Sandy Mosig

Different functions of monocyte subsets in familial hypercholesterolemia: potential function of CD14+CD16+ monocytes in detoxification of oxidized LDL

The study was undertaken to investigate whether the two major monocyte subsets defined by the surface markers CD14+CD16+ and CD14++CD16− show differences in their responses to hypercholesterolemia. Monocytes were rapidly isolated from the blood of hypercholesterolemic, low‐density lipoprotein (LDL) receptor‐defective familial hypercholesterolemia (FH) patients and from control persons. Using flow cytometry and uptake, adhesion, and phagocytosis assays as well as laser scanning microscopy, we found significant differences between the monocyte subsets. FH‐CD14+CD16+ monocytes exhibit an increased uptake of oxidized LDL (oxLDL) via CD36, whereas FH‐CD14++CD16− monocytes preferentially take up native LDL (nLDL). FH‐CD14+CD16+ monocytes have an increased expression of surface proteins CD68, stabilin‐1, and CD11c and a higher adherence to activated endothelial cells in response to oxLDL and nLDL stimulation. In addition, all CD14+CD16+ monocytes have an increased ability for phagocytosis and a higher resistance to phagocytosis impairment by oxLDL compared with CD14++CD16− monocytes. We conclude that FH‐CD14+CD16+ monocytes have specialized functions in the uptake of oxLDL at activated endothelial cell surfaces, and we hypothesize that these functions are critical for the clearance of oxLDL deposits and apoptotic cells from the vessel wall under hyperlipidemic conditions.— Mosig, S., Rennert, K., Krause, S., Kzhyshkowska, J., Neunubel, K., Heller, R., Funke, H. Different functions of monocyte subsets in familial hypercholesterolemia: potential function of CD14+CD16+ monocytes in detoxification of oxidized LDL. FASEB J. 23, 866–874 (2009)

Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

BackgroundElevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals.MethodsCholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL.ResultsUsing microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells.ConclusionOur data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

Gene expression in the detection of autologous blood transfusion in sports - a pilot study

The reinfusion of autologous blood to enhance performanceremains a significant problem in sports. Although allogeneicblood transfusions can be detected since 2003 [1], there is atpresent no detection method for autologous blood transfu-sions, although indirect approaches such as the biologicalpassport [2] might give indications on the illicit use of bloodtransfusions.It is well-documented that several molecular changesoccur in stored red blood cells (RBCs), commonly referred toas the ‘storage lesion’ [3–5]. We therefore hypothesize thatautologous transfusion will lead to a sudden exposure of celldetritus to the immune system causing a cellular and molecularimmune response including gene expression alterations ofwhite blood cells such as T-lymphocytes. Hence, the primaryobjective of this study was to investigate the transcriptionalresponse of T-lymphocytes after reinfusion of autologousRBCs in order to search for a theoretical model for an un-equivocal detection method of autologous blood doping. Themost significant Gene Ontology (GO) clusters of regulatedgenes at 72 h after autologous transfusion included leucocyteimmunoglobulin receptors, toll-like receptor (TLR) pathway[6], adaptive immune response and cell death/apoptosis aswell as regulation of endocytosis of surface receptors and theTLR pathway at 96 h, respectively. The quantitative reversetranscriptase polymerase chain reaction (qRT-PCR) confirmedsignificant up-regulation of TLR4, TLR5, TLR6, apoptosis-associated tyrosine kinase (AATK) [7,8] and low densitylipoprotein receptor related protein (LRP1) [9,10] at 72 h aswell as TLR6 at 96 h. Therefore, the main finding of our pilotstudy is the fact that the transfusion of autologous bloodtriggers a distinct immune reaction within the T-lymphocytesof the recipient and may aid in the development of a practicablemethod to detect autologous blood doping based on molecularimmune response measurements.

Gene expression profiles of T lymphocytes are sensitive to the influence of heavy smoking: A pilot study.

Cigarette smoke components have a proven negative influence on human health. Adverse metabolic effects were observed in tissues and single cells. T lymphocytes get in contact with affected organs (e.g., lung) or cells (e.g., erythrocytes), as well as with smoke components and bioactive molecules, whose production is triggered by tobacco smoke. We therefore compared the gene expression profiles in these cells of the adaptive immune system of three male heavy smokers and three male nonsmokers using rapid T cell isolation and Affymetrix GeneChip® HG U133A 2.0 microarray analysis. Eighty-eight genes were found to be significantly (t test) differentially expressed by a factor of 1.5-fold or larger between smokers and nonsmokers. Using the gene function groups of the gene ontology consortium to categorize the functions of the differentially expressed genes, the group termed “response to stimulus” was found to be most significantly affected by smoking. Our data indicate a prominent role of cytotoxic T lymphocytes in response to smoking. Several genes that are typically expressed in these cells were found regulated although the ratio of cytotoxic and helper T lymphocytes remained unchanged in smokers. Our data show that, in principle, it might be possible to identify health-related biomarkers in the transcriptome of T lymphocytes.

SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

BackgroundGenetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer.MethodsMicroarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction.ResultsOverall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro.ConclusionsThe relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence.