Ceruloplasmin activity in Holstein dairy cows: effects of lactation stages and anticoagulants

The effects of lactation stages and anticoagulants on the ceruloplasmin (Cp) activity were investigated in 240 Holstein dairy cows. The cows were classified according to their lactation stages into four different groups as close-up stage (3–1?week(s) antepartum), fresh-lactation stage (0–1?week postpartum), early-lactation stage (3–5?weeks postpartum), and mid-lactation stage (15–18?weeks postpartum). Each group consisted of 60 multiparous cows. Serum and plasma samples (heparin and EDTA) were obtained from each cow. The activity of ceruloplasmin was measured by p-phenylenediamine oxidation. Cp activity was higher in the group of fresh-lactation stage than other groups (P < 0.05). Pooled data of the four groups revealed higher Cp activities in heparinized plasma than serum and EDTA plasma and in serum than EDTA plasma (P < 0.05). Cp activities in serum and EDTA plasma were approximately 30% and 57%, respectively, lower relative to heparinized plasma. High correlation was found between heparinized plasma Cp and serum Cp (r = 0.82; P < 0.000). Suggestive reference values for serum Cp and heparinized plasma Cp were 88–334 and 143–459?mg/l, respectively. Ceruloplasmin undergoes a physiological increase just after calving and it is recommended to use heparinized plasma for estimation of ceruloplasmin in Holstein dairy cows.     

Long-lived autoreactive plasma cells drive persistent autoimmune inflammation

Aberrant production of autoantibodies by inappropriately self-reactive plasma cells is an inherent characteristic of autoimmune diseases. Several therapeutic strategies aim to deplete the plasma cell pool, or to prevent maturation of B cells into plasma cells. However, accepted views of B-cell biology are changing; recent findings show that long-lived plasma cells refractory to immunosuppressants and B-cell depletion therapies contribute to the maintenance of humoral memory and, in autoimmunity, to autoreactive memory. As a consequence of their longevity and persistence, long-lived plasma cells can support chronic inflammatory processes in autoimmune diseases by continuously secreting pathogenic antibodies, and they can contribute to flares of symptoms. As long-lived plasma cells are not sufficiently eliminated by current therapies, these findings are extremely relevant to the development of novel concepts for the treatment of autoimmune diseases. Thus, long-lived plasma cells appear to be a promising new therapeutic target.     

The effect of filtering on the two-global-flash mfERG: identifying the optimal range of frequency for detecting glaucomatous retinal dysfunction

Purpose

To study the effects of filtering bandwidth on the two-global-flash multifocal electroretinogram (mfERG) responses in primary open-angle glaucoma (POAG) compared with control subjects.

Methods

A two-global-flash mfERG (VERIS 6.06?, FMS III) was recorded in 20 healthy subjects and 22 POAG patients with a band-pass filter (BPF) of 1–300 Hz (103 Hexagons, M-sequence stimulus: Lmax 100 cd/m², Lmin < 1 cd/m², global flash: 200 cd/m²). The root-mean-square average of the central 10° was calculated. Three response epochs were analysed: the response to the focal flash, at 15–45 ms (DC), and the following two components induced by the effects of the preceding focal flash on the response to the global flashes at 45–75 ms (IC1) and at 75–105 ms (IC2). The following BPF settings were analysed: 1–300 Hz, 3–300 Hz, 10–300 Hz, 100–300 Hz, 200–300 Hz, 1–10 Hz, 1–100 Hz and 1–200 Hz.

Results

Filtering at 1–300 Hz showed significantly lower responses in POAG than in control subjects (p < 0.001) for all epochs analysed. At 1–100 Hz, this also held true even though the difference between the groups became smaller. At 1–10 Hz, responses were extremely small and did not differ between POAG and control (p > 0.5). This would suggest a filter setting of 10–300 Hz for mfERG recordings in POAG. However, when a filter setting of 10–300 Hz was compared to 1–300 Hz, with a filter setting of 10–300 Hz, the DC in POAG differed more (p < 0.0001) from normal than with 1–300 Hz (p = 0.0002). For IC1 and IC2, the stronger difference between POAG and control was found with 1–300 Hz (p < 0.0001) rather than with 10–300 Hz (p < 0.0001 and p = 0.0005, respectively). For the ‘oscillatory potentials’ at 100–300 Hz, POAG and control differed significantly in IC1 and IC2 (p < 0.05), but not in DC (p = 0.8). However, filtering at 200–300 Hz did not show a difference between POAG and control (p > 0.5). Thus, we applied a filter setting of 1–200 Hz, which seemed to be most sensitive in detecting glaucomatous retinal dysfunction (p < 0.0001).

Conclusions

A filter setting of 1–200 Hz appears most sensitive to detect glaucomatous damage if using a two-global-flash mfERG: using a band-pass filter a with lower low-frequency cut-off, containing the 10 Hz component, may be especially important in the small induced components that show glaucomatous damage most sensitively. High frequencies of 100–300 Hz also contain information that differentiates glaucoma from normal and thus should be included in the analysis.     

Influence of visual perception on spatial coding of saccadic eye movements and fixation

The Müller-Lyer illusion produces differences in visual length perception when lines of equal length are presented as double-arrows with inward-pointing and outward-pointing arrowheads, respectively. The aim of this study was to investigate the influence of length perception on the amplitude of saccadic eye movements and on postsaccadic fixation positions. Video recordings of eye movements were obtained from 35 healthy volunteers (age: 20-30 years) while they scanned the endpoints of Müller-Lyer targets. The targets were presented in horizontal and vertical orientations with an arrow length of 15°. Fixation periods between the saccades were adjusted at 1 s and 4 s. The final saccadic amplitudes between the endpoints of Müller-Lyer figures reflect the visually perceived length differences: lines with outward-pointing arrowheads were scanned with smaller saccades than lines with inward-pointing arrowheads. This bias in final saccadic amplitude was equal in both the horizontal and vertical target orientations as well as for 1-s and 4-s fixation periods. Postsaccadic drift after initial saccadic movements reduced the effect of the Müller-Lyer illusion depending on arrowhead and target orientation: outward-pointing horizontal arrows caused hypometric pulses, while inward-pointing vertical arrows caused hypermetric pulses. The results of this study demonstrate that spatial coding of both saccadic eye movements and fixation is strongly influenced by visual perception. While the influence of the Müller-Lyer illusion on final saccadic amplitudes did not depend on target orientation or fixation period, an additional pulse-step mismatch appeared in the horizontal (hypometria) and vertical (hypermetria) direction.     

Optimization of culture conditions for osteogenically‐induced mesenchymal stem cells in β‐tricalcium phosphate ceramics with large interconnected channels

The aim of this study was to optimize culture conditions for human mesenchymal stem cells (hMSCs) in β‐tricalcium phosphate ceramics with large interconnected channels. Fully interconnected macrochannels comprising pore diameters of 750 µm and 1400 µm were inserted into microporous β‐tricalcium phosphate (β‐TCP) scaffolds by milling. Human bone marrow‐derived MSCs were seeded into the scaffolds and cultivated for up to 3 weeks in both static and perfusion culture in the presence of osteogenic supplements (dexamethasone, β‐glycerophosphate, ascorbate). It was confirmed by scanning electron microscopic investigations and histological staining that the perfusion culture resulted in uniform distribution of cells inside the whole channel network, whereas the statically cultivated cells were primarily found at the surface of the ceramic samples. It was also determined that perfusion with standard medium containing 10% fetal calf serum (FCS) led to a strong increase (seven‐fold) of cell numbers compared with static cultivation observed after 3 weeks. Perfusion with low‐serum medium (2% FCS) resulted in moderate proliferation rates which were comparable to those achieved in static culture, although the specific alkaline phosphatase (ALP) activity increased by a factor of more than 3 compared to static cultivation. Gene expression analysis of the ALP gene also revealed higher levels of ALP mRNA in low‐serum perfused samples compared to statically cultivated constructs. In contrast, gene expression of the late osteogenic marker bone sialoprotein II (BSPII) was decreased for perfused samples compared to statically cultivated samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway

The triggering receptor expressed on myeloid cells 1 (TREM-1) has been implicated in the production of proinflammatory cytokines and chemokines during bacterial infection and sepsis. For downstream signal transduction, TREM-1 is coupled to the ITAM-containing adaptor DAP12. Here, we demonstrate that Bruton tyrosine kinase (Btk), a member of the Tec kinases, becomes phosphorylated upon TREM-1 triggering. In U937-derived cell lines, in which expression of Btk was diminished by shRNA-mediated knockdown, phosphorylation of Erk1/2 and PLCγ1 and Ca2? mobilization were reduced after TREM-1 stimulation. Importantly, TREM-1-induced production of the pro-inflammatory cytokines, TNF-α and IL-8, and up-regulation of activation/differentiation cell surface markers were impaired in Btk knockdown cells. Similar results were obtained upon TREM-1 stimulation of BMDCs of Btk(-/-) mice. The analysis of cells containing Btk mutants revealed that intact membrane localization and a functional kinase domain were required for TREM-1-mediated signaling. Finally, after TREM-1 engagement, TNF-α production by PBMCs was reduced in the majority of patients suffering from X-linked agammaglobulinemia (XLA), a rare hereditary disease caused by mutations in the BTK gene. In conclusion, our data identify Btk as a positive regulator in the ITAM-mediated TREM-1/DAP12 pathway and suggest its implication in inflammatory processes.     

Mutated RAS and constitutively activated Akt delineate distinct oncogenic pathways, which independently contribute to multiple myeloma cell survival

We have recently shown that approximately half of primary multiple myeloma (MM) samples display constitutive Akt activity, which disposes them for sensitivity to Akt inhibition. The Akt pathway counts among the signaling conduits for oncogenic RAS and activating mutations of K- and N-RAS frequently occur in MM. We therefore analyzed the relation between RAS mutation and Akt dependency in biopsies and CD138-purified cells from MM patients (n = 65) and the function of oncogenic RAS for MM cell survival in a range of MM cell lines with differing RAS status. Whereas RAS mutations do not predict Akt dependency, oncogenic RAS retains an important role for MM cell survival. Knockdown of either K- or N-RAS strongly decreased the viability of MM cells that harbored the respective oncogenic isoform, whereas ablation of wild-type RAS isoforms had little or no effect. Silencing of oncogenic RAS did not affect the Akt pathway, again indicating lack of a direct link. Combined inhibition of RAS and Akt strongly enhanced MM cell death. These data suggest that oncogenic RAS and Akt may independently contribute to MM cell survival. Targeting of both pathways could provide an attractive therapeutic strategy for patients with oncogenic RAS and dysregulated Akt signaling.     

Localization of the motor endplate zone in human skeletal muscles of the lower limb: anatomical guidelines for injection with botulinum toxin

Aim Botulinum toxin gives a local tone reduction by blocking neurotransmission at the motor endplate (MEP). The importance of using MEP‐targeted injections is demonstrated in animal models and in a clinical human study. The goal of this review is to present the available data on the localization of the MEP zone of frequently injected muscles of the lower limb and to compare this with current practice. Method Current knowledge on the localization of the MEP zone is based on some older histological studies, and for some of the more frequently injected muscles also on more recent anatomical dissection. Results We find that for some muscles the MEP zone can be more precisely demarcated, and for many other muscles that its location is somewhat different than the currently injected areas in clinical practice. Optimal injection sites are presented for gastrocnemius, soleus, tibialis posterior, semitendinosus, semimembranosus, gracilis, biceps femoris, rectus femoris, adductor longus, brevis and magnus, and psoas muscles. Interpretation We propose optimal injection sites in relation to external anatomical landmarks for the frequently injected muscles of the human lower limb to facilitate the efficiency of botulinum toxin injections.     

L-DRD4 genotype not associated with sensation seeking, gambling performance and startle reactivity in adolescents: the TRAILS study

The purpose of the present study was to investigate whether a length polymorphism in the dopamine receptor D4 gene (DRD4) was associated with approach related traits in adolescents. Data were used from TRAILS (TRacking Adolescents’ Individual Lives Survey), a population based cohort of Dutch adolescents. Sensation seeking, assessed with personality questionnaires from the participants themselves and their biological father and mother (n = 1282) was not associated with DRD4 genotype. Gambling performance (n = 591) and startle reactivity (n = 432) were not associated with DRD4 genotype either. Explanations for the dissociation might be sought in differences in development of the limbic system and the prefrontal cortex, both with high dopamine receptor D4 densities and both involved in approach related behaviours.