonucleotide-based enzyme capture assay (OECA) technology. Thrombin generation markers and other hemostasis parameters were measured additionally.

Results

All women developed APC resistance as indicated by an increased APC sensitivity ratio compared with baseline after start of OC (p = 0.0003). Simultaneously, plasma levels of thrombin, prothrombin fragment 1+2, and of thrombin-antithrombin complexes did not change, ruling out increased thrombin formation. APC plasma levels were also not influenced by OC use, giving further evidence that increased thrombin formation did not occur.

Conclusions

In the majority of OC users no enhanced thrombin formation occurs despite the development of APC resistance. It cannot be ruled out, however, that thrombin formation might occur to a greater extent in the presence of additional risk factors. If this were the case, endogenous thrombin levels might be a potential biomarker candidate to identify women at high thrombotic risk during OC treatment. Large-scale studies are required to assess the value of plasma levels of thrombin as predictors of OC-associated thrombotic risk.     

Identification of blapsins A and B as potent small-molecule 14-3-3 inhibitors from the insect Blaps japanensis

In this study, we report three novel naturally occurring compounds, blapsins A (1) and B (2), and blapsamide (3) from the ethanol extract of the stink beetle, Blaps japanensis. The structures of these compounds were determined using spectroscopic methods. Compound 3 is a phenolic compound bearing a formamido group in the structure. Functional studies revealed that compounds 1 and 2 potently inhibited 14-3-3 protein-protein interactions (PPIs) with IC(50) values of 9.2 and 10.0 μM as determined by an ELISA assay, and 2.0 and 2.5 μM in an FP assay, respectively. These compounds represent the first example of natural small-molecule 14-3-3 inhibitors.     

In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.     

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.     

Comparison of Different Methods for the Evaluation of Treatment Effects from the Sleep EEG of Patients with Major Depression

In healthy subjects, sleep has a typical structure of three to five cyclic transitions between different sleep states. In major depression, this regular pattern is often destroyed but can be reestablished during successful treatment. The differences between healthy and abnormal sleep are generally assessed in a time-consuming process, which consists of determining the nightly variations of the sleep states (the hypnogram) based on visual inspection of the electroencephalogram (EEG), electrooculogram, and electromyogram. In this study, three different methods of sleep EEG analysis (spectrum, outlier, and recurrence analysis) have been examined with regard to their ability to extract information about treatment effects in patients with major depression. Our data suggest that improved sleep patterns during treatment with antidepressant medication can be identified with an appropriate analysis of the EEG. By comparing different methods, we have found that many treatment effects identified by spectrum analysis can be reproduced by the much simpler technique of outlier analysis. Finally, the cyclic structure of sleep and its modification by antidepressant treatment is best illustrated by a non-linear approach, the so-called recurrence method.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.