Scaffold-cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source.

The properties of osteoblasts (OBs) isolated from the axial skeleton (tOBs) differ from OBs of the orofacial skeleton (mOBs) due to the different embryological origins of the bones. The aim of the study was to assess and compare the regenerative potential of allogenic bone marrow-derived mesenchymal progenitor cells with allogenic tOBs and allogenic mOBs in combination with a mPCL-TCP scaffold in critical-sized segmental bone defects in sheep tibiae. After 6 months, the tibiae were explanted and underwent biomechanical testing, micro-computed tomography (microCT) and histological and immunohistochemical analyses. Allogenic MPCs demonstrated a trend towards a better outcome in biomechanical testing and the mean values of newly formed bone. Biomechanical, microCT and histological analysis showed no significant differences in the bone regeneration potential of tOBs and mOBs in our in vitro study, as well as in the bone regeneration potential of different cell types in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Involvement of 15-lipoxygenase in early stages of atherogenesis.

The arachidonate 15-lipoxygenase which is expressed in atherosclerotic lesions is implicated in the oxidative modification of low density lipoproteins during atherogenesis. To obtain experimental in vivo evidence for this hypothesis, we analyzed the structure of oxygenated lipids isolated from the aorta of rabbits fed with a cholesterol-rich diet for different time periods and compared the pattern of oxygenation products with that isolated from low density lipoproteins treated in vitro with the pure rabbit 15-lipoxygenase and with oxygenated lipids isolated from advanced human atherosclerotic lesions. In early atherosclerotic lesions (12-wk cholesterol feeding), specific lipoxygenase products were detected whose structure was similar to those isolated from lipoxygenase-treated low density lipoproteins. The appearance of these products did coincide with the lipid deposition in the vessel wall. In later stages of atherogenesis (26-wk cholesterol feeding) the degree of oxidative modification of the tissue lipids did increase but the share of specific lipoxygenase products was significantly lower, suggesting an increasing overlay of the specific lipoxygenase products by nonenzymatic lipid peroxidation. In advanced human atherosclerotic lesions, large amounts of oxygenation products were detected whose structure suggests a nonenzymatic origin. These data suggest that the arachidonate 15-lipoxygenase is of pathophysiological importance during the early stages of atherogenesis. In later stages of plaque development nonenzymatic lipid peroxidation becomes more relevant.

Endothelin receptors in cultured renal epithelial cells.

We demonstrated the presence of specific binding sites for endothelin in the renal epithelial cell lines. MDCK and LLC-PK1. Endothelin binding induced mobilisation of intracellular calcium, as shown by an increase in 45Ca2+ efflux. This suggests a direct effect of endothelin on renal reabsorption in addition to the effects on the renal vasculature.

The influence of calcium on the deformability of human granulocytes.

Experiments were carried out to determine the importance of extra- and intracellular calcium for the deformability of granulocytes during filtration tests. At low calcium concentration (0.1 mM), granulocytes are more deformable than at the physiological free-calcium concentration of 1.25 mM. Increasing calcium concentrations up to 10 mM do not further impair the deformability. Parallel measurements of the intracellular calcium concentration by means of the fura fluorescence method were performed to explain this. Extracellular calcium concentrations between 1.25 mM and 10 mM had no influence on the intracellular calcium level. A lower extracellular calcium concentration (0.1 mM), however, decreased the intracellular calcium level. Therefore, the measurements of the intracellular calcium concentrations are consistent with the deformability results. Studies with the calcium entry blocker nifedipine suggested that a low intracellular calcium improves the deformability of granulocytes. It is concluded; (i) the physiological calcium concentration of 1.25 mM is stressful for isolated granulocytes, and (ii) the intracellular calcium level plays a crucial role in granulocyte deformability, i.e. the lower the intracellular calcium concentration the greater the deformability.

Drastic change in idiotypic but not antigen-binding specificity of an antibody by a single amino-acid substitution.

In proliferating B lymphocytes, somatic mutation of rearranged antibody variable (V)-region genes occurs at high frequency and may have a key role in the selection of these cells. It is of interest in this context to learn in which way single mutations can affect antigen binding and/or idiotypic specificity of an antibody. Previous investigations have analysed spontaneous mutants of myeloma and hybridoma cells in which the mutation affected the antigen-binding specificity of the antibody. Here we describe an antibody mutant that has fully retained antigen-binding specificity but has lost or drastically changed all V-region antigenic determinants (idiotopes) of the wild type as defined by monoclonal anti-idiotope antibodies. The mutant phenotype is generated by a glycine to arginine exchange in the middle of the diversity (D) element, at position 103 of the heavy chain.

Myeloma class switch variant X63.2aRI-16 secretes IgG2a and IgG1 with identical VDJ sequence.

The myeloma line X63.2aRI-16 isolated in vitro as spontaneous tertiary class switch variant (gamma 1 leads to gamma 2b leads to gamma 2a,gamma 1) of X63 (IgG1, chi) by fluorescence-activated cell sorting using class-specific antisera expresses two heavy chains. X63.2aRI-16 secretes IgG2a,chi as does the parental cell line X63.2a-25, a hybrid molecule containing gamma 2a and gamma 1 heavy chains and IgG1,chi. The chain of the latter protein is the product of a reverse class switch in regard to the embryonic order of the CH genes. We purified the immunoglobulins of X63.2aRI-16, isolated the CNBr fragments of both heavy chains and determined the complete amino acid sequence of the VH domains and of the CH domains up to the first subclass-specific residues. The remaining CNBr fragments of the CH domains were characterized by amino acid analyses. It was found that both heavy chains of the double producer possess identical VH domains and CH domains characteristic for the subclasses gamma 2a and gamma 1, respectively. The identities of the two VDJ sequences expressed in X63.2aRI-16 cells suggest that the reverse class switch event to gamma 1 cannot simply be explained on the basis of a deletion model involving only a single CH locus.