Beta2-adrenergic receptors mediate the differential effects of catecholamines on cytokine production of PBMC.

We determined characteristics of beta2-adrenergic receptors (beta2R) on peripheral blood mononuclear cells (PBMC) and cytokine production after mitogenic stimulation and coincubation with catecholamines. PBMCs were stimulated with interleukin-2 (IL-2), tetanus toxoid (TT), anti-CD3 antibody, or phytohemagglutinin (PHA). The cytokines interferon-gamma (IFN-gamma), IL-4, and IL-6 were determined by ELISA following coincubation with high-dose (10(-5) M) and low-dose (10(-9) M) epinephrine (EPI) and norepinephrine (NE). Intracellular IFN-gamma and IL-4 were studied by FACS analysis. The beta2R density was investigated using a radioligand binding assay. The stimuli induced various cytokine profiles in PBMCs. Synthesis of IFN-gamma was induced by all mitogens and could be suppressed by catecholamines (26%-85% reduction). In PHA-stimulated PBMCs, IL-4 synthesis was decreased by high-dose catecholamines (24%-28% reduction). Adding a beta-blocking agent attenuated most catecholamine effects. A highly significant negative correlation between the density of beta2R with IFN-gamma and IL-6 levels of PHA-activated PBMCs (r = -0.88 to -0.96, p < 0.01-< 0.001) was observed. The results indicate that the density of beta2R on PBMC plays a role in mediating the differential catecholamine effects on cytokine production of PBMC. Furthermore, changes in cytokine expression induced by catecholamines favor Th2 responses.     

Osteoprotegerin reduces the loss of periarticular bone mass in primary and secondary spongiosa but does not influence inflammation in rat antigen-induced arthritis

Objective: To assess the effect of osteoprotegerin (OPG) on joint swelling, synovial inflammation and cartilage destruction, periarticular and axial bone volume, and bone turnover in rat antigen-induced arthritis (AIA). Design: Rats were treated with OPG (3 mg/kg/day) at regular intervals from day 1 to day 20 of AIA. Disease activity was evaluated by measurement of joint swelling as well as, joint inflammation and destruction by histology. Bone volume and cellular turnover parameters of secondary spongiosa of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. Periarticular bone volume of the primary spongiosa at the right tibia head was measured by linear scanning. The findings were compared with those of PBS-treated AIA and healthy animals. Result: OPG treatment did not reduce joint swelling or histological signs of inflammation. Cartilage destruction was reduced. However, this effect did not reach statistical significance . In the secondary spongiosa OPG treatment reduced the loss of periarticular bone volume. However, the latter did not reach the level of healthy controls. OPG treatment significantly reduced parameters of bone formation and bone resorption. In the primary spongiosa, OPG-treatment led to a higher amount of mineralized tissue and a greater number of trabeculae compared to PBS-treated animals with AIA or healthy controls. In the axial skeleton, OPG treatment reduced bone formation and bone resorption parameters compared to healthy animals. This treatment had no influence on bone volume. Conclusions: In periarticular bone of AIA rats, OPG treatment reduced the loss of bone volume and decreased the bone turnover, thus preventing periarticular bone destruction. OPG treatment had no influence on inflammatory process or on cartilage destruction. Received 2 June 2005; returned for revision 26 July 2005; returned for final revision 9 August 2005; accepted by M. Parnham 24 September 2005 Presented in part at the 66. Annual Meeting of the American College of Rheumatology, New Orleans, U.S.A., October 2002, and at the 25. Annual Meeting of the American Society of Bone and Mineral Research, Minneapolis, USA, September 2003 Supported by grants from the Thuringian Ministry of Science, Research and Art (B307-01025, B378-01017), the Interdisciplinary Center for Clinical Research (IZKF) Jena, and the Deutsche Forschungsgemeinschaft (Br 1372/5-1) Osteoprotegerin was generously provided by Amgen (Thousand Oaks, CA, USA). Drs. Neumann and Oelzner contributed equally to this work.     

A novel locus for autosomal dominant, non-syndromic hearing impairment (DFNA18) maps to chromosome 3q22 immediately adjacent to the DM2 locus

Investigating a large German pedigree with non-syndromic hearing impairment of early onset and autosomal dominant mode of inheritance, linkage to known DFNA loci was excluded and in a subsequent genomic scan the phenotype was mapped to a 10-cM interval on chromosome 3q22; a maximum two-point lod score of 3.77 was obtained for the marker D3S1292. The new locus, DFNA18, is excluded from neighbouring deafness loci, DFNB15 and USH3, and it overlaps with the recently described DM2/PROMM locus. As hearing loss has been described as one feature of the PROMM phenotype, the DFNA18 gene might also be responsible for hearing loss in DM2/PROMM.     

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.     

Sequential expression of functions during macrophage differentiation in murine bone marrow liquid cultures

In the presence of a colony-stimulating factor, murine bone marrow cells proliferate and differentiate into macrophages. This culture system was taken as a model to study the expression of various functions by macrophages in the course of maturation. Several tests were performed daily and in parallel from the same batch of cells. It was found that certain functions were expressed early and were also characteristic for mature macrophages such as Fc receptors, phagocytosis of latex beads and unspecific esterase activity. Other functions appeared and disappeared in an ordered sequence, such as the response to macrophage migration inhibitory factor and chemotactic factor as well as the production of interferon and of plasminogen activator. The time course of functional expression was strongly dependent on proliferation of precursor cells as well as proliferation of differentiated macrophages. It is suggested that the phenotypic expression of functions during differentiation is the basis for the functional heterogeneity of macrophages.     

A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives

A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1–2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca²⁺] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.     

Expression of adhesion molecules in allergic lung diseases

Endothelial adherence and migration of leukocytes into tissue is mediated by different sets of adhesion molecules. The expression of these sets might not only preselect the types of leukocytes that enter the inflammatory sites, but also activate these leukocytes, induce adherence to epithelial cells, and cause the release of cytokines. Atopic asthma, extrinsic allergic alveolitis, and sarcoidosis as examples of immunologic lung diseases were investigated for the expression of adhesion molecules. Bronchial biopsies in chronic obstructive lung disease (COPD) and resected lung tissue of juvenile emphysema were chosen for controls. Immunohistochemistry was done on sections from bronchial and transbronchial biopsies and on smears from bronchoalveolar lavage cells. In all three types of immune disorders, lymphocytes expressed the integrins alpha4/beta1 (VLA4) and ICAM3, whereas lymphocytes in COPD bronchitis and in emphysema controls were unreactive. Eosinophils in atopic asthma bronchitis in contrast to COPD bronchitis also expressed both VLA4 and ICAM3. The expression of VCAM1 on endothelial cells was only seen in atopic asthma and was related to disease activity. The expression of other adhesion molecules was nonspecific. Expression of VCAM1 on endothelial cells and its ligand VLA4 on lymphocytes and eosinophils seems to be a specific event in atopic asthma. Expression of VLA4 and ICAM3 on lymphocytes, however, might be a specific event in all three immune reactions.     

Cyclodextrins in Polymer Synthesis: Free Radical Polymerization of β‐Cyclodextrin Complexes of Photosensitive Mesoionic 6‐Oxo‐1,6‐dihydropyrimidin‐3‐ium‐4‐olates in Aqueous Medium

Solid mesoionic 2‐[2‐(isopropenylcarbonyloxy)ethylthio]‐1‐methyl‐6‐oxo‐3‐phenyl‐5‐propyl‐1,6‐dihydropyrimidin‐3‐ium‐4‐olate was complexed in water using β‐cyclodextrin (β‐CD) and randomly methylated β‐CD, which resulted in polymerizable complexes with 2:1 stoichiometry. The β‐CD complex was characterized using ¹H NMR, ROESY NMR and UV spectroscopy. Polymerization of the complex prepared from methylated β‐CD led to a photosensitive polymer, which precipitated during polymerization and was nearly free of CD. Polymerization was carried out with a water‐soluble redox initiator. In addition, a copolymer with methyl methacrylate was prepared from the complexes, which showed a different mass‐dependent distribution in the incorporation in comparison to a copolymer prepared without CD in organic solvents.

     

A Novel Macroinitiator for the Synthesis of Triblock Copolymers via Atom Transfer Radical Polymerization: Polystyrene‐block‐poly(bisphenol A carbonate)‐block‐polystyrene and Poly(methyl methacrylate)‐block‐poly(bisphenol A carbonate)‐block‐poly(methyl methacrylate)

Summary: Bis(hydroxy)telechelic bisphenol A polycarbonate (PC) was prepared via melt polycondensation of bisphenol A (BPA) and diphenyl carbonate (DPC) using lanthanum(III ) acetylacetonate as a catalyst for transesterification. Subsequently, the polycarbonate was converted to a bifunctional macroinitiator for atom transfer radical polymerization (ATRP) with the reagent, α‐chlorophenylacetyl chloride. The macroinitiator was used for the polymerization of styrene (S) and methyl methacrylate (MMA) to give PS‐block‐PC‐block‐PS and PMMA‐block‐PC‐block‐PMMA triblock copolymers. These block copolymers were characterized by NMR and GPC. When styrene and methyl methacrylate were used in large excess, significant shifts toward high molecular weights were observed with quantitative consumption of the macroinitiator. Several ligands were studied in combination with CuCl as the ATRP catalyst. Kinetic studies reveal the controlled nature of the polymerization reaction for all the ligands used.

Formation of a bifunctional ATRP macroinitiator by esterification of bis(hydroxy)telechelic PC with α‐chlorophenylacetyl chloride.     

Temperature dependence of M pilus formation as demonstrated by electron microscopy

The formation of IncM plasmid encoded pili is dependent on the incubation temperature of the corresponding host strains. By labelling the short, rigid M pili with the donor specific bacteriophage luminal diameter M, the presence of pili at 30 degrees C but not at 37 degrees C incubation temperature could be demonstrated for E. coli K12 substrains carrying different IncM group plasmids. In contrast, such a temperature dependence of M pilus formation is not observed in S. typhimurium substrains.