We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

Peripheral biomarkers play an indispensable role in quick and reliable diagnoses of any kind of disease. With the population ageing, the number of people suffering from age-related diseases is expected to rise dramatically over the coming decades. In particular, all types of cognitive deficits, such as Alzheimer’s disease, will increase. Alzheimer’s disease is characterised mainly by coexistence of amyloid plaques and neurofibrillary tangles in brain. Reliable identification of such molecular characteristics antemortem, however, is problematic due to restricted availability of appropriate sample material and definitive diagnosis is only possible postmortem. Currently, the best molecular biomarkers available for antemortem diagnosis originate from cerebrospinal fluid. Though, this is not convenient for routine diagnosis because of the required invasive lumbar puncture. As a consequence, there is a growing demand for additional peripheral biomarkers in a more readily accessible sample material. Blood platelets, due to shared biochemical properties with neurons, can constitute an attractive alternative as discussed here. This review summarises potential platelet Alzheimer’s disease biomarkers, their role, implication, and alteration in the disease. For easy comparison of their performance, the Hedge effect size was calculated whenever data were available.

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56–100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4–7 and 6–9. Only spots that were reproducibly detectable in at least 90% of all gels (n = 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1–2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance.

During sepsis and major trauma the blood glutamine (Gln) level is reduced. The administration of Gln can improve the outcome of these patients. However, the mechanism of this beneficial effect of Gln is poorly understood. In the course of critical illness leucocytes are confronted with cytotoxic inflammatory mediators. To protect themselves against these factors, cells express heat shock proteins (HSP). Previous studies have shown that the expression of the major inducible HSP (HSP70) is improved by high Gln concentrations above 4 mM. In this study we investigated whether Gln depletion, such as observed during critical illness, has an effect on HSP70 expression. Human lymphocytes exposed for 2 h to 42 degrees C showed a 3-fold increase in HSP70 expression (P<0.01). A preceding Gln starvation period over 3 days had no influence on this increase. However, when Gln is reduced during the stress response, HSP70 expression is impaired. A reduction of Gln from 0.5 mM (physiological) to 0.125 mM (pathological) led to a 40% lower HSP70 level (P<0.002). In contrast, increasing Gln concentrations (up to 2 mM) had only minor stimulatory effects (about 15%). This Gln-dependency of heat mediated HSP70 expression was observed in resting as well as proliferating lymphocytes. Our data indicate that during periods of reduced plasma Gln levels the stress response of human lymphocytes is impaired. Thus, Gln may be essential to minimize the susceptibility of leucocytes to cytotoxic inflammatory mediators. This is a new aspect of the protective effect of Gln supplementation in critically ill patients.