Scavenging of Dickkopf-1 by macromer-based biomaterials covalently decorated with sulfated hyaluronan displays pro-osteogenic effects.

Dickkopf-1 (DKK1), a Wnt inhibitor secreted by bone marrow stromal cells (MSC), is known to play an important role in long-term non-union bone fracture defects and glucocorticoid induced osteoporosis. Mitigating its effects in early bone defects could improve osteogenesis and bone defect healing. Here, we applied a biomaterial strategy to deplete a defect environment from DKK1 by scavenging the protein via a macromer-based biomaterial covalently decorated with sulfated hyaluronan (sHA3). The material consisted of cross-copolymerized three-armed macromers with a small anchor molecule. Using the glycidyl anchor, polyetheramine (ED900) could be grafted to the material to which sHA3 was efficiently coupled in a separate step. For thorough investigation of material modification, flat material surfaces were generated by fabricating them on glass discs. The binding capability of sHA3 for DKK1 was demonstrated in this study by surface plasmon resonance measurements. Furthermore, the surfaces demonstrated the ability to scavenge and inactivate pathologic amounts of DKK1 from complex media. In a combinatory approach with Wnt3a, we were able to demonstrate that DKK1 is the preferred binding partner of our sHA3-functionalized surfaces. We validated our findings in a complex in vitro setting of differentiating SaOS-2 cells and primary hMSC. Here, endogenous DKK-1 was scavenged resulting in increased osteogenic differentiation indicating that this is a consistent biological effect irrespective of the model system used. Our study provides insights in the mechanisms and efficiency of sHA3 surface functionalization for DKK1 scavenging, which may be used in a clinical context in the future.

The killer cell lectin-like receptor B1 (KLRB1) 503T>C polymorphism (rs1135816) and acute rejection after liver transplantation.

The killer cell lectin-like receptor B1 (KLRB1) gene encodes for CD161 expressed by different subsets of leukocytes involved in the development of acute liver transplant rejection. The single nucleotide polymorphism (SNP) 503T>C (rs1135816) in the KLRB1 gene represents a missense mutation modifying functional properties of CD161. The aim of our study is to determine whether the SNP 503T>C is associated with acute liver transplant rejection. We genotyped the SNP for 163 liver recipients without acute rejection, 125 recipients with a single acute rejection, and 53 recipients with multiple acute rejections. The genotype frequencies within the groups did not show any significant difference. Our data suggest that the SNP 503T>C has no impact on the susceptibility of acute liver transplant rejection.

Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function.

[Optimised rate of specific, revascularising therapies in acute stroke--what is the aim? Considerations from the experience of a tertiary centre]. / Optimierte rate spezifischer revaskularisierender therapien beim akuten ischämischen schlaganfall--was ist das ziel? Erwägungen anhand der erfahrungen eines tertiären versorgungszentrums.

BACKGROUND: Recanalisation favourably influences outcome in acute stroke. Improved endovascular approaches seem to have higher recanalisation rates than systemic thrombolysis. Substantial efforts have been undertaken to increase the proportion of patients to whom these therapies can be applied. It is still unclear what rates can be realised in a clinical setting. PATIENTS AND METHODS: This is a retrospective single-centre analysis of patients with acute ischaemic stroke and specific recanalisation therapy primarily admitted to our tertiary care centre from 1/2010 to 3/2012. RESULTS: 20 % of patients received systemic thrombolysis, 20 % of these additional endovascular strategies. Pathological multimodal CT patterns were more common in patients not fulfilling the inclusion criteria for thrombolysis. Short-term clinical outcomes were similar in on-label and off-label applications. CONCLUSION: Structured clinical pathways including multimodal CT imaging are useful in identifying patients likely to profit from revascularisation therapies. Based upon our data, some realistic aims concerning therapy rates in patients with ischaemic stroke treated in everyday practice may be formulated (20/20 in 2020).

Intermittent hyperthermia enhances skeletal muscle regrowth and attenuates oxidative damage following reloading.

Skeletal muscle reloading following disuse is characterized by profound oxidative damage. This study tested the hypothesis that intermittent hyperthermia during reloading attenuates oxidative damage and augments skeletal muscle regrowth following immobilization. Forty animals were randomly divided into four groups: control (Con), immobilized (Im), reloaded (RC), and reloaded and heated (RH). All groups but Con were immobilized for 7 days. Animals in the RC and RH groups were then reloaded for 7 days with (RH) or without (RC) hyperthermia (41-41.5 degrees C for 30 min on alternating days) during reloading. Heating resulted in approximately 25% elevation in heat shock protein expression (P < 0.05) and an approximately 30% greater soleus regrowth (P < 0.05) in RH compared with RC. Furthermore, oxidant damage was lower in the RH group compared with RC because nitrotyrosine and 4-hydroxy-2-nonenol were returned to near baseline when heating was combined with reloading. Reduced oxidant damage was independent of antioxidant enzymes (manganese superoxide dismutase, copper-zinc superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase). In summary, these data suggest that intermittent hyperthermia during reloading attenuates oxidative stress and improves the rate of skeletal muscle regrowth during reloading after immobilization.

Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and Sorghum bicolor (L.) Moench.

We report here an in vitro culture system that provides reliable, highly efficient regeneration from immature embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench]. Immature embryos were isolated 10-20 days after pollination and cultured on various L3 media. The influence of different parameters during the callus induction phase was examined with respect to the regeneration rate: (1) the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and various cytokinins; (2) the addition of AgNO3; (3) the use of maltose or sucrose as a carbon source. Modifications in the phytohormones alone resulted in the regeneration of fertile sorghum plants at high efficiency. Significant increases in the regeneration rates of pearl millet genotypes were achieved by the combination of sucrose as a carbon source and silver nitrate as a potential ethylene inhibitor.

Learning-induced expression of meningeal ependymin mRNA and demonstration of ependymin in neurons and glial cells.

The turnover of a CNS-specific cell adhesion glycoprotein, ependymin, has earlier been found to increase during periods of neuronal plasticity. Here, ependymin mRNA expression was analyzed by semiquantitative in situ hybridization in goldfish. Learning of an active avoidance response resulted in a significant increase in ependymin mRNA expression 20 min to 4 h after acquisition of the task. In contrast, yoked control animals that were exposed to the same numbers of conditioned and unconditioned stimuli in a random, unpaired manner exhibited a strong down-regulation of ependymin mRNA. Hybridization signals were also increased by injection of anti-ependymin antiserum into brain ventricles. Ependymin mRNA was exclusively localized to reticular-shaped fibroblasts of the inner endomeningeal cell layer. Immunoelectron microscopic investigation, however, revealed ependymin also in distinct neuronal and glial cell populations in which no ependymin mRNA had been detected. Uptake of meningeal protein factors into glial and neuronal cells may therefore be of functional importance for plastic adaptations of the CNS.

Inhibition of memory consolidation after active avoidance conditioning by antisense intervention with ependymin gene expression.

A rapid increase in ependymin mRNA expression demonstrated by semiquantitative in situ hybridization after avoidance conditioning on goldfish suggested a molecular demand for newly synthesized ependymin translation product. To inhibit de novo synthesis of ependymin molecules without interference with preexisting ones, 18 mer anti-ependymin mRNA-phosphorothioate oligodeoxynucleotides (S-ODNs) were injected into the perimeningeal brain fluid before active avoidance training. S-ODN-injected animals learned the avoidance response; however, they were amnesic in the test. When injected into overtrained animals, S-ODNs did not interfere with retrieval or performance of the avoidance response. Fish treated with randomized S-ODN sequences served as further controls. Incorporation of S-ODNs was analyzed by injection of fluorescein isothiocyanate (FITC)-conjugated oligodeoxynucleotide probes. Microscopic observation revealed strong FITC-S-ODN fluorescence in reticular-shaped fibroblasts, the only known site of ependymin synthesis. Results demonstrate that selective inhibition of ependymin gene expression in vivo can specifically prevent memory formation. We conclude that in particular the newly synthesized ependymin molecules are involved in memory consolidation, possibly because they have not yet undergone irreversible molecular changes, which have been reported of this glycoprotein in a low-calcium microenvironment.

Neuronal plasticity depending on a glycoprotein synthesized in goldfish leptomeninx.

Transcription of a calcium and zinc binding, nervous system-specific cell adhesion glycoprotein, ependymin, in goldfish leptomeninx was significantly enhanced after active avoidance conditioning, followed by enhanced translation and secretion. Inactivation of secreted ependymin by injected antisera interfered with behavioral adaptations. In addition to the site of synthesis in reticular cells of the leptomeninx electronmicroscopic immunochemistry localized the protein to tectal neurons of the superficial plexiform and the periventricular cell layers. Detection of ependymin in cells where it is not synthesized, namely in neurons, suggests a re-uptake during functional activity of the CNS and assigns a pivotal role to the cerebrospinal and interstitial brain fluids for the distribution of protein factors that support axonal growth and neuronal plasticity.

Arc and Sfr functions of the Escherichia coli K-12 arcA gene product are genetically and physiologically separable.

The Escherichia coli arcA gene product regulates chromosomal gene expression in response to deprivation of oxygen (Arc function; Arc stands for aerobic respiration control) and is required for expression of the F plasmid DNA transfer (tra) genes (Sfr function; Sfr stands for sex factor regulation). Using appropriate lacZ fusions, we have examined the relationship between these two genetic regulatory functions. Arc function in vivo was measured by anaerobic repression of a chromosomal sdh-lacZ operon fusion (sdh stands for succinate dehydrogenase). Sfr function was measured by activation of a plasmid traY-lacZ gene fusion. An eight-codon insertion near the 5' terminus of arcA, designated arcA1, abolished Arc function, as previously reported by S. Iuchi and E.C.C. Lin (Proc. Natl. Acad. Sci. USA 85:1888-1892, 1988), but left Sfr function largely (greater than or equal to 60%) intact. Similarly, the arcB1 mutation, which depressed sdh expression and is thought to act by abolishing the signal input that elicits ArcA function, had little effect (less than or equal to 20%) on the Sfr function of the arcA+ gene product. Conversely, a valine-to-methionine mutation at codon 203 (the sfrA5 allele) essentially abolished Sfr activity without detectably altering Arc activity. These data indicate that Sfr and Arc functions are separately expressed and regulated properties of the same protein.