Molecular classification of tumour cells in a patient with intravascular large B-cell lymphoma.

BACKGROUND: Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal LBCL. It is characterized by the proliferation of tumour cells exclusively intraluminally in small blood vessels of different organs. The clinical manifestation depends on the type of organ affected; additionally, a haemophagocytic syndrome can be observed in some patients. OBJECTIVES: The aim was to further understand the nosology of this lymphoma as, due to its rarity and in spite of detailed immunohistochemical investigations, its exact nosology is only incompletely understood. METHODS: We used microarray-based analysis of gene expression of tumour cells isolated from a patient with primary manifestation of the lymphoma in the skin and compared it with various other diffuse LBCLs (DLBCLs) as well as a previously published DLBCL classifier. RESULTS: In unsupervised analyses, the tumour cells clustered together with non-germinal centre B-cell (non-GCB) DLBCL samples but were clearly distinct from GCB-DLBCL. Analogous to non-GCB DLBCL, molecular cell-of-origin classification revealed similarity to bone-marrow derived plasma cells. CONCLUSIONS: The IVLBCL of this patient showed molecular similarity to non-GCB DLBCL. Due to the prognostic and increasingly also therapeutic relevance of molecular subtyping in DLBCL, this method, in addition to immunohistochemistry, should also be considered for the diagnosis of IVLBCL in the future.

Common dysregulated pathways in obese adipose tissue and atherosclerosis.

BACKGROUND: The metabolic syndrome is becoming increasingly prevalent in the general population that is at simultaneous risk for both type 2 diabetes and cardiovascular disease. The critical pathogenic mechanisms underlying these diseases are obesity-driven insulin resistance and atherosclerosis, respectively. To obtain a better understanding of molecular mechanisms involved in pathogenesis of the metabolic syndrome as a basis for future treatment strategies, studies considering both inherent risks, namely metabolic and cardiovascular, are needed. Hence, the aim of this study was to identify pathways commonly dysregulated in obese adipose tissue and atherosclerotic plaques. METHODS: We carried out a gene set enrichment analysis utilizing data from two microarray experiments with obese white adipose tissue and atherosclerotic aortae as well as respective controls using a combined insulin resistance-atherosclerosis mouse model. RESULTS: We identified 22 dysregulated pathways common to both tissues with p values below 0.05, and selected inflammatory response and oxidative phosphorylation pathways from the Hallmark gene set to conduct a deeper evaluation at the single gene level. This analysis provided evidence of a vast overlap in gene expression alterations in obese adipose tissue and atherosclerosis with Il7r, C3ar1, Tlr1, Rgs1 and Semad4d being the highest ranked genes for the inflammatory response pathway and Maob, Bckdha, Aldh6a1, Echs1 and Cox8a for the oxidative phosphorylation pathway. CONCLUSIONS: In conclusion, this study provides extensive evidence for common pathogenic pathways underlying obesity-driven insulin resistance and atherogenesis which could provide a basis for the development of novel strategies to simultaneously prevent type 2 diabetes and cardiovascular disease in patients with metabolic syndrome.

Identification of matrix metalloproteinase-12 as a candidate molecule for prevention and treatment of cardiometabolic disease.

Obesity is strongly associated with metabolic syndrome, a combination of risk factors that predispose to the development of the cardiometabolic diseases: atherosclerotic cardiovascular disease and type 2 diabetes mellitus. Prevention of metabolic syndrome requires novel interventions to address this health challenge. The objective of this study was the identification of candidate molecules for the prevention and treatment of insulin resistance and atherosclerosis, conditions that underlie type 2 diabetes mellitus and cardiovascular disease, respectively. We used an unbiased bioinformatics approach to identify molecules that are upregulated in both conditions by combining murine and human data from a microarray experiment and meta-analyses. We obtained a pool of eight genes that were upregulated in all the databases analysed. This included well known and novel molecules involved in the pathophysiology of type 2 diabetes mellitus and cardiovascular disease. Notably, matrix metalloproteinase 12 (MMP12) was highly ranked in all analyses and was therefore chosen for further investigation. Analyses of visceral and subcutaneous white adipose tissue from obese compared to lean mice and humans convincingly confirmed the up-regulation of MMP12 in obesity at mRNA, protein and activity levels. In conclusion, using this unbiased approach an interesting pool of candidate molecules was identified, all of which have potential as targets in the treatment and prevention of cardiometabolic diseases.

Strong association of serum- and glucocorticoid-regulated kinase 1 with peripheral and adipose tissue inflammation in obesity.

OBJECTIVE: The serum- and glucocorticoid-regulated kinase 1 (SGK1) is an early transcriptional target of glucocorticoids and is activated via insulin. Here we investigate the regulation of SGK1 expression in human obesity, diet-induced murine obesity and human monocytic cell line THP-1 monocytes. SUBJECTS AND METHODS: SGK1 expression was studied in subcutaneous and omental adipose tissue (AT) of 20 morbidly obese and 20 age- and gender-matched non-obese controls in murine diet-induced obesity and the THP-1 cell line. The regulation of SGK1 by inflammatory signals was tested in THP-1 cells. RESULTS: Murine diet-induced obesity is associated with a significant upregulation of Sgk1 in gonadal AT. Sgk1 expression is highest in the macrophage-rich stromal vascular fraction and lower in adipocytes. In humans, AT SGK1 is predominantly expressed in CD14(+) macrophages and significantly upregulated in omental and subcutaneous AT of obese subjects. SGK1 mRNA expression in both omental and subcutaneous AT correlates with body mass index, circulating leptin and C-reactive protein, and the local expression of inflammatory markers including monocyte chemotactic protein-1 and macrophage inflammatory protein-1α. The expression of SGK1 in THP-1 cells is upregulated by inflammatory signals, such as lipopolysaccharide and tumour necrosis factor-α, as well as during the induction of monocyte-to-macrophage maturation. CONCLUSIONS: Our data present the first link between SGK1 and obesity-associated inflammation. SGK1 expression is stimulated in response to inflammatory signals and increased in AT macrophages. The characterisation of SGK1 functions in obesity and immunity may help identify potential therapeutic targets in the treatment of obesity.

Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue.

OBJECTIVE: Obesity is associated with chronic inflammation of the adipose tissue, which contributes to obesity-associated complications such as insulin resistance and type 2 diabetes. Interleukin (IL)-33 acts via its receptor ST2 and is involved in the pathogenesis of inflammatory disorders including atherosclerosis and heart disease. IL-33 has been demonstrated to promote endothelial cell inflammatory response, but also anti-inflammatory and protective actions such as TH2 and M2 polarization of T cells and macrophages, respectively. IL-33 and ST2 have been shown to be expressed in human and murine adipose tissue. Our objective was to investigate alterations in obesity and a possible role of IL-33 in adipose tissue inflammation. SUBJECTS AND METHODS: We investigated severely obese patients (BMI>40 kg m(-2), n=20) and lean to overweight controls (BMI<30 kg m(-2); n=20) matched for age and sex, as well as diet-induced obese and db/db mice, in order to determine the impact of obesity on IL-33 and ST2 gene and protein expression levels in adipose tissue and blood, and their correlation with inflammatory and metabolic parameters. Furthermore, we examined the cellular source and location of IL-33 and ST2 in situ. RESULTS: IL-33 and ST2 expression levels were markedly elevated in omental and subcutaneous adipose tissue of severely obese humans and in diet-induced obese mice, but not in leptin receptor-deficient db/db mice. In addition, soluble ST2, but not IL-33 serum levels, were elevated in obesity. The main source for IL-33 in adipose tissue were endothelial cells, which, in humans, exclusively expressed ST2 on their surface. IL-33 expression strongly correlated with leptin expression in human adipose tissue. CONCLUSIONS: Expression of IL-33 and its receptor ST2 in human adipose tissue is predominantly detectable in endothelial cells and increased by severe obesity indicating an autocrine action. Thus, the adipose tissue microvasculature could participate in obesity-associated inflammation and related complications via IL-33/ST2.

Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice.

AIMS/HYPOTHESIS: Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism. METHODS: Wild-type (WT) and Opn (also known as Spp1) knockout (Opn (-/-)) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations. RESULTS: We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic-hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn (-/-) mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency. CONCLUSIONS/INTERPRETATION: Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes.

Newly identified adipose tissue macrophage populations in obesity with distinct chemokine and chemokine receptor expression.

OBJECTIVE: Infiltration by macrophages is a hallmark of obesity-related adipose tissue (AT) inflammation that is tightly linked to insulin resistance. Although CD11c+ AT macrophages (ATMs) have recently been shown to promote inflammation in obese mice, the knowledge on phenotype and function of different ATM populations is still very limited. This study aimed at identifying and characterizing ATM populations in obesity. METHODS: Isolation of ATM populations defined by CD11c and mannose receptor (MR) expression and analysis of gene expression in high-fat diet-induced obese mice. RESULTS: Obesity provoked a shift from a predominant MR+CD11c⁻ population ('MR-ATM') to two MR⁻ populations, namely MR⁻CD11c+ ('CD11c-ATM') and MR⁻CD11c⁻ (double negative, 'DN-ATM'). Although CD11c-ATMs were of a clear inflammatory M1 phenotype, DN-ATMs expressed few inflammatory mediators and highly expressed genes for alternative activation (M2) markers involved in tissue repair, such as arginase and YM1. In contrast, MR-ATMs marginally expressed M1 and M2 markers but highly expressed chemokines, including Mcp-1 (Ccl2) and Mcp-3 (Ccl7). Both CD11c-ATMs and DN-ATMs, but not MR-ATM, highly expressed a panel of chemokine receptors (namely Ccr2, Ccr5, Ccr3 and Cx3cr1), whereas the expression of Ccr7 and Ccr9 was selective for CD11c-ATMs and DN-ATMs, respectively. Notably, stressed adipocytes upregulated various chemokines capable of attracting CD11c-ATM and DN-ATM. CONCLUSION: This study identifies a novel ATM population with a putatively beneficial role in AT inflammation. This DN-ATM population could be attracted to the obese AT by similar chemokines such as inflammatory CD11c-ATM, on which only Ccr7 is uniquely expressed.

Diminished upregulation of visceral adipose heme oxygenase-1 correlates with waist-to-hip ratio and insulin resistance.

OBJECTIVE: Insulin resistance in visceral obesity is substantially driven by adipose tissue inflammation, particularly by macrophages accumulating in obese adipose tissue. In contrast, adipose tissue macrophages express the hemoglobin scavenger receptor (CD163) and heme oxygenase-1 (gene: HMOX1) that together protect from oxidative stress. Our aim was to evaluate the expression of CD163 and HMOX1 in intra-abdominal visceral (omental) and subcutaneous adipose tissue as well as circulating soluble CD163 concentrations in human obesity and its association with adipose tissue inflammation, body fat distribution and insulin resistance. METHODS: CD163, HMOX1 and CD68 mRNA expression in visceral and subcutaneous adipose tissue, serum concentration of soluble CD163 in morbidly obese patients (body mass index (BMI) >40 kg m(-2)), who underwent laparoscopic surgery for gastric banding (n=20), matched for age and sex to controls (BMI<30 kg m(-2); n=20) was analyzed. RESULTS: CD163 expression was highly upregulated in human adipose tissue and soluble CD163 serum concentration was elevated in obese vs lean subjects. HMOX1 was upregulated in adipose tissue by obesity as well and expressed predominantly in macrophages. Although CD163 expression strictly correlated with macrophage abundance, HMOX1 was additionally upregulated within macrophages. This upregulation was significantly lower in visceral compared with subcutaneous adipose tissue. Strikingly, relative visceral adipose tissue expression of HMOX1 negatively correlated with waist-to-hip ratio and the homeostasis model assessment of insulin resistance (both P=0.024). CONCLUSIONS: Visceral obesity is associated with defective upregulation of heme oxygenase-1 in visceral adipose tissue. A lack of this antioxidative and anti-inflammatory enzyme in visceral adipose tissue could contribute to the development of insulin resistance.

Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production.

OBJECTIVE: Obesity is associated with a chronic low-grade inflammation and an increased abundance of macrophages in adipose tissue. Adipose tissue macrophages (ATMs) are assumed to interfere with adipocyte function leading to insulin resistance, thereby contributing to the pathogenesis of type 2 diabetes mellitus. Macrophages exist in separate types of differentiation, but the nature of ATMs is largely unknown. DESIGN AND MEASUREMENTS: Stromal vascular cells (SVCs) and ATMs were isolated from human adipose tissues from different locations. We characterized ATMs phenotypically and functionally by flow cytometry, endocytosis assay and determination of secreted cytokines. For comparison, we used macrophages of the 'classical' (M1) and the 'alternative', anti-inflammatory (M2) type differentiated in vitro from peripheral blood monocytes. RESULTS: Like prototypic M2 macrophages, ATMs expressed considerable amounts of mannose receptor, haemoglobin scavenger receptor CD163 and integrin alphavbeta5. The number of cells expressing these molecules correlated significantly with the donors' body mass indices (BMIs). Notably, SVCs positive for the common monocyte/macrophage marker CD14 contained a considerable fraction of blood monocytes, the abundance of which did not correlate with the BMIs, pointing to the requirement of the surface markers identified here for the identification of ATMs. ATMs showed endocytic activities similar to M2 macrophages and accordingly secreted high amounts of IL-10 and IL-1 receptor antagonist. However, basal and induced secretion of pro-inflammatory mediators TNF-alpha, IL-6, IL-1, MCP-1 and MIP-1alpha was even higher in ATMs than in pro-inflammatory M1 macrophages. CONCLUSION: ATMs comprise a particular macrophage type that is M2-like by surface marker expression, but they are competent to produce extensive amounts of inflammatory cytokines, which could considerably contribute to the development of insulin resistance.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids.

OBJECTIVE: Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue. METHODS AND PROCEDURE: Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence. RESULTS: HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes. CONCLUSION: N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.

Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n-3 polyunsaturated fatty acids.

AIMS/HYPOTHESIS: Inflammatory alterations in white adipose tissue appear to underlie complications of obesity including diabetes mellitus. Polyunsaturated fatty acids (PUFA), particularly those of the n-3 series, modulate immune responses and may ameliorate insulin sensitivity. In this study, we investigated how PUFA affect white adipose tissue inflammation and gene expression in obese diabetic animals. MATERIALS AND METHODS: We treated db/db mice as well as lean non-diabetic mice (db/+) with either low-fat standard diet (LF) or high-fat diets rich in (1) saturated/monounsaturated fatty acids (HF/S), (2) n-6 PUFA (HF/6) and (3) the latter including purified marine n-3 PUFA (HF/3). RESULTS: Many genes involved in inflammatory alterations were upregulated in db/db mice on HF/S compared with LF in parallel with phosphorylation of c-Jun N-terminal kinase (JNK). In parallel, adipose tissue infiltration with macrophages was markedly enhanced by HF/S. When compared with HF/S, HF/6 showed only marginal effects on adipose tissue inflammation. However, inclusion of n-3 PUFA in the diet (HF/3) completely prevented macrophage infiltration induced by high-fat diet and changes in inflammatory gene expression, also tending to reduce JNK phosphorylation (p<0.1) in diabetic mice despite unreduced body weight. Moreover, high-fat diets (HF/S, HF/6) downregulated expression and reduced serum concentrations of adiponectin, but this was not the case with n-3 PUFA. CONCLUSIONS/INTERPRETATION: n-3 PUFA prevent adipose tissue inflammation induced by high-fat diet in obese diabetic mice, thereby dissecting obesity from adipose tissue inflammation. These data suggest that beneficial effects of n-3 PUFA on diabetes development could be mediated by their effect on adipose tissue inflammation.

Liver X receptors in cardiovascular and metabolic disease.

Liver X receptors (LXRs) alpha and beta are nuclear oxysterol receptors and metabolic sensors initially found to regulate cholesterol metabolism and lipid biosynthesis. Recent studies have elucidated the importance of LXR in the development of cardiovascular diseases and metabolic disorders. LXR agonists prevent development of atherosclerosis by modulation of metabolic as well as inflammatory gene expression in rodent models. Moreover, LXR activation inhibits hepatic gluconeogenesis and lowers serum glucose levels, indicating possible application of LXR activation in the treatment of diabetes mellitus. However, first-generation LXR agonists elevate hepatic and serum trigylceride levels, making subtype-specific agonists and selective LXR modulators rather than unselective LXR agonists a potential pharmacological strategy. This review summarizes the multiple physiological and pathophysiological implications of LXRs and observations that identify LXRs as potential targets for therapeutic interventions in human cardiovascular and metabolic disease.

The novel immunosuppressant FK778 inhibits formation of the immunologic Synapse.

The malononitrilamide FK778 is a derivative of A77 1726, the active metabolite of the antirheumatic drug leflunomide. A77 1726 inhibits de novo pyrimidine synthesis and activity of Src-family kinases; thus, it may interfere with T-cell proliferation as well as with early T-cell signaling. Formation of a stable interaction between T cells and antigen-presenting cells (APC)--the immunologic synapse--has emerged to be of crucial importance for T-cell activation. Here in we show that FK778 inhibits formation of the immunologic synapse by blocking superantigen-stimulated relocalization of adhesion (LFA-1), and signaling molecules (CD3) to the T-cell/APC contact site. These data show that FK778 affects T-cell/APC interactions, particularly events crucial for T-cell adhesion and formation of stable conjugates underlying sustained and effective T-cell activation. Thus, in this model system close to physiologic T-cell stimulation, FK778 affects critical events in the course of T-cell-mediated immune responses earlier than T-cell proliferation, which may contribute to its immunosuppressive potential.

The malononitrilamide FK778 inhibits activation of NF-kappaB in human dendritic cells.

FK778, a derivative of the active leflunomide-metabolite, A77 1726, has been shown to be a powerful immunosuppressant in several transplantation models, particularly efficient in prevention of chronic allograft rejection. However, the cellular and molecular mechanisms underlying these effects of FK778 have not been investigated yet in detail. Because dendritic cells (DCs) are a crucial cell type in initiation of immune responses including chronic allograft rejection, we investigated whether FK778 affects this peculiar cell population. NF-kappaB is the essential transcription factor involved in DC activation and function. We found that lipopolysaccharide (LPS)-induced activation of NF-kappaB, as assessed using electromobility shift assay, is markedly inhibited by FK778 in human monocyte-derived DCs. Hence, FK778 could exert its immunosuppressive effects via inhibition of activation and thus function of the central antigen-presenting cell, ie, DC.

Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.

The production of Green Fluorescent Protein in recombinant NIH3T3 mouse fibroblast cells was used as a model to determine the optimal conditions for the rapid isolation of high-producing cell lines with a fluorescence-activated cell sorter. "Bulk sorting", that is, sorting of a large number of positive cells, did not result in a stable, high-producing cell line due to overgrowth of high-producing cells by low- or nonproducing cells. The production kinetics and expression of GFP during batch culture was found to differ between NIH3T3 cells and HepG2 hepatoma cells, even though the same plasmid was used for transfection. The kinetics of product formation need therefore to be determined from case to case to select the optimal timepoint for analysis and sorting. Subcloning of sorted cells into microtiter plates only resulted in high-producing subclones when 1 or 2 cells were seeded per well. Higher seeding rates again resulted in overgrowth of low- or nonproducers. By subcloning, two high-producing cells lines could be isolated. They had a 10- and 15-fold higher fluorescent signal compared to the negative control. While one of these subclones started to decrease it's GFP expression after 2 months, the other clone stably expressed GFP for 4 months.