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Detection of serum embryonic miRNAs miR-371a-3p and miR-372-3p has been proposed to aid in diagnosis, prognosis, and management of patients with testicular germ cell tumors (GCTs). This study describes the analytical validation and performance of a laboratory-developed test to detect these miRNA targets by stem loop real-time quantitative RT-PCR (RT-qPCR) in serum from patients with GCTs. The assay was standardized using an exogenous spike-in control of nonhuman miRNA from Caenorhabditis elegans (cel-miR-39-3p) to assess extraction efficiency, and an endogenous housekeeping miRNA, miR-30b-5p, to control for miRNA normalization. miRNA results were expressed as relative expression level, using the comparative threshold cycle method (2

Rapid proliferation of cancer cells is enabled by favoring aerobic glycolysis over mitochondrial oxidative phosphorylation (OXPHOS). P32 (C1QBP/gC1qR) is essential for mitochondrial protein translation and thus indispensable for OXPHOS activity. It is ubiquitously expressed and directed to the mitochondrial matrix in almost all cell types with an excessive up-regulation of p32 expression reported for tumor tissues. We recently demonstrated high levels of non-mitochondrial p32 to be associated with high-grade colorectal carcinoma. Mutations in human p32 are likely to disrupt proper mitochondrial function giving rise to various diseases including cancer. Hence, we aimed to investigate the impact of the most common single nucleotide polymorphism (SNP) rs56014026 in the coding sequence of p32 on tumor cell metabolism. In silico homology modeling of the resulting p.Thr130Met mutated p32 revealed that the single amino acid substitution potentially induces a strong conformational change in the protein, mainly affecting the mitochondrial targeting sequence (MTS). In vitro experiments confirmed an impaired mitochondrial import of mutated p32-T130M, resulting in reduced OXPHOS activity and a shift towards a low metabolic phenotype. Overexpression of p32-T130M maintained terminal differentiation of a goblet cell-like colorectal cancer cell line compared to p32-wt without affecting cell proliferation. Sanger sequencing of tumor samples from 128 CRC patients identified the heterozygous SNP rs56014026 in two well-differentiated, low proliferating adenocarcinomas, supporting our in vitro data. Together, the SNP rs56014026 reduces metabolic activity and proliferation while promoting differentiation in tumor cells.

Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized byTo investigate the feasibility of identifyingThese results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

Introduction: Cancer patients can develop hypercoagulability, leading to stroke. The pathophysiology of cancer-mediated hypercoagulability, however, is poorly understood and biomarkers associated with stroke in cancer patients are uncertain. Methods: We performed a pilot cross-sectional study at a tertiary-care cancer center to evaluate potential hematological biomarkers of stroke in cancer patients. From October 2013 through May 2014, we prospectively identified 15 inpatients with active systemic cancer and acute ischemic stroke and 15 inpatients with active systemic cancer and no stroke. Using collected plasma, we compared six potential biomarkers of stroke, selected in advance based on expert opinion and prior data, which included markers of coagulation (thrombin-antithrombin complex and D-dimer), platelet function (P-selectin), and endothelial activation state (sICAM-1, vICAM-1, and thrombomodulin). We used the Wilcoxon rank-sum test to compare biomarker levels between groups. Results: Each group comprised 14 patients with solid cancer and one patient with hematological cancer. All patients in the cancer and stroke group had stage 4 disease, while only 60% of the cancer and no stroke group had stage 4 disease. Age and demographics were similar between groups. Median levels of thrombin-antithrombin complex, D-dimer, P-selectin, sICAM-1, and vICAM-1 were significantly increased in cancer patients with stroke, but thrombomodulin was not (Table). Our results were substantially unchanged in a sensitivity analysis limited to patients with only stage 4 cancer. Conclusion: These preliminary data suggest that thrombin-antithrombin complex, D-dimer, P-selectin, sICAM-1, and vICAM-1 could serve as the focus of future prospective studies to elucidate the potential predictive, diagnostic, or etiologic role of hematological biomarkers for stroke in the cancer population.

Successful peripheral blood stem cell transplantation (PBSCT) depends on the collection and infusion of adequate numbers of peripheral blood progenitor cells (PBPCs). Several predictors of PBPC yield are used currently, including white blood cell (WBC) count and CD34 analysis. This study evaluated the utility of the new automated hematopoietic progenitor cell count available on Sysmex XN hematology analyzers (XN-HPCs) in PBSCT.The performance characteristics of XN-HPC, CD34+, and WBC analysis were compared using 107 matched peripheral blood and apheresis samples.Good correlation was observed between XN-HPC and CD34+ cell counts in peripheral blood (r = 0.88; slope, 0.81) and apheresis collections (r = 0.91; slope, 0.89). Moreover, peripheral blood XN-HPC and CD34 analysis showed comparable ability to predict successful PBPC harvests (≥2 × 10(6) CD34+ cells/kg). At a cutoff of 20 × 10(6) progenitor cells/L, peripheral blood XN- HPC and CD34 analysis both showed negative predictive values (NPVs) of 100% and positive predictive values (PPVs) of 55.4 and 63%, respectively. Using an optimized cutoff of 38 × 10(6) progenitor cells/L, derived from receiver operating characteristic analysis, the PPV for XN-HPC and CD34 analysis increased to 71.4 and 78.9%, respectively, with relatively unchanged NPVs (XN-HPC 97.7%, CD34+ 98.0%). In contrast, the correlation between peripheral blood WBC and CD34 analysis was poor (r = 0.48; slope, 669.85), and the peripheral blood WBC count (cutoff, 10 × 10(9) /L) was a poor predictor of PBPC harvest (NPV 60%, PPV 43.1%).XN-HPC compares favorably with CD34 analysis and may be a surrogate for CD34 analysis to predict optimal timing of PBPC collections.

Endovascular infections with Staphylococcus aureus (S. aureus) are associated with high mortality. gC1qR/p33 (gC1qR), a receptor for the complement component C1q expressed on endothelial cells, interacts with protein A of S. aureus and gC1qR blockade reduces S. aureus colonization during infective endocarditis. The aim of this study was to analyze in vivo whether this observation is due to a decreased interaction of S. aureus with the microvascular endothelium. A dorsal skinfold chamber was prepared in Syrian golden hamsters, which were treated with the monoclonal antibody (MAb) 74.5.2 directed against gC1qR or vehicle. The interaction of fluorescein isothiocyanate (FITC)-labeled staphylococci and leukocytes with the endothelium was analyzed under physiological conditions as well as after TNF-α-induced inflammation using intravital fluorescence microscopy. Administration of MAb 74.5.2 significantly reduced adherence of S. aureus to the endothelium in untreated and TNF-α-exposed tissue. In addition, we could demonstrate in vitro that S. aureus adherence to human endothelial cells was inhibited by MAb 74.5.2. Blockade of gC1qR did not affect leukocyte-endothelial cell interaction. In conclusion, our findings indicate that immunological inhibition of gC1qR may be therapeutically used to decrease the interaction of S. aureus with the microvascular endothelium.

The association between C1q and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE) is well established. Deficiency in C1q is considered to be a strong susceptibility factor and is corroborated by the fact thator = 92% of the known cases of hereditary deficiency in C1q develop rheumatic disease. Furthermore, the observation of the presence of high-affinity autoantibodies against C1q antibodies in patients with SLE provides a strong correlation between these antibodies and the inflammatory processes that occur in this disease. Recent evidence using C1q-deficient mice has shown the presence of glomerulonephritis with immune deposits and a large number of apoptotic bodies in the diseased glomeruli suggesting a defect in the clearance of apoptotic cell by macrophages and dendritic cells (DCs). Although these data are consistent with the hypothesis that C1q deficiency may induce a generalized failure to clear immune complexes and apoptotic cells, this concept alone cannot wholly explain why individuals with C1q deficiency are prone to develop SLE. Therefore, C1q alone or in conjunction with other surface molecules must play a much more fundamental role in immunoregulation, especially those processes that regulate T cell function and tolerance. In support of this hypothesis is the finding that C1q causes inhibition of mitrogen-induced T cell-proliferative response by interaction with C1q receptors. Furthermore, macrophages and possibly DCs not only synthesize but also display C1q as a type II cell surface molecule, especially at sites of inflammation. Although it is not yet known what role the surface-expressed C1q plays, it is tempting to assume that it plays a role in the priming of naïve T cells by DCs. This work will review the current concepts of the role of C1q and C1q receptors in autoimmunity.

Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

45Calcium uptake was studied with aspirin-treated platelets that were gel-filtered through a column of Sepharose 2B equilibrated with divalent cation-free modified Tyrode's solution to remove readily exchangeable surface-associated calcium. These platelets aggregated almost immediately when exposed to ADP, fibrinogen and at least 30 microM CaCl2. At this calcium ion concentration, 10(8) platelets took up 36.6 +/- SEM 2.7 pmol of 45calcium within 1-2 min. The presence of ADP and fibrinogen did not affect the amount of calcium bound. Over 90% of this platelet-associated calcium was removed by EDTA in 5 min suggesting that it was surface-bound. Calcium uptake increased rapidly for 10 min, then more slowly for up to 2 h. At 60 min, maximal uptake was approached at CaCl2 concentrations between 250 and 300 microM when an average of 276 +/- SEM 18 pmol of calcium was associated with 10(8) platelets. Only 50-60% of this calcium could be removed by EDTA in 5 min, and about 70% in 20 min, suggesting that some of it had been internalized. Platelets from two patients with thrombasthenia that were unable to aggregate took up 50% less calcium than platelets from normal volunteers. Similarly, platelets that had been incubated with EDTA at 37 degrees C, pH 7.8 for 8 min lost the ability to aggregate despite recalcification and took up 40-60% less calcium than CaEDTA-treated controls. Platelets from a patient with the Bernard-Soulier syndrome aggregated and bound calcium normally. Thus the platelets' ability to take up calcium after removal of surface-associated calcium correlates with their ability to aggregate. Since thrombasthenic platelets and platelets rendered incapable of aggregating after prolonged calcium deprivation with EDTA do not bind fibrinogen, we postulate that some of the surface-associated calcium normally binds to the fibrinogen receptors.

The distribution of surface sialic acid and its possible alteration after ADP-induced shape change and aggregation were studied. Gel-filtered platelets that were aggregated, disaggregated, and centrifuged showed decreased total protein and a significant decrease in sialic acid/mg protein. Platelet surface sialic acid was labeled by periodate oxidation followed by borotritiide reduction. Tritium was apparently incorporated only into sialic acid, since the label was removed by mild acid hydrolysis, had a mobility similar to that of the seven-carbon derivative of sialic acid, and was not taken up when periodate was omitted. When reduced platelet proteins were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, at least three radioactive peaks (1, 2, and 3) were consistently observed, with apparent molecular weights of about 200,000, 150,000, and 100,000 daltons, respectively. Peak 2 had the greatest radioactivity. A shoulder of slightly lower molecular weight than peak 2, or a small peak between peaks 2 and 3, was also seen. Peaks 2 and 3 corresponded to glycoproteins I and III identified with periodic acid-Schiff (PAS) staining, and the material between them to glycoprotein II. Peak 1, and two to four smaller radioactive peaks with apparent molecular weights between 25,000 and 60,000 daltons, were not usually associated with PAS staining. All radioactive peaks were much smaller in platelets labeled after treatment with neuraminidase, indicating that much of the labeled sialic acid was on the platelet surface. The amount of tritium incorporated and its labeling pattern were unaffected when shape change alone was induced with ADP. However, platelets that were aggregated and disaggregated with ADP had reduced tritium uptake with a uniformly decreased labeling pattern, suggesting that the decrease in sialic acid content/mg protein resulted from a generalized loss of surface sialic acid or glycoprotein.

ADP-induced platelet aggregation is associated with the specific binding of fibrinogen to receptors. Since fibrinogen is also a cofactor for the spontaneous aggregation of chilled human platelets during rewarming, we examined the ability of these platelets to bind purified 126l-labeled fibrinogen. Specific binding similar to ADP-induced binding at room temperature was observed after the platelets had been chilled for 20 min at 0–2°C. Aggregation, however, occurred only when the ambient temperature was increased and was followed within 15–30 sec by loss of most of the bound fibrinogen and disaggregation. ADP- and cold-induced fibrinogen binding and aggregation were inhibited by 0.3 µM PGE“ 1 mM dibutyryl cyclic AMP, and 2.5 mM EDTA, unaffected by 208 µM colchicine, and absent from thrombasthenic platelets. Platelets change shape from discs to spiny spheres when they are exposed to ADP or cold. PGE1 and dibutyryl cyclic AMP prevented the shape change induced by ADP but not by chilling. Shape change, fibrinogen binding, and aggregation induced by ADP. but not by chilling, were inhibited by incubating platelets with 7.3 µM antimycin A and 4.9 mM 2-deoxy-D-glucose. Fixed platelets bound fibrinogen only if they were exposed to ADP before fixation but they failed to aggregate. Studies with fixed platelets showed that EDTA and a low pH prevent the interaction of fibrinogen with exposed receptors, whereas platelets stimulated with ADP in the absence of divalent cations or at low pH prior to fixation bound fibrinogen at physiologic pH, and this binding was inhibited by EDTA. The present studies provide the following clues about the requirements for fibrinogen receptor exposure and platelet aggregation. (1) ADP and chilling activate platelets by exposing fibrinogen receptors via a common pathway that is blocked by elevating cyclic AMP. (2) Chilling can bypass the seemingly ATP-dependent step for ADP-induced fibrinogen receptor exposure. (3) Receptor exposure by both ADP and chilling is independent of platelet shape. (4) Increased cyclic AMP inhibits the shape change induced by ADP but not that caused by chilling. (5) EDTA and low pH prevent aggregation by interfering with the binding of fibrinogen to exposed receptors. (6) Receptor mobility may be as important as fibrinogen binding in supporting platelet aggregation.

SummaryThe sialic acid of human gelfiltered platelets was studied before and after ADP-induced shape change. Neuraminidase cleaved 47% of the total sialic acid from both discoid control platelets and platelets that had become spiny spheres after treatment with 5 μM ADP.We are grateful to the following colleagues for materials used in these experiments: Thomas H. Finlay, Ph.D., for the radiolabeled TAMe, Joel U. Harris, Ph.D., for the radiolabeled fibrinogen, Henriette Lackner, M. D., for the connective tissue suspension, and John Fenton, II, Ph.D. for the purified human thrombin.

SummaryADP causes human, aspirin-treated, gel-filtered platelets to change from their native discoid shape to spiny spheres with pseudopods, bind 125I-labeled fibrinogen, and aggregate if shaken with sufficient fibrinogen. After destruction of the added ADP with the enzyme apyrase, the platelets revert to a disc shape and lose much of their bound fibrinogen. Colchicine (208 μM or 83 μg/ml) added to ADP-treated platelets before apyrase prevented restoration of the discoid shape but not the loss of bound fibrinogen. It did not inhibit ADP-induced shape change, aggregation, or fibrinogen binding. Cytochalasin B (0.02–0.2 μM or 0.01–0.10 μg/ml) prevented ADP-induced shape change but not ADP-induced fibrinogen binding or aggregation. Thus, these findings support earlier studies with thrombasthenic and EDTA-treated platelets and with normal platelets at low pH, or in the presence of EDTA to indicate that fibrinogen binding is associated with aggregability but not with platelet shape.