Vascular endothelial microparticles-incorporated microRNAs are altered in patients with diabetes mellitus.

BACKGROUND: Circulating microRNAs (miRs) are differentially regulated and selectively packaged into microparticles (MPs). We evaluated whether diabetes mellitus alters circulating vascular and endothelial MP-incorporated miRs expression levels. METHODS AND RESULTS: Circulating MPs were isolated from 135 patients with or without diabetes mellitus type II and characterized using flow cytometer and electron microscope. Nine miRs involved in the regulation of vascular performance-miR-126, miR-222, miR-let7d, miR-21, miR-30, miR-92a, miR-139, miR-199a and miR-26a-were quantified in circulating MPs by reverse transcription polymerase chain reaction. Among those, miR-126 and miR-26a were significantly reduced in diabetic patients compared to non-diabetic patients. Patients with low miR-26a and miR-126 levels were at higher risk for a concomitant coronary artery disease. MP-sorting experiments showed that endothelial cells were the major cell sources of MPs containing miR-126 and miR-26a, respectively. Finally, in accordance with our clinical results, in vitro experiments revealed that hyperglycemia reduces the packaging of miR-126 and miR-26a into EMPs. CONCLUSION: Diabetes mellitus significantly alters the expression of vascular endothelial miRs in circulating endothelial MPs with potential implications on vascular heath.

Isolation and Flow Cytometry Analysis of Macrophages from White Adipose Tissue.

Macrophages are one of the prominent leukocyte populations in white adipose tissue (WAT) and play an important role during WAT homeostasis and remodeling. Macrophage function in WAT is determined by ontogeny and the local tissue environment. Here, we present a protocol to analyze different macrophage populations from murine WAT using flow cytometry.

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.

Differential polarization and activation dynamics of systemic T helper cell subsets after aneurysmal subarachnoid hemorrhage (SAH) and during post-SAH complications.

Aneurysmal subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. Devastating post-SAH complications, such as cerebral vasospasm (CVS), delayed cerebral ischemia or seizures to mention a few, are mainly responsible for the poor clinical outcome. Inflammation plays an indispensable role during early brain injury (EBI) and delayed brain injury (DBI) phases over which these complications arise. T helper cells are the major cytokine secreting cells of adaptive immunity that can polarize to multiple functionally unique sub-populations. Here, we investigate different CD4+ T cell subsets during EBI and DBI phases after SAH, and their dynamics during post-SAH complications. Peripheral venous blood from 15 SAH patients during EBI and DBI phases, was analyzed by multicolour flowcytometry. Different subsets of CD3+ CD4+ T cells were characterized by differential cell surface expression of CXCR3 and CCR6 into Th1, Th2, Th17, whereas Tregs were defined by CD25hiCD127lo. The analysis of activation states was done by the expression of stable activation markers CD38 and HLA-DR. Interestingly, compared to healthy controls, Tregs were significantly increased during both EBI and DBI phases. Different activation states of Tregs showed differential significant increase during EBI and DBI phases compared to controls. HLA-DR- CD38+ Tregs were significantly increased during DBI phase compared to EBI phase in SAH patients developing CVS, seizures and infections. However, HLA-DR- CD38- Tregs were significantly reduced during EBI phase in patients with cerebral ischemia (CI) compared to those without CI. HLA-DR- CD38- Th2 cells were significantly increased during EBI phase compared to controls. A significant reduction in Th17/Tregs and HLA-DR- CD38+ Th17/Tregs ratios was observed during both EBI and DBI phases compared to controls. While HLA-DR- CD38- Th17/Tregs and HLA-DR- CD38- Th1/Th2 ratios were impaired only during EBI phase compared to controls. In conclusion, CD4+ T cell subsets display dynamic and unique activation patterns after SAH and during the course of the manifestation of post-SAH complications, which may be helpful for the development of precision neurovascular care. However, to claim this, confirmatory studies with larger patient cohorts, ideally from different ethnic backgrounds, are required. Moreover, our descriptive study may be the grounds for subsequent lab endeavors to explore the underlying mechanisms of our observations.

Inhibition of Intercellular Cytosolic Traffic via Gap Junctions Reinforces Lomustine-Induced Toxicity in Glioblastoma Independent of MGMT Promoter Methylation Status.

Glioblastoma is a malignant brain tumor and one of the most lethal cancers in human. Temozolomide constitutes the standard chemotherapeutic agent, but only shows limited efficacy in glioblastoma patients with unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter status. Recently, it has been shown that glioblastoma cells communicate via particular ion-channels-so-called gap junctions. Interestingly, inhibition of these ion channels has been reported to render MGMT promoter-methylated glioblastoma cells more susceptible for a therapy with temozolomide. However, given the percentage of about 65% of glioblastoma patients with an unmethylated MGMT promoter methylation status, this treatment strategy is limited to only a minority of glioblastoma patients. In the present study we show that-in contrast to temozolomide-pharmacological inhibition of intercellular cytosolic traffic via gap junctions reinforces the antitumoral effects of chemotherapeutic agent lomustine, independent of MGMT promoter methylation status. In view of the growing interest of lomustine in glioblastoma first and second line therapy, these findings might provide a clinically-feasible way to profoundly augment chemotherapeutic effects for all glioblastoma patients.

Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

BACKGROUND: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. METHODS: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. RESULTS: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. CONCLUSION: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.

Flow cytometric purification of Colletotrichum higginsianum biotrophic hyphae from Arabidopsis leaves for stage-specific transcriptome analysis.

Generation of stage-specific cDNA libraries is a powerful approach to identify pathogen genes that are differentially expressed during plant infection. Biotrophic pathogens develop specialized infection structures inside living plant cells, but sampling the transcriptome of these structures is problematic due to the low ratio of fungal to plant RNA, and the lack of efficient methods to isolate them from infected plants. Here we established a method, based on fluorescence-activated cell sorting (FACS), to purify the intracellular biotrophic hyphae of Colletotrichum higginsianum from homogenates of infected Arabidopsis leaves. Specific selection of viable hyphae using a fluorescent vital marker provided intact RNA for cDNA library construction. Pilot-scale sequencing showed that the library was enriched with plant-induced and pathogenicity-related fungal genes, including some encoding small, soluble secreted proteins that represent candidate fungal effectors. The high purity of the hyphae (94%) prevented contamination of the library by sequences derived from host cells or other fungal cell types. RT-PCR confirmed that genes identified in the FACS-purified hyphae were also expressed in planta. The method has wide applicability for isolating the infection structures of other plant pathogens, and will facilitate cell-specific transcriptome analysis via deep sequencing and microarray hybridization, as well as proteomic analyses.

Distinct kinetics and dynamics of cross-presentation in liver sinusoidal endothelial cells compared to dendritic cells.

UNLABELLED: Cross-presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ-resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross-presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross-presenting circulating soluble antigen ex vivo as DCs at a per-cell basis. However, antigen uptake in vivo was 100-fold more pronounced in LSECs, indicating distinct mechanisms of cross-presentation. In contrast to mannose-receptor-mediated antigen uptake and routing into stable endosomes dedicated to cross-presentation in DCs, we observed distinct antigen-uptake and endosomal routing with high antigen turnover in LSECs that resulted in short-lived cross-presentation. Receptor-mediated endocytosis did not always lead to cross-presentation, because immune-complexed antigen taken up by the Fc-receptor was not cross-presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity. CONCLUSION: These results provide a mechanistic explanation how organ-resident LSECs accommodate continuous scavenger function with the capacity to cross-present circulating antigens using distinct kinetics and dynamics of antigen-uptake, routing and cross-presentation compared to DCs.

High-Dimensional Analysis of Immune Cell Composition Predicts Periprosthetic Joint Infections and Dissects Its Pathophysiology.

Accurate diagnosis of periprosthetic joint infections (PJI) is one of the most widely researched areas in modern orthopedic endoprosthesis. However, our understanding of the immunological basis of this severe complication is still limited. In this study, we developed a flow cytometric approach to precisely characterize the immune cell composition in periprosthetic joints. Using high-dimensional multi-parametric data, we defined, for the first time, the local immune cell populations of artificial joints. We identified significant differences in the cellular distribution between infected and non-infected samples, and revealed that myeloid-derived suppressor cells (MDSCs) act as potential regulators of infiltrating immune cells in PJI. Further, we developed an algorithm to predict septic and aseptic samples with high sensitivity and specificity, that may serve as an indispensable addition to the current criteria of the Musculoskeletal Infection Society. This study describes a novel approach to flow cytometrically analyze the immune cell infiltrate of joint fluid that not only improves our understanding of the pathophysiology of PJI, but also enables the development of a novel screening tool to predict infection status. Our data further suggest that pharmacological targeting of MDSCs represents a novel strategy for addressing PJI.

Inhibition of Gap Junctions Sensitizes Primary Glioblastoma Cells for Temozolomide.

Gap junctions have recently been shown to interconnect glioblastoma cells to a multicellular syncytial network, thereby allowing intercellular communication over long distances as well as enabling glioblastoma cells to form routes for brain microinvasion. Against this backdrop gap junction-targeted therapies might provide for an essential contribution to isolate cancer cells within the brain, thus increasing the tumor cells' vulnerability to the standard chemotherapeutic agent temozolomide. By utilizing INI-0602-a novel gap junction inhibitor optimized for crossing the blood brain barrier-in an oncological setting, the present study was aimed at evaluating the potential of gap junction-targeted therapy on primary human glioblastoma cell populations. Pharmacological inhibition of gap junctions profoundly sensitized primary glioblastoma cells to temozolomide-mediated cell death. On the molecular level, gap junction inhibition was associated with elevated activity of the JNK signaling pathway. With the use of a novel gap junction inhibitor capable of crossing the blood-brain barrier-thus constituting an auspicious drug for clinical applicability-these results may constitute a promising new therapeutic strategy in the field of current translational glioblastoma research.

Brain endothelial PPARgamma controls inflammation-induced CD4+ T cell adhesion and transmigration in vitro.

An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells (EC) which strongly relies on interaction with EC-expressed adhesion molecules. We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of brain EC inflammation. The PPARgamma agonist pioglitazone reduces transendothelial migration of encephalitogenic T cells across TNFalpha-stimulated brain EC. This effect is clearly PPARgamma mediated, as lentiviral PPARgamma overexpression in brain EC results in selective abrogation of inflammation-induced ICAM-1 and VCAM-1 upregulation and subsequent adhesion and transmigration of T cells. We therefore propose that PPARgamma in brain EC may be exploited to target detrimental EC-T cell interactions under inflammatory conditions.

Further analysis of delayed luminescence of plants.

The delayed luminescence curves of biological tissues, in particular the "hyperbolic" oscillations of the form exp(-iln (1 + lambda t)) with t as the time and lambda as a constant, were subjects of further experimental and theoretical investigations. It turned out that the oscillations are solutions of a Hamiltonian that keeps coherent states coherent. In agreement to the expectations the oscillations disappear as soon as the biological system looses its integrity or its collective structure. The photocount statistics (PCS) confirms these results. The illumination itself influences the biological state under consideration. The temperature dependence displays small deviations from the Arrhenius-law with a mean activation energy of about 0.5 eV in case of plants. The results may be useful for investigating supermolecular interactions as well as for assessing the quality or quality changes of biological tissues.

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% CIAUTHOR: Is 95% CI correct?: 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.

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(+) Burkitt's 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.

Laser-assisted photoablation of human pluripotent stem cells from differentiating cultures.

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 +/- 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.