Hemoglobin and Hematocrit Levels in the Prediction of Complicated Crohn's Disease Behavior – A Cohort Study

Background

Markers that predict the occurrence of a complicated disease behavior in patients with Crohn''s disease (CD) can permit a more aggressive therapeutic regimen for patients at risk. The aim of this cohort study was to test the blood levels of hemoglobin (Hgb) and hematocrit (Hct) for the prediction of complicated CD behavior and CD related surgery in an adult patient population.

Methods

Blood samples of 62 CD patients of the German Inflammatory Bowel Disease-network “Kompetenznetz CED” were tested for the levels of Hgb and Hct prior to the occurrence of complicated disease behavior or CD related surgery. The relation of these markers and clinical events was studied using Kaplan-Meier survival analysis and adjusted COX-proportional hazard regression models.

Results

The median follow-up time was 55.8 months. Of the 62 CD patients without any previous complication or surgery 34% developed a complication and/or underwent CD related surgery. Low Hgb or Hct levels were independent predictors of a shorter time to occurrence of the first complication or CD related surgery. This was true for early as well as late occurring complications. Stable low Hgb or Hct during serial follow-up measurements had a higher frequency of complications compared to patients with a stable normal Hgb or Hct, respectively.

Conclusions

Determination of Hgb or Hct in complication and surgery naïve CD patients might serve as an additional tool for the prediction of complicated disease behavior.     

Biodistribution and Efficacy Studies of the Proteasome Inhibitor BSc2118 in a Mouse Melanoma Model

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

The chromosomal passenger complex activates Polo kinase at centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.     

Identification of low-frequency TRAF3IP2 coding variants in psoriatic arthritis patients and functional characterization

Introduction

In recent genome-wide association studies for psoriatic arthritis (PsA) and psoriasis vulgaris, common coding variants in the TRAF3IP2 gene were identified to contribute to susceptibility to both disease entities. The risk allele of p.Asp10Asn (rs33980500) proved to be most significantly associated and to encode a mutant protein with an almost completely disrupted binding property to TRAF6, supporting its impact as a main disease-causing variant and modulator of IL-17 signaling.

Methods

To identify further variants, exons 2-4 encoding both known TNF-receptor-associated factor (TRAF) binding domains were sequenced in 871 PsA patients. Seven missense variants and one three-base-pair insertion were identified in 0.06% to 1.02% of alleles. Five of these variants were also present in 931 control individuals at comparable frequency. Constructs containing full-length wild-type or mutant TRAF3IP2 were generated and used to analyze functionally all variants for TRAF6-binding in a mammalian two-hybrid assay.

Results

None of the newly found alleles, though, encoded proteins with different binding properties to TRAF6, or to the cytoplasmic tail of the IL-17-receptor α-chain, suggesting that they do not contribute to susceptibility.

Conclusions

Thus, the TRAF3IP2-variant p.Asp10Asn is the only susceptibility allele with functional impact on TRAF6 binding, at least in the German population.     

Quantotypic properties of QconCAT peptides targeting bovine host response to Streptococcus uberis

Mammalian host response to pathogens is associated with fluctuations in high abundant proteins in body fluids as well as in regulation of proteins expressed in relatively low copy numbers like cytokines secreted from immune cells and endothelium. Hence, efficient monitoring of proteins associated with host response to pathogens remains a challenging task. In this paper, we present a targeted proteome analysis of a panel of 20 proteins that are widely believed to be key players and indicators of bovine host response to mastitis pathogens. Stable isotope-labeled variants of two concordant proteotypic peptides from each of these 20 proteins were obtained through the QconCAT method. We present the quantotypic properties of these 40 proteotypic peptides and discuss their application to research in host-pathogen interactions. Our results clearly demonstrate a robust monitoring of 17 targeted host-response proteins. Twelve of these were readily quantified in a simple extraction of mammary gland tissues, while the expression levels of the remaining proteins were too low for direct and stable quantification; hence, their accurate quantification requires further fractionation of mammary gland tissues.     

JNK Pathway Activation Is Controlled by Tao/TAOK3 to Modulate Ethanol Sensitivity

Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.