Elmar Endl

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO

CpG‐rich oligonucleotides (CpG‐ODN) bind to Toll‐like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG‐ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG‐ODN resulted in antigen‐specific T cell activation in local lymph nodes. In contrast, systemic application of CpG‐ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3‐dioxygenase (IDO) as indicated by the observation that CpG‐ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1‐methyl‐tryptophan (1‐MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG‐ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG‐ODN‐induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG‐ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down‐modulation of immune responses by CpG‐ODN may be possible in certain pathological conditions.

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19+ Burkitts lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.

Enrichment of cell-targeting and population-specific aptamers by fluorescence-activated cell sorting

In a sophisticated approach to the systematic evolution of ligands by exponential enrichment (SELEX) of cell-type-specific nucleic acid aptamers, the title technique was combined with the use of combinatorial nucleic acid libraries to target a defined subpopulation within composite mixtures of cells (see picture). The ssDNA library after 10 selection cycles bound vital Burkitt lymphoma cells more effectively than the starting library.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (Treg cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff cell) function and gain of suppressive activity by Treg cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of Treg cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3′ untranslated region. Release of SATB1 from the control of Foxp3 in Treg cells caused loss of suppressive function, establishment of transcriptional Teff cell programs and induction of Teff cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining Treg cell functionality.

Phosphatidylinositol 3′-Kinase/AKT Signaling Is Activated in Medulloblastoma Cell Proliferation and Is Associated with Reduced Expression of PTEN

Purpose: Medulloblastomas represent the most frequent malignant brain tumors of childhood. They are supposed to originate from cerebellar neural precursor cells. Recently, it has been shown that Sonic Hedgehog–induced formation of medulloblastoma in an animal model is significantly enhanced by activation of the phosphatidylinositol 3′-kinase (PI3K) signaling pathway. Experimental Design: To examine a role for PI3K/AKT signaling in the molecular pathogenesis of human medulloblastoma, we did an immunohistochemical study of the expression of Ser473-phosphorylated (p)-AKT protein in 22 medulloblastoma samples: All samples displayed p-AKT expression. To investigate if an activated PI3K/AKT pathway is required for medulloblastoma cell growth, we treated five human medulloblastoma cell lines with increasing concentrations of the PI3K inhibitor LY294002 and analyzed cellular proliferation and apoptosis. The antiproliferative effect could be antagonized by overexpressing constitutively active AKT. As the activation of PI3K/AKT signaling may be associated with alterations of the PTEN gene located at 10q23.3, a chromosomal region subject to frequent allelic losses in medulloblastoma, we screened PTEN for mutations and mRNA expression. Results: Proliferation of all of the medulloblastoma cell lines was dependent on PI3K/AKT signaling, whereas apoptosis was not prominently affected. Allelic loss was detected in 16% of the cases. One medulloblastoma cell line was found to carry a truncating mutation in the PTEN coding sequence. Even more important, PTEN mRNA and protein levels were found to be significantly lower in medulloblastomas compared with normal cerebellar tissue of different developmental stages. Reduction of PTEN expression was found to be associated with PTEN promoter hypermethylation in 50% of the tumor samples. Conclusions: We conclude that activation of the PI3K/AKT pathway constitutes an important step in the molecular pathogenesis of medulloblastoma and that dysregulation of PTEN may play a significant role in this context.

Postnatal development of the murine cerebellar cortex: formation and early dispersal of basket, stellate and Golgi neurons.

The cerebellar cortex consists of a small set of neuronal cell types interconnected in a highly stereotyped way. While the development of cerebellar cortical projection neurons, i.e. Purkinje cells, and that of granule cells has been elucidated in considerable detail, that of cerebellar cortical inhibitory interneurons is still rather fragmentarily understood. Here, we use mice expressing green fluorescent protein (GFP) from the Pax2 locus to analyse the ontogenesis of these cells. Numbers of Pax2‐positive inhibitory interneuronal precursors increase following a classical sigmoidal growth curve to yield a total of some 905.000 ± 77.000 cells. Maximal cell increase occurs at about postnatal day (P)5.4, and some 75% of all inhibitory interneurons are generated prior to P7. Conjoint analysis of the developmental accruement of Pax2‐GFP‐positive cells and their cell cycle distribution reveals that, at least at P0 and P3, the numerical increase of these cells results primarily from proliferation of a Pax2‐negative precursor population and suggests that Pax2 expression begins at or around the final mitosis. Following their terminal mitosis, inhibitory cerebellar cortical interneurons go through a protracted quiescent phase in which they maintain expression of the cell cycle marker Ki‐67. During this phase, they translocate into the nascent molecular layer, where they stall next to premigratory granule cell precursors without penetrating this population of cells. These observations provide a quantitative description of cerebellar cortical inhibitory interneuron genesis and early differentiation, and define Pax2 as a marker expressed in basket and stellate cells, from around their final mitosis to their incipient histogenetic integration.

MicroRNA Expression in Circulating Microvesicles Predicts Cardiovascular Events in Patients With Coronary Artery Disease

Background Circulating microRNAs (miRNAs) are differentially regulated and selectively packaged in microvesicles (MVs). We evaluated whether circulating vascular and endothelial miRNAs in patients with stable coronary artery disease have prognostic value for the occurrence of cardiovascular (CV) events. Methods and Results Ten miRNAs involved in the regulation of vascular performance—miR‐126, miR‐222, miR‐let7d, miR‐21, miR‐20a, miR‐27a, miR‐92a, miR‐17, miR‐130, and miR‐199a—were quantified in plasma and circulating MVs by reverse transcription polymerase chain reaction in 181 patients with stable coronary artery disease. The median duration of follow‐up for major adverse CV event–free survival was 6.1 years (range: 6.0–6.4 years). Events occurred in 55 patients (31.3%). There was no significant association between CV events and plasma level of the selected miRNAs. In contrast, increased expression of miR‐126 and miR‐199a in circulating MVs was significantly associated with a lower major adverse CV event rate. In univariate analysis, above‐median levels of miR‐126 in circulating MVs were predictors of major adverse CV event–free survival (hazard ratio: 0.485 [95% CI: 0.278 to 0.846]; P=0.007) and percutaneous coronary interventions (hazard ratio: 0.458 [95% CI: 0.222 to 0.945]; P=0.03). Likewise, an increased level of miR‐199a in circulating MVs was associated with a reduced risk of major adverse CV events (hazard ratio: 0.518 [95% CI: 0.299 to 0.898]; P=0.01) and revascularization (hazard ratio: 0.439 [95% CI: 0.232 to 0.832]; P=0.01) in univariate analysis. miRNA expression analysis in plasma compartments revealed that miR‐126 and miR‐199a are present mainly in circulating MVs. MV‐sorting experiments showed that endothelial cells and platelets were found to be the major cell sources of MVs containing miR‐126 and miR‐199a, respectively. Conclusion MVs containing miR‐126 and miR‐199a but not freely circulating miRNA expression predict the occurrence of CV events in patients with stable coronary artery disease.

The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure

The expression of the nuclear protein Ki‐67 (pKi‐67) is strictly correlated with cell proliferation. Because of this, anti‐Ki‐67 antibodies can be used as operational markers to estimate the growth fraction of human neoplasia in situ. For a variety of tumours, the assessment of pKi‐67 expression has repeatedly been proven to be of prognostic value for survival and tumour recurrence, but no cellular function has yet been ascribed to the Ki‐67 protein. This study shows that a C‐terminal domain of pKi‐67 (Kon21) is able to bind to all three members of the mammalian heterochromatin protein 1 (HP1) family in vitro and in vivo. This interaction can be manipulated in living cells, as evidenced by ectopic expression of GFP‐tagged HP1 proteins in HeLa cells, which results in a dramatic relocalization of endogenous pKi‐67. Taken together, the data presented in this study suggest a role for pKi‐67 in the control of higher‐order chromatin structure. Copyright

Bypassing genomic imprinting allows seed development

In developing progeny of mammals the two parental genomes are differentially expressed according to imprinting marks, and embryos with only a uniparental genetic contribution die. Gene expression that is dependent on the parent of origin has also been observed in the offspring of flowering plants, and mutations in the imprinting machinery lead to embryonic lethality, primarily affecting the development of the endosperm—a structure in the seed that nourishes the embryo, analogous to the function of the mammalian placenta. Here we have generated Arabidopsis thaliana seeds in which the endosperm is of uniparental, that is, maternal, origin. We demonstrate that imprinting in developing seeds can be bypassed and viable albeit smaller seedlings can develop from seeds lacking a paternal contribution to the endosperm. Bypassing is only possible if the mother is mutant for any of the FIS-class genes, which encode Polycomb group chromatin-modifying factors. Thus, these data provide functional evidence that the action of the FIS complex balances the contribution of the paternal genome. As flowering plants have evolved a special reproduction system with a parallel fusion of two female with two male gametes, our findings support the hypothesis that only with the evolution of double fertilization did the action of the FIS genes become a requirement for seed development. Furthermore, our data argue for a gametophytic origin of endosperm in flowering plants, thereby supporting a hypothesis raised in 1900 by Eduard Strasburger.

Elevation of plasma membrane permeability by laser irradiation of selectively bound nanoparticles.

Irradiation of nanoabsorbers with pico- and nanosecond laser pulses could result in thermal effects with a spatial confinement of less than 50 nm. Therefore absorbing nanoparticles could be used to create controlled cellular effects. We describe a combination of laser irradiation with nanoparticles, which changes the plasma membrane permeability. We demonstrate that the system enables molecules to penetrate impermeable cell membranes. Laser light at 532 nm is used to irradiate conjugates of colloidal gold, which are delivered by antibodies to the plasma membrane of the Hodgkins disease cell line L428 and/or the human large-cell anaplastic lymphoma cell line Karpas 299. After irradiation, membrane permeability is evaluated by fluorescence microscopy and flow cytometry using propidium iodide (PI) and fluorescein isothiocyanate (FITC) dextran. The fraction of transiently permeabilized and then resealed cells is affected by the laser parameter, the gold concentration, and the membrane protein of the different cell lines to which the nanoparticles are bound. Furthermore, a dependence on particle size is found for these interactions in the different cell lines. The results suggest that after optimization, this method could be used for gene transfection and gene therapy.