Maaike Joerink

Methylation of cytosines at CpG sites is a common epigenetic DNA modification that can be measured by a large number of methods, now even in a genome-wide manner for hundreds of thousands of sites. The application of DNA methylation analysis is becoming widely popular in complex disorders, for example, to understand part of the "missing heritability". The DNA samples most readily available for methylation studies are derived from whole blood. However, blood consists of many functionally and developmentally distinct cell populations in varying proportions. We studied whether such variation might affect the interpretation of methylation studies based on whole blood DNA. We found in healthy male blood donors there is important variation in the methylation profiles of whole blood, mononuclear cells, granulocytes, and cells from seven selected purified lineages. CpG methylation between mononuclear cells and granulocytes differed for 22% of the 8252 probes covering the selected 343 genes implicated in immune-related disorders by genome-wide association studies, and at least one probe was differentially methylated for 85% of the genes, indicating that whole blood methylation results might be unintelligible. For individual genes, even if the overall methylation patterns might appear similar, a few CpG sites in the regulatory regions may have opposite methylation patterns (i.e., hypo/hyper) in the main blood cell types. We conclude that interpretation of whole blood methylation profiles should be performed with great caution and for any differences implicated in a disorder, the differences resulting from varying proportions of white blood cell types should be considered.

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.

Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.

Abstract Background. Aging-associated noncommunicable comorbidities are more prevalent among human immunodeficiency virus type 1 (HIV)–infected individuals than among HIV-uninfected individuals. Residual HIV-related chronic immune activation and senescence may increase the risk of developing comorbidities. Methods. Immune phenotyping, thymic output, and telomere length were assessed in 94 HIV-infected individuals who were aged >45 years and receiving antiretroviral therapy (ART; cases) and 95 age-matched uninfected controls. Results. Cases had lower CD4+ T-cell counts, higher CD8+ T-cell counts, and increased levels of immune activation (ie, increased soluble CD14 [sCD14] level and increased percentages of CD38+HLA-DR+ cells among both CD4+ and CD8+ T cells), regulatory T cells, and percentage of programmed cell death 1 (PD-1)–expressing cells among CD4+ T cells. Immune senescence levels (ie, percentages of CD27−CD28− cells or CD57+ cells) were comparable between cases and controls. Peripheral blood mononuclear cells from cases had shorter telomeres but increased single-joint T-cell receptor excision circle content and CD31+ naive CD4+ T cells. Although cytomegalovirus (CMV) antibody titers were higher in cases, CMV-specific T-cell responses were comparable between cases and controls. T-cell senescence in cases was independently associated with T-cell activation but not with CMV-specific immune responses. Conclusions. Despite long-term receipt of ART, HIV-infected adults had higher levels of immune activation, regulatory T cells, and PD-1–expressing CD4+ cells and shorter telomeres. The increased soluble CD14 levels and percentage of CD38+HLA-DR+ cells among CD4+ T cells correlated with shorter telomeres and increased regulatory T-cell levels. This suggests that HIV influences immune function irreversibly, with several pathways that are persistently abnormal during effective ART. Therapies aimed at improving immune health during ART are needed.