The understanding of mechanisms linking psychological stress to disease risk depend on reliable stress biomarkers. Circulating cell-free DNA (cfDNA) has emerged as a potential biomarker of cellular ...stress, aging, inflammatory processes, and cell death. Recent studies indicated that psychosocial stress and physical exercise might also influence its release. We compared the effects of acute psychosocial and physical exercise stress on cfDNA release by exposing 20 young, healthy men to both an acute psychosocial laboratory stressor and an acute physical exercise stressor. Venous blood and saliva samples were collected before and after stress exposure. Cell-free DNA was extracted from plasma and quantified by qPCR. Furthermore, cfDNA fragment length was analyzed and cfDNA methylation patterns were assayed across time. In addition, release of stress hormones and subjective stress responses were measured. Results showed a twofold increase of cfDNA after TSST and fivefold increase after exhaustive treadmill exercise, with an overabundance of shorter cfDNA fragments after physical exhaustion. Interestingly, cell-free mitochondrial DNA showed similar increase after both stress paradigms. Furthermore, cfDNA methylation signatures-used here as a marker for diverse cellular origin-were significantly different post stress tests. While DNA methylation decreased immediately after psychosocial stress, it increased after physical stress, suggesting different cellular sources of active DNA release. In summary, our results suggest stimulus and cell-specific regulation of cfDNA release. Whereas the functional role of stress-associated cfDNA release remains elusive, it might serve as a valuable biomarker in molecular stress research as a part of the psychophysiological stress response.
Background & AimCurrently, it remains a challenge to prepare extracellular vesicles (EVs) to high purity. Neither fractionation by density nor by size alone is sufficient to separate EVs from all ...contaminants e.g. high and low density lipoprotein (HDL/LDL) and adherent proteins. For now, a time consuming combination of two methods (density and size separation) is required to enrich EVs to high purity at the expense of time and low recovery. A fast and efficient alternative is represented by Free Flow Electrophoresis (FFE). It is a well-established (semi-) preparative method to separate analytes with inherent difference of charge density and/or difference of pI-value.Methods, Results & ConclusionA Free Flow Interval Zone Electrophoresis (FF-IZE) method has been developed, for the purification and isolation of EVs as well DNA and RNA from human plasma samples, using a set of buffer media of different pH-values ranging from pH 8.0 to pH 4.8. Upon processing supernatants of mesenchymal stem/stromal cells (MSCs) cultured in the presence of 10% human platelet lysate, EVs are recovered in a small number of FFE fractions lacking most proteins of the conditioned media. Currently, we characterize the identified fractions in more detail. Prepared EVs are quantified by Nano Particle Tracking Analysis (NTA) and imaging flow cytometry. Furthermore, the presence of several EV markers and the absence of contaminants are analyzed by Western Blot and mass spectrometry-based proteomics. We conclude FFE is a feasible and quick method to highly purify EVs in an accurate manner.
Background & AimExtracellular vesicles (EVs), such as exosomes and microvesicles, are shed by all cell types and found in all body fluids. EVs transmit specific information from their cells of origin ...to specific target cells and are key factors in a novel form of intercellular communication. Depending on their origin, EVs can modulate immune responses and either act proinflammatory (e.g. mature DC-EVs) or anti-inflammatory (e.g. mesenchymal stem cell (MSC) and many tumor cell-derived EVs).Methods, Results & ConclusionAiming to analyze immune-modulating properties of EVs from different sources, in vitro, we established a novel form of a mixed lymphocyte reaction (MLR) assay. Here, human peripheral blood-derived mononuclear cells (MNCs) were pooled from up to 12 different healthy donors warranting high cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed MNCs are cultured for 5 days in the absence or presence of EVs. Thereafter, cell morphologies are documented and cells are phenotypically characterized by flow cytometry. By analyzing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by therapeutically active MSC-EVs. For example we observed that in the presence of active MSC-EVs more CD14+ (monocytes) and CD56+ (natural killer cells) are recovered from the MLR than in corresponding control samples. In contrast, in the presence of active MSC-EVs contents of CD4+ and CD8+ T cells got slightly decreased. Focusing on T cells, we learned that active MSC-EVs reduced the content of CD4 and CD8 T cells expressing T cell activation markers like CD54 and CD25. Currently, we compare the immunomodulatory capabilities of EVs of different cell types. Furthermore, we proceed in optimizing the marker panel to distinguish immune cell subtypes such as the different types of CD4+ cell types (T 1, T 2, T 17 and T).
The HLA‐G molecule belongs to the family of nonclassical human leukocyte antigen (HLA) class I. At variance to classical HLA class I, HLA‐G displays (i) a low number of nucleotide variations within ...the coding region, (ii) a high structural diversity, (iii) a restricted peptide repertoire, (iv) a limited tissue distribution and (v) strong immune‐suppressive properties. The physiological HLA‐G surface expression is restricted to the maternal–fetal interface and to immune‐privileged adult tissues. Soluble forms of HLA‐G (sHLA‐G) are detectable in various body fluids. Cellular activation and pathological processes are associated with an aberrant or a neo‐expression of HLA‐G/sHLA‐G. Functionally, HLA‐G and its secreted forms are considered to be key players in the induction of short‐ and long‐term tolerance. Thus, its unique expression profile and tolerance‐inducing functions render HLA‐G/sHLA‐G an attractive biomarker to monitor the systemic health/disease status and disease activity/progression for clinical approaches in disease management and treatments. Here, we place emphasis on (i) the current status of the tolerance‐inducing functions by HLA‐G/sHLA‐G, (ii) the current complexity to implement this molecule as a meaningful clinical biomarker regarding the three dimensions of structural diversity (monomers, dimers and HLA‐G‐expressing extracellular vesicles) with its functional implications, and (iii) novel and future approaches to detect and quantify sHLA‐G structures and functions.
Background & AimHuman mesenchymal stem/stromal cells (MSCs) have been applied in more than 900 NIH-registered clinical trials with different outcomes. In a proportion of studies pro-regenerative ...and/or immunomodulatory therapeutic effects were observed. Against the initial assumption, preclinical data demonstrated that – if functional - MSCs act in a paracrine rather than in a cellular manner. Efforts to search for the active paracrine components ended in the identification of extracellular vesicle (EVs). Indeed, in several preclinical models EVs harvested from MSC conditioned media exerted comparable therapeutic effects than the MSCs themselves.Methods, Results & ConclusionEVs released by virtually all cell types represent a heterogenous population of vesicles of different origin, including exosomes (70-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (> 500 nm). Classically, they are prepared by differential centrifugation. Due to volume restricitions during ultracentrifugation only smaller amounts of EV containing liquids can be processed with this method. To efficiently translate MSC-EVs into the clinics, they need to be produced in a scaled manner. Previously, we established and successfully used a polyehtylen glycol (PEG) precipitation procedure allowing to process up to 10 l conditioned medium. Intending to further scale the MSC-EV production, optimally in a closed system, we have started to optimize tangential flow filtration (TFF) protocols, in principal being scalable up to several 1000 litres. At first, we compared different TFF devices and selected the holofiber-based TFF system. Although EVs can efficiently be prepared from serum-free conditioned media, the processing of serum or human platelet lysate (hPL) containing conditioned media remains challenging. The high protein content of serum and hPL supplemented media quickly results in protein aggregate formation and clogging of the pores of the TFF system, which negatively affect the EV purification process. Currently, by changing flow rates and and re-addition of buffers, we optimize the TFF protocols to enable us obtaining MSC-EV preparations with comparable purities than with the PEG method.
Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting ...histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.
Background & AimExtracellular vesicles (EVs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress acute inflammatory cues in a variety of ...different diseases, including Graft-versus-Host Disease (GvHD) and ischemic stroke, and promote regeneration of affected tissues. Following a successful clinical treatment attempt of a steroid refractory GvHD patient, we intend to optimize MSC-EV production strategies for further clinical applications.Methods, Results & ConclusionAs we observed functional differences of independent MSC-EV preparations in vitro, we aimed to set up an in vivo GvHD model for the more advanced functional testing of different MSC-EV preparations. To this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed severe GvHD symptoms. The GvHD symptoms were effectively suppressed if MSC-EV preparations, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay, were applied at 3 consecutive days. MSC-EV preparations lacking in vitro immune modulating activities within the MLR assay, however, hardly improved the symptoms of the GvHD mice. Thus, our results demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived MSCs are functional equivalent. Thus, successful transplantation of MSC-EVs into the clinics requires a platform allowing identification of MSC-EV preparations with sufficient therapeutic, most probably immune modulating activities.
Extracellular vesicles (EVs) are shed by nearly all cells and provide a novel way of intercellular communication. In addition to their increasing scientific recognition regarding their involvement in ...multiple biological processes, EVs of certain cell types are discussed as a novel class of biomarkers and can serve as potential therapeutic agents. Therefore, the composition of the EV containing sample reflects the heterogeneity of the origin and the EVs need to be chosen wisely dependent on the purpose of use. Due to the novelty of the field and the lack of well-established single EV analysis tools, the heterogeneity of single EVs has not been well analyzed, yet. Recently, our group revealed that imaging flow cytometry allows multi-parametric analyses of EVs at the single vesicle level. With the objective to investigate the heterogeneity of EVs in more detail, we aim to analyze EV-subpopulations from different EV containing samples (e.g. body fluids or different cell types). For the dissection, well qualified antibodies are essential because many of the tested antibodies showed a low efficiency or a background in the region of interest.
Now, we have optimized the single EV analysis protocols, especially for EVs being smaller than 200 nm. By improving the gating strategy and optimizing the staining protocol for the detection of small EVs, we are currently able to dissect heterogeneity of EVs with different antibodies at a certain level. This allows us not only to determine subpopulations to question isolation or purification protocols, but also makes diagnostic statements possible.
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