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Background: Late-onset or sporadic Alzheimer’s disease (sAD) is a neurodegenerative disease leading to cognitive impairment and memory loss. The underlying pathological changes take place several years prior to the appearance of the first clinical symptoms, however, the early diagnosis of sAD remains obscure. Objective: To identify changes in circulating microRNA (miR) expression in an effort to detect early biomarkers of underlying sAD pathology. Methods: A set of candidate miRs, earlier detected in biofluids from subjects at early stage of sAD, was linked to the proposed tau-driven adverse outcome pathway for memory loss. The relative expression of the selected miRs in serum of 12 cases (mild cognitive impairment, MCI) and 27 cognitively normal subjects, recruited within the ongoing Aiginition Longitudinal Biomarker Investigation Of Neurodegeneration (ALBION) study, was measured by RT-qPCR. Data on the protein levels of amyloid-β (Aβ42) and total/phosphorylated tau (t-tau/p-tau), in cerebrospinal fluid (CSF), and the cognitive z-scores of the participants were also retrieved. Results: Each doubling in relative expression of 13 miRs in serum changed the odds of either having MCI (versus control), or having pathological Aβ42 or pathological Aβ42 and tau (versus normal) proteins in their CSF, or was associated with the global composite z-score. Conclusion: These candidate human circulating miRs may be of great importance in early diagnosis of sAD. There is an urgent need for confirming these proposed early predictive biomarkers for sAD, contributing not only to societal but also to economic benefits.

ObjectivemiRNAs are small non-coding RNAs that control gene expression. Specific intra- and extracellular miRNA signatures have been identified in various diseases. Whether certain miRNA signatures are associated with psoriasis (PsO) and PsA is currently unknown. We aimed to search for circulating miRNA signatures associated with PsO and PsA patients.MethodsExpression of miRNAs was analysed by reverse transcription quantitative real-time PCR (RT-qPCR) in the serum of PsA, PsO patients and healthy controls. Demographic and disease-specific characteristics and imaging data from hand MRI were recorded. In the discovery phase, 192 miRNA assays were analysed in 48 samples (PsA, PsO, controls: each N = 16). For validation, 17 selected miRNAs were measured in the total population.ResultsA total of 141 patients and controls were analysed (51 PsA, 40 PsO, 50 controls). In the discovery phase 51 miRNAs in PsO and 64 miRNAs in PsA were down- or upregulated compared with controls, with 33 miRNAs being changed in both (adj. P ConclusionmiRNA signatures in PsA and PsO patients differ from controls. Nine miRNAs were differentially regulated in PsA and PsO patients, five of them previously reported to be involved in bone and cartilage metabolism, indicating an intimate association of psoriatic inflammation and bone/cartilage changes.

The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established microRNA Next-Generation-Sequencing Discovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.

Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression information from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment-specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopathology. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates.

There is an urgent need for an easily assessable preoperative test to predict postoperative liver function recovery and thereby determine the optimal time point of liver resection, specifically as current markers are often expensive, time consuming, and invasive. Emerging evidence suggests that microRNA (miRNA) signatures represent potent diagnostic, prognostic, and treatment‐response biomarkers for several diseases. Using next‐generation sequencing as an unbiased systematic approach, 554 miRNAs were detected in preoperative plasma of 21 patients suffering from postoperative liver dysfunction (LD) after liver resection and 27 matched controls. Subsequently, we identified a miRNA signature—consisting of miRNAs 151a‐5p, 192‐5p, and 122‐5p—that highly correlated with patients developing postoperative LD after liver resection. The predictive potential for postoperative LD was subsequently confirmed using real‐time PCR in an independent validation cohort of 98 patients. Ultimately, a regression model of the two miRNA ratios 151a‐5p to 192‐5p and 122‐5p to 151a‐5p was found to reliably predict postoperative LD, severe morbidity, prolonged intensive care unit and hospital stays, and even mortality before an operation with a remarkable accuracy, thereby outperforming established markers of postoperative LD. Ultimately, we documented that miRNA ratios closely followed liver function recovery after partial hepatectomy. Conclusion: Our data demonstrate the clinical utility of an miRNA‐based biomarker to support the selection of patients undergoing partial hepatectomy. The dynamical changes during liver function recovery indicate a possible role in individualized patient treatment. Thereby, our data might help to tailor surgical strategies to the specific risk profile of patients.

Blood circulating microRNAs (c‐miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti‐aging therapies. Based on a cross‐sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c‐miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR‐19a‐3p and miR‐19b‐3p, were found increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT‐qPCR, but only with a high‐resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR‐19a/b‐3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age‐related increase of plasma miR‐19a/b‐3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c‐miRs and isomiRs as additional parameter to track the onset of aging and age‐related diseases using new potential biomarkers., Small RNA deep sequencing on plasma‐derived RNA from 12 donors (young and old subjects, healthy, and unhealthy centenarians) was performed: 79 differentially expressed c‐miRs were identified. MiR‐19a/b‐3p increased at old age but decreased at extreme age, as confirmed by RT‐qPCR in 49 subjects. The decrease was more relevant in healthy centenarians, also confirmed by the modified pattern of isomiRs. Three common targets of miR‐19a/b‐3p were identified converging into the FoxO signaling pathway.

A specific signature of 19 circulating miRNAs (osteomiRs) has been reported to be associated with fragility fractures due to postmenopausal osteoporosis. However, it is unknown whether osteoporotic fractures or low BMD phenotypes are independently contributing to changes in osteomiR serum levels. The first aim was to characterize the abundance, sensitivity to hemolysis, and correlation of osteomiR serum levels, the second objective to evaluate the diagnostic accuracy of osteomiRs for osteoporosis according to the WHO criteria and on basis of major osteoporotic fracture history. Fifty postmenopausal women with osteoporosis (with or without fragility fracture) and 50 non-osteoporotic women were included in this cross-sectional study. The diagnostic performance of osteomiRs for osteoporosis based on the WHO definition or fracture history was evaluated using multiple logistic regression and receiver-operator curve (AUC) analysis. The osteomiR® signature is composed of four clusters of miRNAs providing good performance for the diagnosis of osteoporosis in postmenopausal women defined by WHO criteria (AUC = 0.830) and based on history of major osteoporotic fractures (AUC = 0.834). The classification performance for the WHO criteria and for fracture risk is driven by miR-375 and miR-203a, respectively. OsteomiRs, a signature of 19 emerging miRNA bone biomarkers, are measurable in human serum samples. They constitute a panel of independent bone and muscle biomarkers, which in combination could serve as diagnostic biomarkers for osteoporosis in postmenopausal women. Supplementary Information The online version of this article (doi:10.1007/s00223-020-00802-3) contains supplementary material, which is available to authorized users.

Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of > 80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescencespecific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15–20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest. Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far—demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy—potentially building on insights generated here., Glioblastoma: screening for survival-related factors Glioblastoma is an aggressive form of brain cancer and effective immunotherapeutics are limited, with treatment currently based on chemotherapy and radiotherapy. A recent phase II clinical trial tested a personalized, targeted dendritic cell-based immunotherapy but there was no observed improvement in patient survival or progression-free survival compared to standard-of-care therapy. Here, Carmen Visus and colleagues have used tumor tissue samples from glioblastoma patients involved in this trial and receiving immunotherapy. Using a combination of mass spectrometry-based proteomics, microRNA sequencing and RT-qPCR they identified factors associated with survival or poor prognosis. Proteomics associated poor prognosis with various proteins including focal adhesion kinase 2 (FAK2), whilst microRNAs, miR-216b, miR-216a, miR-708 and let-7i, were associated with longer survival. Focussing on one pathway, FAK2, they integrated the proteomic and microRNA datasets and saw a negative association with overall survival across all patients. To test this, they added an FAK inhibitor to glioblastoma cell lines, including cells isolated from trial patients, and observed inhibition of gliomaspheres in treated cells, providing insights into potential immunotherapy targets.

The mycotoxin zearalenone (ZEN) poses a risk to animal health because of its estrogenic effects. Diagnosis of ZEN-induced disorders remains challenging due to the lack of appropriate biomarkers. In this regard, circulating microRNAs (small non-coding RNAs) have remarkable potential, as they can serve as indicators for pathological processes in tissue. Thus, we combined untargeted and targeted transcriptomics approaches to investigate the effects of ZEN on the microRNA expression in porcine uterus, jejunum and serum, respectively. To this end, twenty-four piglets received uncontaminated feed (Control) or feed containing 0.17 mg/kg ZEN (ZEN low), 1.46 mg/kg ZEN (ZEN medium) and 4.58 mg/kg ZEN (ZEN high). After 28 days, the microRNA expression in the jejunum remained unaffected, while significant changes in the uterine microRNA profile were observed. Importantly, 14 microRNAs were commonly and dose-dependently affected in both the ZEN medium and ZEN high group, including microRNAs from the miR-503 cluster (i.e. ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-503 and ssc-miR-542-3p). Predicted target genes for those microRNAs are associated with regulation of gene expression and signal transduction (e.g. cell cycle). Although the effects in serum were less pronounced, receiver operating characteristic analysis revealed that several microRNA ratios were able to discriminate properly between non-exposed and ZEN-exposed pigs (e.g. ssc-miR-135a-5p/ssc-miR-432-5p, ssc-miR-542-3p/ssc-miR-493-3p). This work sheds new light on the molecular mechanisms of ZEN, and fosters biomarker discovery.

MicroRNAs control the activity of a variety of genes that are pivotal to bone metabolism. Therefore, the clinical utility of miRNAs as biomarkers and drug targets for bone diseases certainly merits further investigation. This study describes the use of an animal model of postmenopausal osteoporosis to generate a comprehensive dataset on miRNA regulation in bone tissue and peripheral blood during bone loss and specifically anti-resorptive and osteo-anabolic treatment.Forty-two Sprague-Dawley rats were randomized to SHAM surgery (n=10) or ovariectomy (OVX, n=32). Eight weeks after surgery, OVX animals were further randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). After 12 weeks of treatment, bone and serum samples were used for microRNA analysis using next-generation sequencing (NGS), mRNA levels using RT-qPCR, and bone microarchitecture analysis using nanoCT.Ovariectomy resulted in loss of trabecular bone, which was fully rescued using osteo-anabolic treatment, and partially rescued using anti-resorptive treatment. NGS revealed that both, anti-resorptive and anabolic treatment had a significant impact on miRNA levels in bone tissue and serum: out of 426 detected miRNAs, 46 miRNAs were regulated by teriparatide treatment an d 10 by zoledronate treatment (p-adj. < 0.1). Interestingly, teriparatide and zoledronate treatment were able to revert miRNA changes in tissue and serum of untreated OVX animals, such as the up-regulation of miR-203a-3p, a known osteo-inhibitory miRNA. We confirmed previously established mechanisms of miR-203a by analyzing its direct target Dlx5 in femoral head.Our data reveal a significant effect of ovariectomy-induced bone loss, as well as the two major types of anti-osteoporotic treatment on miRNA transcription in femoral head tissue. These changes are associated with altered activity of target genes relevant to bone formation, such as Dlx5. The observed effects of bone loss and treatment response on miRNA levels in bone are also reflected in the peripheral blood, suggesting the possibility of minimally-invasive monitoring of bone-derived miRNAs using liquid biopsies.HighlightsmicroRNA expression in bone tissue is altered by osteo-anabolic and anti-resorptive therapy in OVX rats.microRNA changes in untreated OVX rats are reverted by anti-osteoporotic therapy.miR-203a is up-regulated during bone loss and down-regulated following therapy.Bone tissue and serum levels of miR-203a are highly correlated.

Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well-characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA-qPCR-arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes-related fractures with high specificity and sensitivity (area under the receiver-operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture-indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue-derived mesenchymal stem cells to investigate the effect of miR-550a-5p, miR-188-3p, and miR-382-3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR-382-3p significantly enhanced osteogenic differentiation (p

Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level and are known to take part in the control of bone formation and bone resorption. In addition, it is known that miRNAs are secreted by many cell types and can transfer “messages” to recipient cells. Thus, circulating miRNAs might not only be useful as surrogate biomarkers for the diagnosis or prognosis of pathological conditions, but could be actively modulating tissue physiology.Therefore, the aim of this study was to test whether circulating miRNAs that exhibit changes in recent osteoporotic fracture patients could be causally related to bone metabolism.In the first step we performed an explorative analysis of 175 miRNAs in serum samples obtained from 7 female patients with recent osteoporotic fractures at the femoral neck, and 7 age-matched female controls. Unsupervised cluster analysis revealed a high discriminatory power of the top 10 circulating miRNAs for patients with recent osteoporotic fractures. In total 6 miRNAs, miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p exhibited significantly different serum levels in response to fracture (adjusted p-value

Context: Established bone turnover markers do not reflect fracture risk in idiopathic male and premenopausal osteoporosis and the role of microRNAs (miRNAs) in these patients is currently unclear. miRNAs are a class of small non-coding RNAs that regulate gene expression and bone tissue homeostasis. They are considered a new class of endocrine regulators with promising potential as biomarkers. Objective: Evaluation of circulating miRNA signatures in male and female subjects with idiopathic and postmenopausal osteoporotic low-traumatic fractures. Design, Setting, and Patients: This was a case-control study of cross-sectional design of 36 patients with prevalent low-traumatic fractures and 39 control subjects Main Outcome Measures: One hundred eighty-seven miRNAs were quantified in serum by qPCR, compared between groups and correlated with established bone turnover markers. Results: Significant differences in serum levels of circulating miRNAs were identified in all three subgroups (46 in premenopausal, 52 in postmenopausal, 55 in male). A set of 19 miRNAs was consistently regulated in all three subgroups. Eight miRNAs [miR-152-3p, miR-30e-5p, miR-140-5p, miR-324-3p, miR-19b-3p, miR-335-5p, miR-19a-3p, miR-550a-3p] were excellent discriminators of patients with low-traumatic fractures, regardless of age and sex, with area under the curve values > 0.9. The 11 remaining miRNAs showed area under the curve values between 0.81 and 0.89. Correlation analysis identified significant correlations between miR-29b-3p and P1NP, and miR-365-5p and iPTH, TRAP5b, P1NP and Osteocalcin, as well as BMDL1–L4 and miR-19b-3p, miR-324-3p, miR-532-5p, and miR-93-5p. Conclusions: Specific serum miRNA profiles are strongly related to bone pathologies. Therefore miRNAs might be directly linked to bone tissue homeostasis. In particular, miR-29b-3p has previously been reported as regulator of osteogenic differentiation and could serve as a novel marker of bone turnover in osteoporotic patients as a member of a miRNA signature.

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF-cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty-six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF-cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.

Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplaninhigh and podoplaninlow cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed differences in genes specific for signaling and vascular tissue. Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis in vivo.

Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD41 T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD41 cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs. Fil: Reynoso, Rita Paola. University of Natural Resources and Life Sciences; Austria Fil: Laufer, Natalia Lorna. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina. Universidad de Buenos Aires; Argentina Fil: Hackl, Matthias. University of Natural Resources and Life Sciences; Austria. Tamirna Gmbh; Austria Fil: Skalicky, Susanna. Evercyte Gmbh; Austria Fil: Monteforte, Rossella. University of Natural Resources and Life Sciences; Austria Fil: Turk, Gabriela Julia Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Universidad de Buenos Aires; Argentina Fil: Carobene, Mauricio. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Quarleri, Jorge Fabian. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Cahn, Pedro. Fundación Huésped; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; Argentina Fil: Werner, Roland. Medizinische Universitat Innsbruck; Austria Fil: Stoiber, Heribert. Medizinische Univesitat Innsbruck; Austria Fil: Grillari Voglauer, Regina. University of Natural Resources and Life Sciences; Austria. Evercyte Gmbh; Austria Fil: Grillari, Johannes. University of Natural Resources and Life Sciences; Austria. Evercyte Gmbh; Austria

Summary Background Several static Bethesda-type assays are routinely used to determine ADAMTS-13-neutralizing autoantibodies in acquired thrombotic thrombocytopenic purpura (TTP), but the inhibitory activity of these antibodies has not been thoroughly evaluated under the more physiologic condition of flow. Objectives We investigated whether ADAMTS-13 inhibitor assessment with the FRETS-VWF73 assay is predictive for evaluation under flow. Methods Anti-ADAMTS-13 autoantibodies were purified from patients with acquired TTP by chromatography involving an ADAMTS-13 affinity matrix and/or protein G. ADAMTS-13 activity was measured with the FRETS-VWF73 assay and a novel flow assay determining the ADAMTS-13-mediated decrease in platelet aggregate surface coverage, caused by perfusion of a suspension containing platelets, erythrocytes and von Willebrand factor (VWF) over a surface coated with extracellular matrix components. The neutralizing activities of ADAMTS-13 inhibitors were compared under static conditions and under flow by use of the two assays. Results The suitability of the flow-based ADAMTS-13 activity assay for quantification of ADAMTS-13 inhibitors could be demonstrated by reversibility of the ADAMTS-13-dependent decrease in surface coverage upon addition of goat ADAMTS-13 antiserum. Testing the neutralizing activity of purified autoantibodies from six patients in the flow assay according to their FRETS-VWF73-based inhibitor titers gave rise to vastly different inhibitory effects, indicating a discrepancy in inhibitor assessment between static and flow conditions. Conclusions Anti-ADAMTS-13 autoantibodies may show inhibitory properties in vivo that are not consistent with the ADAMTS-13 inhibitor levels determined in routine static assays, possibly because certain epitopes are selectively exposed under shear. Consequently, the course of disease and treatment efficacy may vary among TTP patients, despite common inhibitor titers.

The platelet dependent-clotting process is a highly complex pathway that needs to be tightly controlled by a variety of soluble and cellular components. Failure to regulate the activity of multimeric von Willebrand factor (VWF) by ADAMTS13 for instance causes thrombotic thrombocytopenia purpura (TTP). Regulation is achieved through the established function of ADAMTS13 as a VWF-cleaving protease; however, it has been reported that ADAMTS13 may additionally curtail VWF multimer fibrillation by forming disulfide bridges with VWF. Since the latter mechanism remained ill-defined, we aimed to directly visualize disulfide bond formation between ADAMTS13 and VWF using multimer gel electrophoresis. Covalent interaction between ADAMTS13 and VWF was time-, concentration-, temperature-, and shear-stress dependent. The covalent linkage was independent of the proteolytic activity of ADAMTS13 and could be assigned to a C-terminal fragment comprising TSP1 domains 5 to 8 plus the CUB domains. This interaction was blocked by thiol-reactive agents, indicating that association was accomplished by a thiol/disulfide exchange reaction. Furthermore, a polyclonal anti-ADAMTS13 antibody that inhibited the protease in a FRETS-VWF73 activity assay also inhibited the formation of VWF-ADAMTS13 adducts. Using a coupled reversed phase HPLC-ESI-QTOF-MS system, several partially reduced free thiols were identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent. Together with the observation that a C1275S point mutation retained the ability to form covalent linkages with VWF, this result indicated that ADAMTS13 has disulfide plasticity. The same conditions that led to VWF complex formation also caused a hitherto undetected homo-oligomerization of ADAMTS13. An even more pronounced oligomerization was noted in the presence of thiol-sensitive agents such as glutathione. We propose a dynamic network of redundant, partially free thiols in ADAMTS13 that undergo intra- and inter-molecular redox reactions capable not only of depolymerizing VWF, but also to prevent further VWF multimerization by facilitating the aggregation of ADAMTS13 around the monomers. Disclosures Rottensteiner: Baxalta Innovations GmbH: Employment. Skalicky:Baxalta Innovations GmbH: Employment. Seyfried:Baxalta Innovations GmbH: Employment. Kaufmann:Baxalta Innovations GmbH: Employment. Fiedler:Baxalta Innovations GmbH: Employment. Hasslacher:Baxalta Innovations GmbH: Employment. Plaimauer:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Thrombotic thrombocytopenic purpura (TTP) is characterized by systemic microvascular thrombosis leading to life-threatening ischemia of multiple organs, and is associated with a severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13, which permits highly adhesive ultra-large VWF multimers to accumulate in the circulation. Inhibitory and less frequent non-inhibitory anti-ADAMTS13 autoantibodies have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies while concomitantly supplying plasma with the deficient protease. Plasma therapy greatly reduces mortality, nevertheless, still about 10% of the patients die from refractory TTP. To explore the cause of treatment failure in a fatal case of idiopathic acquired TTP we investigated retrospectively the anti-ADAMTS13 immunological profile of a 70-year-old female patient during the course of disease progression. At admittance, she presented with schistocytes, thrombocytopenia and severe neurological disturbances. ADAMTS13 antigen and activity were borderline low in the presence of initially non-inhibiting anti-ADAMTS13 IgG and IgM antibodies, no ADAMTS13 gene aberrations were detected. She had had no previous episodes of TTP. During treatment, the patient received repeated plasma exchanges, supportive extracorporal immunoadsorption and corticosteroid therapy. Despite ongoing treatment anti-ADAMTS13 antibodies still developed and ADAMTS13 activity remained less than 0.1U/ml. After 5 weeks of therapy, during which time the patient was seriously ill with striking neurological limitations, her platelet count dropped and a splenectomy was performed. The patient’s state improved despite a complicated postoperative phase. During the next 4 weeks, her platelet count increased gradually up to 780 G/L, a moderately low ADAMTS13 activity of 0.24U/ml (0.57ng/ml ADAMTS13 antigen) was detected and ADAMTS13-specific antibodies were not measurable. However, within a few days her platelet count dramatically dropped to 20G/L, anti-ADAMTS13 antibodies reappeared and ADAMTS13 activity again was undetectable. The patient’s state deteriorated and she died 2 days later from multiple organ failure. We show the results of the fluctuating anti-ADAMTS13 antibody profile (functional inhibitors and total IgG, IgG subtypes, IgA and IgM) and the pattern of the ADAMTS13 domain-specific reactivity superimposed with the common clinical laboratory data over the patient’s 9-week clinical course.

A severe functional deficiency in the ADAMTS13 protease, characteristic for patients suffering from thrombotic thrombocytopenic purpura (TTP), results in persisting prothrombotic ultra-large von Willebrand factor (ULVWF) multimers, which can lead to systemic microvascular thrombosis. The congenital form of TTP is attributed to the presence of homozygous or double heterozygous mutations in the ADAMTS13 gene leading to impaired secretion and/or a functional defect of the protease. Decreased ADAMTS13 activity has been also described for several physiological and pathological conditions. Emerging evidence indicates mild ADAMTS13 deficiency might also increase risk for arterial thrombosis such as for ischemic stroke. We therefore investigated a possible causal relation between moderate ADAMTS13 deficiency and ischemic stroke in one patient selected with the lowest ADAMTS13 activity amongst a cohort of 70 patients with ischemic stroke. At age 34 and 46 years the patient had suffered from idiopathic ischemic strokes with negative common prothrombotic risk factors. We found slightly elevated VWF antigen (161%), ULVWF multimers and reduced ADAMTS13 activity (17%). Genetic analysis identified five nucleotide exchanges in the ADAMTS13 gene leading to one heterozygous missense mutation P353L and four homozygous amino acid polymorphisms (R7W, Q448E, P618A, A732V). Her asymptomatic son presented with normal VWF antigen (99%) and an ADAMTS13 activity of 58% transmitted by a mutant maternal allele and a paternal allele harboring the single Q448E polymorphism. Recombinant expression studies indicated P353L alone causes an impact on ADAMTS13 secretion that is further pronounced in the context of the four polymorphisms. Secreted ADAMTS13 containing P353L or P353L combined with the four polymorphisms presented with similar binding interaction in a binding assay with immobilized multimeric VWF, however, the binding affinity was slightly less than that of wild-type ADAMTS13 or polymorphic rADAMTS13 proteins containing either Q448E alone or all four polymorphisms combined. ADAMTS13 activity analysis suggested that mainly the secretion process or the protein stability rather than the proteolytic activity was affected by the mutations. Co-transfections mimicking the patient’s genotype indicated homozygosity of the four polymorphisms in the patient led to an attenuation of the adverse effect of the heterozygous P353L point mutation resulting in the moderate ADAMTS13 deficiency in the patient. Our results suggest moderate familial ADAMTS13 deficiency might be causally involved in the pathogenesis of ischemic stroke.