Cell motility and migration as determinants of stem cell efficacy.

BACKGROUND: Stem cells` (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. METHODS: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. FINDINGS: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. INTERPRETATION: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. FUNDING: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant.

Modulating endothelial adhesion and migration impacts stem cell therapies efficacy.

BACKGROUND: Limited knowledge of stem cell therapies` mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. METHODS: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. FINDINGS: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. INTERPRETATION: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. FUNDING: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health.

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

BACKGROUND: Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). STUDY DESIGN AND METHODS: ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. RESULTS: ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. CONCLUSIONS: Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.

Aerobic exercise inhibits acute lung injury: from mouse to human evidence Exercise reduced lung injury markers in mouse and in cells.

Acute respiratory distress syndrome (ARDS) is defined as hypoxemic respiratory failure with intense pulmonary inflammation, involving hyperactivation of endothelial cells and neutrophils. Given the anti-inflammatory effects of aerobic exercise (AE), this study investigated whether AE performed daily for 5 weeks would inhibit extra-pulmonary LPS-induced ARDS. C57Bl/6 mice were distributed into Control, Exercise, LPS and Exercise+LPS groups. AE was performed on a treadmill for 5x/week for four weeks before LPS administration. 24hours after the final AE physical test, animals received 100ug of LPS intra-peritoneally. In addition, whole blood cell culture, neutrophils and human endothelial cells were preincubated with IL-10, an anti-inflammatory cytokine induced by exercise. AE reduced total protein levels (p<0.01) and neutrophil accumulation in bronchoalveolar lavage (BAL) (p<0.01) and lung parenchyma (p<0.01). AE reduced BAL inflammatory cytokines IL-1ß, IL-6 and GM-CSF (p<0.001), CXCL1/KC, IL-17, TNF-alpha and IGF-1 (p<0.01). Systemically, AE reduced IL-1ß, IL-6 and IFN-gamma (p<0.001), CXCL1/KC (p<0.01) and TNF-alpha (p<0.05). AE increased IL-10 levels in serum (p<0.001) and BAL (p<0.001). Furthermore, AE increased superoxide dismutase SOD (p<0.01) and decreased superoxide anion accumulation in the lungs (p<0.01). Lastly, pre-incubation with IL-10 significantly reduced LPS-induced activation of whole blood cells, neutrophils and HUVECs, as observed by reduced production of IL-1ß, IL-6, IL-8 and TNF-alpha. Our data suggest that AE inhibited LPS-induced lung inflammation by attenuating inflammatory cytokines and oxidative stress markers in mice and human cell culture via enhanced IL-10 production.

QCM-D surpassing clinical standard for the dose administration of new oral anticoagulant in the patient of coagulation disorders.

The study focuses the dose administration of dabigatran to avoid the deaths due to hemorrhagic complications and thromboembolic stroke in clinics worldwide. To target the issue, a novel emerging acoustic technology, namely ''Quartz Crystal Microbalance with Dissipation'' (QCM-D) has been applied, while the acoustic assays namely ''activated Partial Thromboplastin Time'' (aPTT) and ''Prothrombinase complex-induced Clotting Test'' (PiCT) have been compared with the standard methods in parallel. Both techniques have been applied to 300 samples, including 220 plasma samples of patients suffering coagulation disorders and 80 plasma samples of non-patients. In comparison, the coagulation times of the acoustic aPTT and PiCT yielded an excellent correlation with the standard methods with in analytical standard deviation limits. Finally, the acoustic aPTT assay is the ''gold standard'' for a dose administration of the new oral anticoagulant, where the Δf/ΔΓ ratio of the acoustic assay demonstrates that dabigatran with FEIBA 50 combination could be a safe remedy to avoid the deaths in clinics.

Good Feasibility of the New German Blood Donor Questionnaire.

BACKGROUND: We assessed the effect of the uniform donor questionnaire (UDQ) on deferral rates in first-time and repeat donors. We focused on the introduced question about unprotected sexual contact with a new partner. Another goal was a stratified comparison of the deferral rates of the donor questionnaire (DQ) and UDQ. METHODS: Data on donors and deferrals using the DQ and UDQ were collected at four blood establishments. The comparison included a 2-year period by questionnaire version. For the comparison of the questionnaires, an adjusted multinomial logistic regression was performed. RESULTS: The analysis included 260,848 donations. First-time (FTD) and repeat donations (RD) showed higher deferral rates with the UDQ (FTD +5.4%, RD +1.4%). Deferral due to a new partner was 3.0% in first-time and 0.4% in repeat donors. The majority of these occurred in the youngest age groups. The most frequent deferral criterion was 'disease' (5.1%). CONCLUSION: The regression revealed stronger predictors for deferral than the questionnaire version. Especially younger age carried a higher and independent risk for deferral. The additional deferrals of mainly young first-time donors due to a new sexual partner may identify those donors with potential heterosexual risk behavior who would otherwise not be identified.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Defined Structure-Activity Relationships of Boswellic Acids Determine Modulation of Ca2+ Mobilization and Aggregation of Human Platelets by Boswellia serrata Extracts.

Boswellic acids constitute a group of unique pentacyclic triterpene acids from Boswellia serrata with multiple pharmacological activities that confer them anti-inflammatory and anti-tumoral properties. A subgroup of boswellic acids, characterized by an 11-keto group, elevates intracellular Ca2+ concentrations [Ca2+]i and causes moderate aggregation of human platelets. How different BAs and their mixtures in pharmacological preparations affect these parameters in activated platelets has not been addressed, so far. Here, we show that boswellic acids either antagonize or induce Ca2+ mobilization and platelet aggregation depending on defined structural determinants with inductive effects predominating for a B. serrata gum resin extract. 3-O-Acetyl-11-keto-ß-boswellic acid potently suppressed Ca2+ mobilization (IC50 = 6 µM) and aggregation (IC50 = 1 µM) when platelets were activated by collagen or the thromboxane A2 receptor agonist U-46619, but not upon thrombin. In contrast, ß-boswellic acid and 3-O-acetyl-ß-boswellic acid, which lack the 11-keto moiety, were weak inhibitors of agonist-induced platelet responses, but instead they elicited elevation of [Ca2+]i and aggregation of platelets (≥ 3 µM). 11-Keto-ß-boswellic acid, the structural intermediate between 3-O-acetyl-11-keto-ß-boswellic acid and ß-boswellic acid, was essentially inactive independent of the experimental conditions. Together, our study unravels the complex agonizing and antagonizing properties of boswellic acids on human platelets in pharmacologically relevant preparations of B. serrata gum extracts and prompts for careful evaluation of the safety of such extracts as herbal medicine in cardiovascular risk patients.

Sex-specific variation in signaling pathways and gene expression patterns in human leukocytes in response to endotoxin and exercise.

BACKGROUND: While exercise effects on the immune system have received increasing attention in recent years, it remains unclear to what extent gender and fluctuations in sex hormones during menstrual cycle influence immunological responses to exercise. METHODS: We investigated mRNA changes induced through exhaustive exercise (half-marathon; pre-exercise and post-exercise [30 min, 3 h, 24 h] on whole blood cultures ± lipopolysaccharide [LPS] [1 h]) with a specific focus on sex differences (men vs women in luteal phase) as an extension of our previous study. RESULTS: Inflammation related signaling pathways, TLRs, cytosolic DNA sensing and RIG-I like receptors were differentially activated between sexes in LPS-stimulated cultures. Genes differentially regulated between sexes included TNIP-1, TNIP-3, IL-6, HIVEP1, CXCL3, CCR3, IL-8, and CD69, revealing a bias towards less anti-inflammatory gene regulation in women compared to men. In addition, several genes relevant to brain function (KMO, DDIT4, VEGFA, IGF1R, IGF2R, and FGD4) showed differential activation between sexes. Some of these genes (e.g., KMO in women, DDIT4 in both sexes) potentially constitute neuroprotective mechanisms. CONCLUSIONS: These data reveal that the exercise-induced change in gene expression might be gender and menstrual cycle phase dependent.

Aerobic Exercise Reduces Asthma Phenotype by Modulation of the Leukotriene Pathway.

INTRODUCTION: Leukotrienes (LTs) play a central role in asthma. Low- to moderate-intensity aerobic exercise (AE) reduces asthmatic inflammation in clinical studies and in experimental models. This study investigated whether AE attenuates LT pathway activation in an ovalbumin (OVA) model of asthma. METHODS: Sixty-four male, BALB/c mice were distributed into Control, Exercise (Exe), OVA, and OVA + Exe groups. Treadmill training was performed at moderate intensity, 5×/week, 1 h/session for 4 weeks. Quantification of bronchoalveolar lavage (BAL) cellularity, leukocytes, airway remodeling, interleukin (IL)-5, IL-13, cysteinyl leukotriene (CysLT), and leukotriene B4 (LTB4) in BAL was performed. In addition, quantitative analyses on peribronchial leukocytes and airway epithelium for LT pathway agents: 5-lypoxygenase (5-LO), LTA4 hydrolase (LTA4H), CysLT1 receptor, CysLT2 receptor, LTC4 synthase, and LTB4 receptor 2 (BLT2) were performed. Airway hyperresponsiveness (AHR) to methacholine (MCh) was assessed via whole body plethysmography. RESULTS: AE decreased eosinophils (p < 0.001), neutrophils (p > 0.001), lymphocytes (p < 0.001), and macrophages (p < 0.01) in BAL, as well as eosinophils (p < 0.01), lymphocytes (p < 0.001), and macrophages (p > 0.001) in airway walls. Collagen (p < 0.01), elastic fibers (p < 0.01), mucus production (p < 0.01), and smooth muscle thickness (p < 0.01), as well as IL-5 (p < 0.01), IL-13 (p < 0.01), CysLT (p < 0.01), and LTB4 (p < 0.01) in BAL were reduced. 5-LO (p < 0.05), LTA4H (p < 0.05), CysLT1 receptor (p < 0.001), CysLT2 receptor (p < 0.001), LTC4 synthase (p < 0.001), and BLT2 (p < 0.01) expression by peribronchial leukocytes and airway epithelium were reduced. Lastly, AHR to MCh 25 mg/mL (p < 0.05) and 50 mg/mL (p < 0.01) was reduced. CONCLUSION: Moderate-intensity AE attenuated asthma phenotype and LT production in both pulmonary leukocytes and airway epithelium of OVA-treated mice.

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BACKGROUND: An important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions. METHODS: Binding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-L-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes. RESULTS: Frequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells. CONCLUSIONS: In this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.

Utilisation of Quartz Crystal Microbalance Sensors with Dissipation (QCM-D) for a Clauss Fibrinogen Assay in Comparison with Common Coagulation Reference Methods.

The determination of fibrinogen levels is one of the most important coagulation measurements in medicine. It plays a crucial part in diagnostic and therapeutic decisions, often associated with time-critical conditions. The commonly used measurement is the Clauss fibrinogen assay (CFA) where plasma is activated by thrombin reagent and which is conducted by mechanical/turbidimetric devices. As quartz crystal microbalance sensors with dissipation (QCM-D) based devices have a small footprint, can be operated easily and allow measurements independently from sample transportation time, laboratory location, availability and opening hours, they offer a great opportunity to complement laboratory CFA measurements. Therefore, the objective of the work was to (1) transfer the CFA to the QCM-D method; (2) develop an easy, time- and cost-effective procedure and (3) compare the results with references. Different sensor coatings (donor's own plasma; gold surface) and different QCM-D parameters (frequency signal shift; its calculated turning point; dissipation signal shift) were sampled. The results demonstrate the suitability for a QCM-D-based CFA in physiological fibrinogen ranges. Results were obtained in less than 1 min and in very good agreement with a standardized reference (Merlin coagulometer). The results provide a good basis for further investigation and pave the way to a possible application of QCM-D in clinical and non-clinical routine in the medical field.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. STUDY DESIGN: Activated THP-1 cells were exposed to .5 to 50 µM of nitrogen-containing bisphosphonates and .5 to 500 µM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. RESULTS: All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 µM and 500 µM; P < .05). Low concentrations (0.5 µM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations. CONCLUSIONS: Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Detection of HIT antibody dependent platelet aggregation using novel surface imprinting approach.

We present a fast, robust and straightforward spin force assisted surface imprinting approach for activated platelets and demonstrate that Heparin induced thrombocytopenia (HIT) platelet aggregation can be measured by this approach. A critical and challenging step in functional assays for HIT is platelet separation from the healthy donor's platelet-rich plasma (PRP). Our approach using surface imprinted polymer (MIP) for measurements on a quartz crystal microbalance with dissipation (QCM-D) enables monitoring of platelet aggregation directly in PRP thus eliminating the challenge of platelet separation. This is the first report of platelet imprinting. We also provide proof of principle that QCM-D technology can be applied for functional measurements of HIT antibodies.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

Zoledronate but not denosumab suppresses macrophagic differentiation of THP-1 cells. An aetiologic model of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Bisphosphonates and denosumab are antiresorptive drugs used for the treatment of osteoporosis and oncological tumors. A severe side effect is osteonecrosis of the jaw. Monocyte/macrophage dysfunction is considered to play a distinct role in osteonecrosis. THP-1 monocytic cells were used in this study to elucidate the influence of zoledronate and denosumab on phorbol-12-myrisate-13-acetate (PMA)-induced macrophage differentiation and function in real-time. MATERIALS AND METHODS: Macrophagic differentiation of the THP-1 suspension cells was measured by cell adherence in the presence or absence of different concentrations of zoledronate (0.5, 5, 50 µM) and denosumab (1, 10, 20, 40 µg/mL) using the real-time xCELLigence system. Additionally, a live/dead staining was performed by fluorescence microscopy. RESULTS: THP-1 cells demonstrated a regular initial PMA-induced differentiation to macrophages by live measurements of cell adherence and by an increase in CD68 surface expression as detected by flow cytometry. The addition of zoledronate led to cell detachment of the THP-1-derived macrophages in a dose-dependent manner in contrast to denosumab. Cell detachment was based on cell death as confirmed by live/dead staining, revealing elevated numbers of dead cells following addition of high zoledronate concentrations. However, denosumab did not deteriorate THP-1 cell viability. CONCLUSION: Our results demonstrate that zoledronate but not denosumab suppresses monocytic THP-1 cell viability after macrophagic differentiation dose-dependently. CLINICAL RELEVANCE: This is the first real-time study providing evidence for a dose-dependent immunosuppressive effect of zoledronate in contrast to denosumab on local macrophages.

QCM-D providing new horizon in the domain of sensitivity range and information for haemostasis of human plasma.

Monitoring of the haemostasis status is significant for proper therapeutic directions and decisions in surgery and innate coagulation disorders. In this regard, to gain a general overview of the plasmatic coagulation, prothrombin time (PT) tests are frequently combined with tests for activated partial thromboplastin time (aPTT). For aPTT we report for the first time that a QCM-D (Quartz Crystal Microbalances with Dissipation) based technique offers a better alternative to the standard coagulometer method in the perspective of range and information. We used heparin as anticoagulant to generate different coagulation times for human plasma. QCM-D astonishingly proved to be more sensitive and reliable than the standard coagulometer for aPTT range of upper limits of coagulation times. The established platform can monitor the fibrinogen concentration ranging from 1-6g/L (yielding R(2)=0.98 in calibration curves) along with aPTT from frequency and dissipation shifts together in a single set of measurements. Additionally the sensor layers have been tested for reusability, demonstrating no loss in sensor characteristics up to ten times measurements.

Exercise immunology meets MiRNAs.

A large body of evidence indicates modified expression of protein-coding genes in response to different kinds of physical activity. Recent years have exposed another level of regulation of cellular processes mediated by non-coding RNAs. MicroRNAs (miRNAs) are one of the largest families of non-coding RNAs. MiRNAs mediate post-transcriptional regulation of gene expression. The amount of data supporting the key role of miRNAs in the adaptation of the immune and other body systems to exercise steadily grows. MiRNAs change their expression profiles after exercise and seem to be involved in regulation of exercise-responsive genes in immune and other cell types. Here we discuss existing data and future directions in the field.