NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox^-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox^-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox^-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox^-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox^-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction.     

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne’s disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold’s egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne’s) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.     

Impact of early applied upper limb stimulation: The EXPLICIT-stroke programme design

Background  

Main claims of the literature are that functional recovery of the paretic upper limb is mainly defined within the first month post stroke and that rehabilitation services should preferably be applied intensively and in a task-oriented way within this particular time window. EXplaining PLastICITy after stroke (acronym EXPLICIT-stroke) aims to explore the underlying mechanisms of post stroke upper limb recovery. Two randomized single blinded trials form the core of the programme, investigating the effects of early modified Constraint-Induced Movement Therapy (modified CIMT) and EMG-triggered Neuro-Muscular Stimulation (EMG-NMS) in patients with respectively a favourable or poor probability for recovery of dexterity.     

Cell death induced by the Jak2 inhibitor, G6, correlates with cleavage of vimentin filaments

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 μM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.     

Identification of a novel inhibitor of JAK2 tyrosine kinase by structure-based virtual screening

Janus kinase 2 (JAK2) plays a crucial role in the pathomechanism of myeloproliferative disorders and hematologic malignancies. A somatic mutation of JAK2 (Val617Phe) was previously shown to occur in 98% of patients with polycythemia vera and 50% of patients with essential thrombocythemia and primary myelofibrosis. Thus, effective JAK2 kinase inhibitors may be of significant therapeutic importance. Here, we applied a structure-based virtual screen to identify novel JAK2 inhibitors. One JAK2 inhibitor in particular, G6, demonstrated remarkable potency as well as specificity, which makes it as a potential lead candidate against diseases related to elevated JAK2 tyrosine kinase activity.     

Dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats

The hypothesis was tested that dietary fructose vs glucose lowers copper solubility in the digesta in the small intestine of rats, which in turn causes a decreased copper absorption. Male rats were fed adequate-copper (5 mg Cu/kg) diets containing either fructose or glucose (709.4 g monosaccharide/kg) for a period of 5 wk. Fructose vs glucose significantly lowered copper concentrations in plasma and the liver, but did not alter hepatic copper mass. Fructose feeding resulted in a significantly lesser intestinal solubility of copper as based on either a smaller soluble fraction of copper in the liquid phase of small intestinal contents or a lower copper concentration in the liquid phase. The latter fructose effect can be explained by the observed fructose-induced increase in volume of liquid phase of intestinal digesta. After administration of a restricted amount of diet extrinsically labeled with⁶⁴Cu, rats fed fructose also had significantly lower soluble⁶⁴Cu fraction in the digesta of the small intestine. Although this study shows that fructose lowered intestinal copper solubility, only a slight reduction of apparent copper absorption was observed. It is suggested that the fructose-induced lowering of copper status in part counteracted the fructose effect on copper absorption at the level of the intestinal lumen.     

Effect of consumption of phenols from olives and extra virgin olive oil on LDL oxidizability in healthy humans

A high intake of olive oil has been proposed as an explanation for the low incidence of coronary heart disease in Mediterranean countries, but it is unclear whether olive oil offers specific benefits beyond a low content of saturated fat. Some types of extra virgin olive oil are rich in non-polar phenols, which might be taken up by plasma LDL particles and protect these from becoming atherogenic by oxidative modification. In a pilot study we found that consumption of 47 g fortified olive oil containing 31 mg phenols significantly increased the lag time of LDL oxidation from 112 ± 5 min before to 130 ± 7 min 2h after the meal. However, this study was not controlled, and in the current study we therefore investigated whether olive oil phenols increase the lag time of LDL oxidation in postprandial samples when compared with a control group.

Twelve healthy men and women consumed four different olive oil supplements with a meal on four separate occasions: one similar to the supplement in the pilot study (positive control); one containing mainly non-polar olive oil phenols; one containing mainly polar olive oil phenols; and one without phenols (placebo). Lag time significantly increased 2 h after the meals with the positive control (8 ± 2 min), the polar phenols (8 ± 2 min), and the placebo (8 ± 2 min), but not after the non-polar phenols (-0.4 ± 3 min). Increases were not statistically different between supplements.

These results indicate that the lag time of LDL-oxidation is increased after consumption of a meal. This increase is probably due to non-specific meal or time effects and not to phenols from olives or olive oil. Furthermore, these findings stress the need for adequate controlled studies to avoid misinterpretations of the data.     

Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.     

A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.