IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice.

BACKGROUND: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO). METHODS: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO. RESULTS: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45(+) leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4(+) and CD8(+) T cells, as well as macrophages, was significantly decreased. CONCLUSIONS: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Formyl peptide receptor 2 orchestrates mucosal protection against Citrobacter rodentium infection.

Citrobacter rodentium is an attaching and effacing intestinal murine pathogen which shares similar virulence strategies with the human pathogens enteropathogenic- and enterohemorrhagic Escherichia coli to infect their host. C. rodentium is spontaneously cleared by healthy wild-type (WT) mice whereas mice lacking Muc2 or specific immune regulatory genes demonstrate an impaired ability to combat the pathogen. Here we demonstrate that apical formyl peptide receptor 2 (Fpr2) expression increases in colonic epithelial cells during C. rodentium infection. Using a conventional inoculum dose of C. rodentium, both WT and Fpr2-/- mice were infected and displayed similar signs of disease, although Fpr2-/- mice recovered more slowly than WT mice. However, Fpr2-/- mice exhibited increased susceptibility to C. rodentium colonization in response to low dose infection: 100% of the Fpr2-/- and 30% of the WT mice became colonized and Fpr2-/- mice developed more severe colitis and more C. rodentium were in contact with the colonic epithelial cells. In line with the larger amount of C. rodentium detected in the spleen in Fpr2-/- mice, more C. rodentium and enteropathogenic Escherichia coli translocated across an in vitro mucosal surface to the basolateral compartment following FPR2 inhibitor treatment. Fpr2-/- mice also lacked the striated inner mucus layer that was present in WT mice. Fpr2-/- mice had decreased mucus production and different mucin O-glycosylation in the colon compared to WT mice, which may contribute to their defect inner mucus layer. Thus, Fpr2 contributes to protection against infection and influence mucus production, secretion and organization.

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Leukocyte CD40L deficiency affects the CD25(+) CD4 T cell population but does not affect atherosclerosis.

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on cholesterol levels and atherosclerosis in ApoE-deficient mice.

In the arterial wall, scavenger receptor class A (SRA) is implicated in pathological lipid deposition. In contrast, in the liver, SRA is suggested to remove modified lipoproteins from the circulation, thereby protecting the body from their pathological action. The role of SRA on bone marrow-derived cells in lipid metabolism and atherogenesis was assessed in vivo by transplantation of bone marrow cells overexpressing human SRA (MSR1) to apoE-deficient mice. In vitro studies with peritoneal macrophages from the transplanted mice showed that macrophage scavenger receptor function, as measured by cell association and degradation studies with acetylated LDL, was approximately 3-fold increased on overexpression of MSR1 in bone marrow-derived cells as compared with control mice. Despite the increased macrophage scavenger receptor function in vitro, no significant effect of MSR1 overexpression in bone marrow-derived cells on the in vivo atherosclerotic lesion development was found. In addition to arterial wall macrophages, liver sinusoidal Kupffer cells also overexpress MSR1 after bone marrow transplantation, which may scavenge atherogenic particles more efficiently from the blood compartment. Introduction of bone marrow cells overexpressing human MSR1 in apoE-deficient mice induced a significant reduction in serum cholesterol levels of approximately 20% (P:<0.001, 2-way ANOVA) as the result of a decrease in VLDL cholesterol. It is suggested that the reduction in VLDL cholesterol levels is due to increased clearance of modified lipoproteins by the overexpressed MSR1 in Kupffer cells of the liver, thereby protecting the arterial wall against the proatherogenic action of modified lipoproteins.

A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans.

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.

Lithium gamma-linolenate-induced cytotoxicity against cells chronically infected with HIV-1.

Lithium gamma-linolenate (Li-GLA), was evaluated for its activity in selectively killing H9 cells chronically infected with HIV-1RF. After 4 days incubation with Li-GLA approximately 90% of the H9RF cells were non-viable compared to 20% of uninfected H9 cells. The efficacy of the Li-GLA, in preferentially killing HIV infected cells also correlates with lipid peroxidation, as measured by the intracellular thiobarbituric acid-reactive material content. The addition of an antioxidant (vitamin E) to the culture medium reduced the toxicity of Li-GLA. These data indicate that this selective killing effect of cells chronically infected with HIV may be due to the enhanced extent of lipid peroxidation of the added Li-GLA.

Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines.

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.

Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids.

Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively. Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.

Selective cytotoxicity of lithium gamma-linolenic acid in human T cells chronically and productively infected with HIV.

We sought to extend observations of lithium gamma-linolenic acid (LiGLA)-associated selective cytotoxicity in different models of chronic HIV infection in vitro. In our initial experiments, 8E5, 8E5L and A3.01 cells were allowed to proliferate in the presence of 0-20 microg/ml LiGLA for 4 days. Similarly, OM-10.1 cells (with or without prior stimulation with tumour necrosis factor-alpha (TNF-alpha)) were grown with 0-5 microg/ml LiGLA for 10 days. Significant cytotoxicity was observed in productively infected 8E5 cells (100% cell death by day 2 at the highest concentration) as compared with latently infected 8E5L cells (50% cell death on days 2-4) and uninfected A3.01 cells. No drug-induced viral stimulation was observed in surviving cells. In fact, a mild direct antiviral effect may be present, independent of cytotoxicity. Maximum cytotoxicity to OM-10.1 cells was only observed when active viral replication was induced by TNF-alpha. Our preliminary results are encouraging and suggest that LiGLA should be retained as a candidate antiretroviral agent. Further work is underway to identify the specific mechanism underlying our observations.

Scavenger receptor deficiency leads to more complex atherosclerotic lesions in APOE3Leiden transgenic mice.

Apolipoprotein (apo) E3Leiden is a dysfunctional apo E variant associated with familial dysbetalipoproteinemia in humans. Transgenic mice carrying the APOE3Leiden gene develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. An early step in atherosclerosis is foam cell formation, which is thought to result from the unrestricted uptake of modified lipoproteins by macrophages. To investigate the role of the macrophage scavenger receptor type I and II (MSR-A) in this process, APOE3Leiden transgenic mice were crossed onto a MSR-A deficient background and the development of atherosclerosis was examined. In view of recent results with apo E deficient mice (Suzuki H et al., A role for the macrophage scavenger receptors in atherosclerosis. Nature 1997; 386(6622):292-296), absence of the MSR-A in APOE3Leiden mice was expected to lead to a reduction of atherosclerosis. In our study we compared APOE3Leiden/MSR-A deficient mice (E3L MSR-A -/-) to APOE3Leiden/MSR-A wild-type mice (E3L MSR-A +/+). These animals were fed an atherogenic diet for 10 weeks. Quantification of the lesion area showed no significant difference between E3L MSR-A -/- and E3L MSR-A +/+ mice although there was a trend towards the development of larger lesions in the E3L MSR-A -/- mice. All lesions were typed according to their cellular composition. In both male and female E3L MSR-A -/- mice, significantly more severe lesions developed as compared to E3L MSR-A +/+ mice. These results indicate that the effect of MSR-A deficiency on atherogenesis may depend on the presence or absence of apo E.

Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages.

Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene (MSR1) with 60- and 40-kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination. This 180-kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells. Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein. In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice. However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages. Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.

Role of thiol homeostasis and adenine nucleotide metabolism in the protective effects of fructose in quinone-induced cytotoxicity in rat hepatocytes.

Freshly-isolated rat hepatocytes were exposed in glucose (15 mM) or fructose (5 mM) medium to menadione (2-methyl-1,4-naphthoquinone) (85 microM) or 1,4-naphthoquinone (NQ) (50 microM). Menadione and NQ are closely related quinones and have an approximately equal potential to induce redox cycling. However, NQ has a higher potential to arylate and is more toxic than menadione. During 2 hr of incubation, cell viability, thiol status, adenine nucleotide level and lactate production were determined. LDH-leakage was used as a measure of cell viability. In glucose medium, exposure of hepatocytes to menadione or NQ resulted in a faster excretion rate of oxidized glutathione as compared to those cells in fructose medium. As a result, quinone-exposed hepatocytes in fructose medium retained higher amounts of oxidized glutathione. Menadione-exposed hepatocytes in fructose medium exhibited a diminished rate of transthiolation of protein thiols with oxidized glutathione as compared to those cells in glucose medium. The adenine nucleotide level of hepatocytes in glucose medium was markedly higher than in fructose medium. This was caused by an ATP decrease in hepatocytes in fructose medium resulting in a low energy charge (E.C.) (0.6) as compared to hepatocytes in glucose medium (0.9). Only menadione caused a decrease in the E.C. in glucose medium while NQ caused a decrease of all three adenine nucleotides. In fructose medium, quinone-exposed hepatocytes showed no change in their adenine nucleotides as compared to control cells. Despite the higher oxidized glutathione content and the lower ATP level of NQ-exposed hepatocytes in fructose medium, they had a better viability than those cells in glucose medium. From our results we conclude that a high ATP content is not always beneficial for cell survival.

A pilot study to investigate the treatment of anogenital warts with Topical Lithium Succinate cream (8% lithium succinate, 0.05% zinc sulphate).

The incidence of anogenital warts (condyloma acuminatum) is rapidly increasing while there is still no totally satisfactory treatment available. In light of the emphasis of experimental approaches toward the prevention of viral replication and evidence of the antiviral action of lithium salts it was proposed to investigate the efficacy of Topical Lithium Succinate cream (LSC) in the treatment of anogenital warts. A total of 101 patients (42 women, 59 men) were randomized to receive either active or placebo treatment for a period of 4 weeks. Assessment of the number, location, size and area of coverage of the warts was made by the clinician at baseline, weeks 2, 4, 6 and 12. Compliance to the study protocol following cessation of treatment at week 4 was extremely poor. The high drop-out rate after this was felt to invalidate data collected after that point. It was therefore decided that the analysis should concentrate on the treatment period. Of 101 patients entering the trial 51 received active (30 male and 21 female) and 50 received placebo (29 male, 21 female). The primary efficacy variable was percentage change from baseline in the overall coverage of lesions. Over all patients LSC treatment resulted in a reduction of 42% (P<0.02) in the overall coverage of lesions. Separate analyses for male and female patients showed that for males there was a highly significant reduction in the coverage of lesions of 65% (P<0.02). However for females the reduction of 11% was not significant. A possible explanation for this difference between the sexes is that as many of the lesions in the female patients were internal therefore this could lead to difficulty in both application of the cream, and subsequent lesion assessment.

Transgenic mouse models to study the role of the macrophage scavenger receptor class A in atherosclerosis.

Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice. The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the athero-susceptible background. To study the role of SR-A in a different model, we generated a transgenic mouse model with high level expression of the human SR-A gene using a 180 Kb yeast artificial chromosome (MSR1 transgenic mice). These mice show increased expression of SR-A according to the natural expression pattern. The MSR1 transgenic mice were crossed onto a low-density lipoprotein receptor deficient background and were fed a high fat diet for 10 weeks. After this period, the size of the atherosclerotic lesions in the proximal aorta was measured. Surprisingly, atherosclerosis was significantly reduced in the MSR1 transgenic mice. In a second study, the effect of SR-A was examined in APOE-3 Leiden mice providing a different athero-susceptible background. To exclude nonmacrophage effects, bone marrow was transplanted from MSR1 mice and wild-type littermates to APOE-3 Leiden transgenic mice. After 8 weeks on a high fat diet, atherosclerosis in the mice that had received MSR1 bone marrow was reduced compared with mice that had received wild-type bone marrow. This difference reached statistical significance when individual cholesterol exposure of the mice was taken into account. Both experiments indicated an antiatherogenic role of the SR-A. This observation cannot be explained easily by SR-A function in foam cell formation because in MSR1 macrophages in vitro foam cell formation is increased. Alternatively, however, SR-A may affect the activation of macrophages. Hence the response to lipopolysaccharide was measured in MSR1-transgenic macrophages. These macrophages showed a reduction in their activation in response to lipopolysaccharide, as measured by nitric oxide production. These data show that an elevated level of SR-A expression reduces atherosclerosis, potentially by modifying the response of macrophages to activation signals in the plaque.

The impact of new technologies on vaccine development.

The ability to move genetic determinants between species using in vitro gene-manipulation techniques has opened up new approaches to vaccine development. This has rapidly grown into an exciting area of research in both academic and industrial laboratories. There are numerous scientific challenges which require multidisciplinary teams to solve problems in creating new immunogens. This has challenged our existing knowledge about protein structure and conformation, microbial pathogenicity and the immune system. Recombinant-DNA techniques are invaluable as tools of analysis and antigen production. The surface of micro-organisms can also be minutely explored with the use of synthetic peptides and monoclonal antibodies. Nevertheless, these new technologies do not allow us to circumvent the need for detailed understanding of pathogens and the disease process. What is apparent from the work carried out so far is that there are few easy answers to vaccine development and it is not realistic to expect rapid solutions to these problems. As there are many potential targets for constructing novel vaccines for both human and animal diseases, it is helpful to establish some priorities. There is a tendency to look at the existing effective vaccines and simply direct research at producing them more economically or with enhanced safety and stability. The advantage of this approach is that considerable background work will have already been carried out establishing the basis for the application of recombinant DNA techniques. However, this can also lead to conflicts (often within the same institute or company) between the new and old technologies. This could be to the detriment of the new technologies which are still only partly developed and may not be good enough yet to compete with existing vaccines in cost or efficacy. The more ambitious, and eventually more rewarding, approach is to attempt to develop new vaccines where none had existed before. There is a vast untapped market, especially in the parasitic diseases, but the scientific problems may be considerable and much more background work is likely to be necessary. Indeed, most of the work in this area is more accurately referred to as basic research rather than vaccine development as totally new, effective vaccines are still some way off. Having directed research towards a specific organism or disease there are still many options available as to the scientific strategy to adopt. As discussed in this review it may be possible to consider subunits, synthetic antigens and live (attenuated or heterologous) organisms as possible vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation.

The phenotype of macrophages in atherosclerotic lesions can vary dramatically, from a large lipid laden foam cell to a small inflammatory cell. Classically, the concept of macrophage heterogeneity discriminates between two extremes called either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Polarisation of plaque macrophages is predominantly determined by the local micro-environment present in the atherosclerotic lesion and is rather more complex than typically described by the M1/M2 paradigm. In this review we will discuss the role of various polarising factors in regulating the phenotypical state of plaque macrophages. We will focus on two main levels of phenotype regulation, one determined by differentiation factors produced in the lesion and the other determined by T-cell-derived polarising cytokines. With foam cell formation being a key characteristic of macrophages during atherosclerosis initiation and progression, these polarisation factors will also be linked to lipid handling of macrophages.