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Background and Aims: Ultra-microangiography (UMA) is a novel Doppler technique with optimized wall filtering that provides high sensitivity to low-velocity blood flows and optimized visualization of microcirculation. The aim of this pilot study was to compare intestinal vascularization assessed by color Doppler signals (CDS) and UMA., Methods: We investigated intestinal vascularization using UMA and CDS in 13 patients with confirmed inflammatory bowel disease (IBD). A cohort of 28 patients without structural bowel disease served as the control., Results: Microcirculation and dysregulated microcirculation in patients without and with inflammatory bowel disease can be visualized and quantified using UMA. In 83 % of IBD patients and 76% of non-IBD patients, a high resolution of intestinal perfusion could be achieved using UMA., Conclusions: To the best of our knowledge, this is the first study to investigate intestinal vascularization using UMA in patients with and without structural bowel disease. Quantification and visualization of intestinal vascularization should be further investigated in prospective studies and could help guide our therapy of patients with IBD.

Objective: Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells., Methods: Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors., Results: Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent., Conclusion: Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.

Aims/hypothesis: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D)., Methods: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments., Results: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 microg/ml) significantly (p<0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 microg/ml) significantly (p=0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p=0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients., Conclusions: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Compared with bone marrow-derived mesenchymal stem cells, adipose tissue-derived stromal cells (ADSC) do have an equal potential to differentiate into cells and tissues of mesodermal origin, such as adipocytes, cartilage, bone, and skeletal muscle. However, the easy and repeatable access to subcutaneous adipose tissue and the simple isolation procedures provide a clear advantage. Since extensive reviews focusing exclusively on ADSC are rare, it is the aim of this review to describe the preparation and isolation procedures for ADSC, to summarize the molecular characterization of ADSC, to describe the differentiation capacity of ADSC, and to discuss the mechanisms and future role of ADSC in cell therapy and tissue engineering. An initial effort has also been made to differentiate ADSC into hepatocytes, endocrine pancreatic cells, neurons, cardiomyocytes, hepatocytes, and endothelial/vascular cells. Whereas the lineage-specific differentiation into cells of mesodermal origin is well understood on a molecular basis, the molecular key events and transcription factors that initially allocate the ADSC to a lineage-specific differentiation are almost completely unknown. Decoding these molecular mechanisms is a prerequisite for developing novel cell therapies.

Heterogeneity of peripheral blood monocytes is characterized by specific patterns in the membrane expression of Fc gamma-receptor III (FcgammaRIII/CD16) and the lipopolysaccharide receptor (LPS receptor CD14), allowing discrimination of distinct subpopulations. The aim was to analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with modified lipoproteins by the use of either enzymatically degraded low density lipoprotein (E-LDL), acetylated low density lipoprotein (ac-LDL), oxidized low density lipoprotein (ox-LDL), or native low density lipoprotein. Highest E-LDL binding was observed on CD14(high) CD16(+) monocytes as determined by flow cytometry, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly isolated peripheral blood monocytes. This was accompanied by upregulation of the 2 class B scavenger receptors CLA-1/SR-BI (CD36 and LIMPII Analogous-1/scavenger receptor type B class I) and CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger-receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. In predifferentiated monocyte-derived macrophages, an anti-CD36 antibody inhibited cellular binding and uptake of E-LDL by approximately 20%, suggesting that recognition of these hydrolase-modified low density lipoprotein particles is mediated only in part by the class B scavenger receptor CD36.

Background and Aims: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism., Methods: ABCA1-deficient mice (ABCA1-/-) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays., Results: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1-/- mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1-/- mice occurs in peripheral tissues rather than the liver., Conclusions: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function.

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL(3), suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL(3)-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL(3)-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.

After transformation of the Streptomyces lividans chloramphenicol-sensitive, arginine-auxotrophic mutant strain AJ100 with a derivative of plasmid SCP2, some of the regenerated protoplasts contained an 8.2 kb DNA sequence amplified to several hundred copies per chromosome. The corresponding non-amplified sequence, called AUD4, was isolated from a lambda phage genomic library of S. lividans 1326. Two cytosine residues were the only directly repeated nucleotides at the ends of the element, indicating that AUD4 is a class I amplifiable sequence. The element mapped in the AseI-D fragment of the S. lividans chromosome, where other class I amplifications have been described. The complete element was sequenced and 10 ORFs were identified. Some of the deduced proteins are highly conserved in other organisms but a putative function could be attributed to only a few of them. Duplication of AUD4 by integration of an Escherichia coli plasmid carrying various parts of AUD4 and a thiostrepton-resistance gene in S. lividans AJ100, ZX7 or TK64 induced amplification of the integrated plasmid, AUD4 or both at high frequency.

Cholesteryl ester storage disease (CESD) and Wolman disease are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). We provide evidence that the strikingly more severe course of Wolman disease is caused by genetic defects of LAL that leave no residual enzyme activity. In a CESD patient, a G --> A mutation at position -1 of the exon 8 splice donor site results in skipping of exon 8 in 97% of the LAL hnRNA originating from this allele, while 3% are spliced correctly, resulting in full-length LAL enzyme. The mutant LAL mRNA codes for a protein lacking amino acids 254 to 277. On the other allele, a G --> T mutation leads to a premature stop codon at Gly245, resulting in inactive LAL enzyme. In addition, the previously identified Leu179 --> Pro mutation is present on this allele, and the LAL mRNA is rendered unstable by the premature stop codon. Analysis of two children with Wolman disease showed that both were homozygous for a G --> A mutation at position +1 of the same splice donor site as for the CESD patient, leading to skipping of exon 8. In contrast to the CESD patient, no correctly spliced mRNA was detectable. We have also expressed a wildtype LAL cDNA and the mutant LAL cDNA from one Wolman patient in Sf9 and H5 insect cells. We demonstrate that the LAL enzyme generated from the wildtype LAL cDNA was active in homogenates from Sf9 and H5 cells, while the enzyme with the internal deletion of 24 amino acids originating from the LAL cDNA of the Wolman patient was not. The combined data provide evidence that the only functionally relevant genetic difference between the Wolman patients and the CESD patient is that the splice defect in Wolman, which affects one of the invariable nucleotides of the splice consensus sequences (position +1), does not permit any correct splicing, whereas the defect observed in CESD (position -1) allows some correct splicing (3% of total LAL mRNA) and therefore the synthesis of functional enzyme.