Julian Kamhieh-Milz

Differentially Expressed MicroRNAs in Maternal Plasma for the Noninvasive Prenatal Diagnosis of Down Syndrome (Trisomy 21)

Objectives. Most developmental processes are under the control of small regulatory RNAs called microRNAs (miRNAs). We hypothesize that different fetal developmental processes might be reflected by extracellular miRNAs in maternal plasma and may be utilized as biomarkers for the noninvasive prenatal diagnosis of chromosomal aneuploidies. In this proof-of-concept study, we report on the identification of extracellular miRNAs in maternal plasma of Down syndrome (DS) pregnancies. Methods. Using high-throughput quantitative PCR (HT-qPCR), 1043 miRNAs were investigated in maternal plasma via comparison of seven DS pregnancies with age and fetal sex matched controls. Results. Six hundred and ninety-five miRNAs were identified. Thirty-six significantly differentially expressed mature miRNAs were identified as potential biomarkers. Hierarchical cluster analysis of these miRNAs resulted in the clear discrimination of DS from euploid pregnancies. Gene targets of the differentially expressed miRNAs were enriched in signaling pathways such as mucin type-O-glycans, ECM-receptor interactions, TGF-beta, and endocytosis, which have been previously associated with DS. Conclusions. miRNAs are promising and stable biomarkers for a broad range of diseases and may allow a reliable, cost-efficient diagnostic tool for the noninvasive prenatal diagnosis of DS.

Oxidative stress is predominant in female but not in male patients with autoimmune thrombocytopenia.

As the involvement of oxidative stress (OS) in autoimmune thrombocytopenia (AITP) has been reported, a fast and rapid test for the reliable measurement of OS and antioxidant capacities (AOCs) might be a useful tool in extending current diagnostic possibilities. The free oxygen radical test (FORT) and free oxygen radical defence (FORD) assay (Callegari, Italy) are easy to perform and reliable, with results available within 15 minutes. Thirty-seven AITP patients and 37 matched healthy individuals were included in this study. All participants responded to a standard questionnaire provided by these assays. Female patients with AITP were observed to demonstrate significantly higher OS in comparison to female controls (P = 0.0027) and male AITP patients (P = 0.0018). The AOCs were not reduced in patients with AITP (P = 0.7648). Correlation of OS with platelet count identified a weak positive correlation (P = 0.0327, Spearman R = 0.4672). The questionnaire revealed that ITP patients in comparison to healthy controls are more stressed, feel exhausted and fatigued, and eat a healthier diet. In conclusion, OS is predominant in female but not in male patients with AITP suggesting gender-specific differences in the pathomechanisms of AITP. Identification of patients with high levels of OS might be beneficial in the management of AITP.

Reduced antioxidant capacities in platelets from patients with autoimmune thrombocytopenia purpura (ITP)

Autoimmune thrombocytopenic purpura (ITP) is characterized by an abnormally low platelet count and bleeding risks. The exact triggering event remains elusive. Oxidative stress may play a role in several autoimmune diseases. A direct link between platelets in ITP and oxidative stress has not yet been addressed. The intracellular platelet antioxidant capacity (AOC) in ITP patients in the active phase (n = 24) and remission (n = 12), and 44 healthy controls were analysed with 2′,7′-dichlorodihydrofluorescein diacetate, and in combination with hydrogen peroxide. Enzyme activities (EA) of serum glutathione peroxidase (GPx), glutathione reductase (GRed) and catalase (CAT) were investigated colourimetrically in patients and controls. The AOC of ITP patients in the active phase was drastically reduced, with significantly high mean fluorescence intensity values. Higher GPx activity was observed in both active phase and remission in comparison to healthy controls (p < 0.001), with greater activity observed in active ITP than remission (p = 0.001). However, GRed EA was not elevated indicating that reduced glutathione (GSH) is not comparably recovered as consumed, leading to a decreased bioavailability of GSH and increased oxidative stress. These results suggest that oxidative stress is implicated in active ITP and may play a crucial role in its pathophysiology.

Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour‐suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti‐nuclear autoantibodies in a subset of ITP patients: anti‐PCNA, anti‐SmD, anti‐Ro/SSA60, anti‐Ro/SSA52, anti‐La/SSB and anti‐RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

Regular blood donation may help in the management of hypertension: an observational study on 292 blood donors

Hypertension is one of the leading global risks for cardiovascular events worldwide. There is preliminary evidence that regular blood donation may be beneficial.

Proteomic profiling of secreted proteins for the hematopoietic support of interleukin-stimulated human umbilical vein endothelial cells.

Human umbilical cord vein endothelial cells (HUVECs) secrete a number of factors that greatly impact the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). These factors remain largely unknown. Here, we report on the most comprehensive proteomic profiling of the HUVEC secretome and identified 827 different secreted proteins. Two hundred and thirty-one proteins were found in all conditions, whereas 369 proteins were identified only under proinflammatory conditions following IL-1β, IL-3, and IL-6 stimulation. Thirteen proteins including complement factor b (CFb) were identified only under IL-1β and IL-3 conditions and may potentially represent HSPC proliferation factors. The combination of bioinformatics and gene ontology annotations indicates the role of the complement system and its activation. Furthermore, CFb was found to be transcriptionally strongly upregulated. Addition of complement component 5b-9 (C5b-9) monoclonal antibody to the stem cell expansion assay was capable of significantly reducing their proliferation. This study suggests a complement-mediated cross-talk between endothelial cells and HSPCs under proinflammatory conditions.

Transcriptional Profiling of the Hematopoietic Support of Interleukin-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs)

Endothelial cells can be successfully used to maintain or increase the number of hematopoietic stem cells in vitro. Previously we identified hematopoietic progenitor cell (HPC) expansion or survival benefit induced by IL-1β-, IL-3-, and IL-6-stimulated human umbilical vein endothelial cell (HUVEC) supernatants. In order to identify molecular mechanisms that support hematopoiesis, we examined the time-dependent expression profiles of IL-1β-, IL-3-, and IL-6-stimulated HUVECs via microarray. Here, we present 24 common upregulated elements and three common downregulated elements of IL-1β- and IL-3-stimulated HUVECs, with these factors exhibiting great potential for the observed HPC expansion. Furthermore, metabolic pathway analysis resulted in the identification of nonproteinogenic factors such as prostaglandin E2 (PGE2) and nitric oxide (NO) and determined their HPC expansion potential via delta, methylcellulose, and cobblestone assays. We confirmed PGE2 and spermine as hematopoietic expansion factors. Furthermore, we identified several factors such as SSAT, extracellular matrix components, microRNA21, and a microvesicle-mediated cross-talk between the endothelium and HPCs that may play a crucial role in determining stem cell fate. Our results suggest that microarray in combination with functional annotations is a convenient method to identify novel factors with great impact on HPC proliferation and differentiation.

Analysis of platelet-derived extracellular vesicles in plateletpheresis concentrates: a multicenter study

Routine quantification of platelet‐derived extracellular vesicles (PL‐EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL‐EV quantification using conventional flow cytometers.

Secretome profiling of apheresis platelet supernatants during routine storage via antibody-based microarray

Platelet storage lesions (PSLs) occur during platelet concentrate (PC) storage. Adverse transfusion reactions (ATRs) have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Proteomic profiling of PC supernatants was thus performed to identify proteins associated with PSLs and ATRs. Twenty-four PCs were investigated daily from day 0 to day 9 for platelet pre-activation (PPA), platelet-derived extracellular vesicles (PEVs), and platelet function. Using antibody microarrays, 673 extracellular proteins were analysed in PC supernatants on days 0, 3, 5, 7, and 9. During 5days of storage, PPA and PEVs continuously increased (P<0.0001). Platelet function was observed to remain stable within the first 5days (P=0.1751) and decreased thereafter. Comparison of all time points to day 0 revealed the identification of 136 proteins that were significantly changed in abundance during storage, of which 72 were expressed by platelets. Network analysis identified these proteins to be predominantly associated with exosomes (P=4.61×10-8, n=45 genes) and two clusters with distinct functions were found with one being associated with haemostasis and the other with RNA binding. These findings may provide an explanation for ATRs. SIGNIFICANCE Changes in platelet concentrate (PC) supernatants during storage have been so far only poorly addressed and high abundant proteins burden the identification of quantitative changes in the secretome. We applied a high-throughput antibody microarray allowing for the sensitive quantification of 673 extracellular factors. PCs account for the highest number of adverse transfusion reactions (ATRs). ATRs have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Comprehensive interpretation of the changing proteins in the secretome during platelet storage under blood banking conditions may help to identify mechanisms leading to the occurrence of adverse transfusion reactions.

Efficacy and safety of erythrocytapheresis and low-dose erythropoietin for treatment of hemochromatosis

The aim of this study was to determine retrospectively the efficacy of combined therapy using erythropoietin (EPO) and erythrocytapheresis (EA) in patients with hereditary hemochromatosis (HH) who did not tolerate phlebotomy.