A flow cytometric screening test for detergent-resistant surface antigens in monocytes.

Rafts resemble cholesterol- and glycosphingolipid-enriched, liquid-ordered plasma membrane microdomains, showing resistance to nonionic detergents, and are involved in various cellular processes. In the present study, we have tested surface antigens on resting and lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes for their detergent resistance (i.e. raft-association), by flow cytometry. Constitutive (CD14, CD32, CD55), or LPS-induced (CD81) raft-association, and detergent solubility (i.e. exclusion of rafts) (CD71) of monocyte antigens in the presence of 0.01% Triton X-100 are clearly demonstrated. Flow cytometric detergent insolubility is a powerful tool for rapid screening the raft-association of monocyte antigens in a whole-blood assay.     

Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction

BackgroundAdipose tissue is an abundant source of mesenchymal stem cells (MSC), which can be used for tissue-engineering purposes. The aim of our study was to determine the more suitable procedure, surgical resection or liposuction, for harvesting human adipose tissue-derived stem cells (hASC) with regard to viability, cell count and differentiation potential.MethodsAfter harvesting hASC, trypan blue staining and cell counting were carried out. Subsequently, hASC were cultured, analyzed by fluorescence-activated cell sorting (FACS) and differentiated under adipogenic, osteogenic and chondrogenic conditions. Histologic and functional analyzes were performed at the end of the differentiation period.ResultsNo significant difference was found with regard to the cell counts of hASC from liposuction and surgically resected material (P = 0.086). The percentage of viable cells was significantly higher for liposuction aspirates than for resection material (P = 0.002). No significant difference was found in the adipogenic differentiation potential (P = 0.179). A significantly lower number of cultures obtained from liposuction material than from resection material could be differentiated into osteocytes (P = 0.049) and chondrocytes (P = 0.012).DiscussionEven though some lineages from lipoaspirated hASC can not be differentiated as frequently as those from surgically resected material, liposuction may be superior for some tissue-engineering purposes, particularly because of the less invasive harvesting procedure, the higher percentage of viable cells and the fact that there is no significant difference between lipoaspirated and resected hASC with regard to adipogenic differentiation potential.     

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.     

Exploring strategies to prevent post-lobectomy space: transient diaphragmatic paralysis using Botulinum Toxin Type A (BTX-A)

Objective

Various techniques to reduce air space after pulmonary lobectomy especially for lung cancer have been an important concern in thoracic surgical practice. The aim of this study was to assess the effectiveness of Botulinum toxin A (BTX-A) injection into the diaphragm to reduce air space after right lower pulmonary lobectomy in an animal model.

Methods

Twelve male New Zealand rabbits were randomly allocated into two groups. All animals underwent right lower lobectomy. Then, normal saline of 0,1 ml and 10 units of 0,1 ml Botulinum toxin type A were injected into the muscular part of the right hemidiaphragm in control (n = 6) and BTX-A groups (n = 6) respectively. Residual air space and diaphragmatic elevation were evaluated with chest X-ray pre- and postoperatively. Diaphragmatic elevation was measured as a distance in millimetre from the line connecting the 10th ribs to the midpoint of the right hemidiaphragm.

Results

The mean diaphragmatic elevation in BTX-A and control groups were 7.0 ± 2.5 and 1.3 ± 1.2 millimetres respectively. Diaphragmatic elevations were significantly higher in BTX-A group (p = 0.0035).

Conclusion

Intraoperative Botulinum toxin type A injection may reduce postlobectomy spaces effectively via hemidiaphragmatic paralysis in rabbits. Further studies are needed to validate the safe use of Botulinum toxin type A in human beings.

     

The satiety factor apolipoprotein A-IV modulates intestinal epithelial permeability through its interaction with alpha-catenin: implications for inflammatory bowel diseases.

INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.     

Ischaemia-modified albumin levels in newborn jaundice before and after phototherapy

The aim of our study was to assess the effect of phototherapy (PT) on ischaemia‐modified albumin (IMA) and malondialdehyde (MDA) levels in hyperbilirubinemic full‐term newborns. The study was performed on 36 full‐term infants exposed to PT. The babies were aged 3 to 13 days. IMA and MDA levels of the babies were determined before and after PT, by a colorimetric assay. IMA levels before and after PT were found as 0.424 ± 0.290 and 0.531 ± 0.262 absorbance units, respectively. Although IMA levels after PT were slightly higher, the difference was not statistically significant (p > 0.131). MDA levels before and after PT were found as 8.4 ± 1.8 µmol/l and 9.4 ± 1.5 µmol/l, respectively. Serum MDA concentrations were significantly higher after PT than before PT (p < 0.000). In previous studies, conflicting findings have been reported about the effect of PT on oxidant and antioxidant systems. However, we have found no study investigating IMA levels in hyperbilirubinaemia in newborns before and after PT. Our results shows that PT does not affect IMA levels significantly. IMA increases as a result of oxidative stress. We believe that the lack of significant difference between our IMA levels before and after PT may resulted from hyperbilirubinaemia, which has antioxidant effect. Copyright © 2011 John Wiley & Sons, Ltd.     

Ezetimib influences the expression of raft-associated antigens in human monocytes.

Aminopeptidase N (CD13) was recently identified as a molecular target of the cholesterol absorption inhibitor Ezetimib. Regarding that CD13 is expressed in lipid rafts of monocytic cells, we have investigated whether Ezetimib influences raft function in these cells. Expression of raft-associated antigens (CD11b, CD13, CD14, CD16, CD36, and CD64) was followed by flow cytometry and/or immunoblot in human monocyte-derived macrophages in response to in vitro administration of Ezetimib. Cellular redistribution of CD13 was assessed by confocal imaging. Ezetimib significantly decreased the surface expression of CD13, CD16, CD64, and CD36; furthermore, it induced a shift of CD13 from plasma membrane to intracellular vesicles, and thus it quite likely modulated monocytic raft-assembly.