Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Kinetics and topology of DNA associated with circulating extracellular vesicles released during exercise

Although it is widely accepted that cancer derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12 % of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20 % are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Canine Neutrophil Extracellular Traps Have Pro-Coagulant and Anti-Fibrinolytic Effects

Pet dogs experience similar environmental influences to their owners, and thus provide valuable spontaneous models for investigation of novel therapies for human disease. DNase and treatments targeting neutrophil extracellular trap (NET) proteins show promise in murine thrombosis models. Dogs at risk of thrombosis could provide evidence for the efficacy of these treatments in a second species, but the pro-thrombotic effects of NETs on canine plasma must be confirmed. We isolated neutrophils from healthy canine blood by density gradient and stimulated NET release using phorbol 12-myristate 13-acetate (PMA). To release NETs into suspension, culture media was replaced by the restriction enzyme Alu1. Supernatants were centrifuged to remove any intact cells. By gel electrophoresis, NET fragments were predominantly 600-1000 base pairs which is sufficient to simultaneously bind plasmin and fibrin (Arterioscler Thromb Vasc Biol., 2015, 35: 2544-53). To distinguish the effects of DNA and NET proteins, a subset of NET fragments were further digested with DNase. Supernatants were identically prepared from non-stimulated neutrophils. DNA concentration was 106ng/ml (sd 49, n=5 dogs) for non-stimulated supernatants; 401ng/ml (sd 94) for NET supernatants and reduced to 113ng/ml (sd 18) by DNase. The effect of NETs on clot formation and lysis were measured spectrophotometrically. To bring fibrinogen into the range typically found in dogs suffering from pro-thrombotic disease, 4.2mg/ml of plasminogen-depleted fibrinogen was mixed with pooled platelet poor plasma from healthy dogs. Plasma was incubated with non-stimulated supernatants; NETs +/- DNase or controls for effects of restriction enzymes, PMA and culture media. We added tissue factor, 5mM calcium chloride and 100µg/ml tissue plasminogen activator and measured absorption at 405nm q. 10 seconds for 25 minutes. NETs increased maximum formation velocity (MFV) (192mOD/min sd. 12, p=0.0011), measured by a line of best fit for the upswing of the curve, and delayed time to 50% clot lysis (448s sd. 43) compared with non-stimulated supernatants (165mOD/min sd. 12; 395s sd 38; n=5). Using a repeated measures ANOVA followed by paired t-tests with Tukey's correction, the effect on MFV (p=0.0089) but not time to 50% clot lysis (p=0.08) remained significant after DNase digestion. NETs did not have detectable effects on area under the curve (AUC) or peak (pANOVA >0.2) (Fig. 1). Reagent controls had no detectable effects on any parameter (pANOVA≥0.717; 5-6 technical replicates). Reduction in fibrinolysis by DNA replicates its effect on human plasma (Arterioscler Thromb Vasc Biol., 2015, 35: 2544-53). The lack of effect of DNA on canine clot formation was surprising given the ability of DNA to increase human plasma thrombin generation (Arterioscler Thromb Vasc Biol. 2014, 34:1977-84). To determine if this was a species difference or a concentration dependent effect, DNA from canine leukocytes was added to plasma at a final concentration of 200ng/ml (approximately equal to the final concentration in our NET studies) or 1000ng/ml. At 1000ng/ml but not 200ng/ml, DNA increased MFV (p=0.038). To determine if NET proteins were increasing MFV by a direct effect on fibrinogen or thrombin, we next studied effects of NETs on thrombin induced clotting of plasminogen-depleted fibrinogen. NETs with or without DNase digestion had no detectable effects on peak, AUC, MFV or fibrin fiber diameter estimated by turbidity. This suggests NET proteins increase plasma clot formation rate through interactions with coagulation components other than thrombin or fibrinogen. Based on human and murine studies showing NET proteins inhibit endogenous anticoagulants, we selected histones and elastase as candidate proteins for further investigation. Purified histones from calf thymus added to plasma at approximately the concentration present in NET supernatants (150ng/ml) had no effect on MFV. Pre-incubation of elastase with canine plasma abolished elastase activity measured by N-Succinyl-Ala-Ala-Ala-p-nitroanilide. Therefore, it is unlikely that the ability of canine NET proteins to increase MFV is mediated by elastase or histones alone. In conclusion, NETs increase clot formation rate and delay lysis in canine platelet poor plasma. Since effects were mediated by both proteins and DNA, dogs are a promising model for trials of both DNase and therapies targeting NET proteins. Disclosures No relevant conflicts of interest to declare.

Plasma viral DNA as a marker of tumor response in EBV(+) Hodgkin lymphoma in a phase III study (E2496).

8003 Background: Epstein-Barr virus (EBV) is associated with Hodgkin lymphoma (HL) and can be detected by in situ hybridization (ISH) of viral nucleic acid (EBER) in tumor cells. Studies have suggested a correlation in HL between plasma EBV DNA and EBER ISH. We previously studied the DNase sensitivity of plasma EBV DNA and found plasma EBV of patients with EBV(+) HL was not protected from DNase digestion, consistent with tumor-derived DNA, while plasma EBV of patients with HIV without EBV(+) tumors was protected from DNase digestion, consistent with virion DNA. We sought to determine whether plasma EBV could serve as a surrogate for EBER ISH and whether reappearance of plasma EBV predicts treatment failure. Methods: Specimens from a Cancer Cooperative Intergroup Trial (E2496/Stanford V versus ABVD for HL) were used to compare pretreatment plasma EBV DNA copy number, assessed by real-time quantitative PCR, with EBV status by EBER ISH. An ROC analysis was performed using patients with both pretreatment plasma EBV and EBER results (n=121), identifying a cutoff of 60 viral copies/100 µL plasma (95% concordance, 92% sensitivity, 96% specificity for EBV status by EBER). Using this cutoff, pretreatment plasma specimens (n=274) were designated EBV(+) (n=54) or EBV(-) (n=220), as were serial follow-up specimens. Cox proportional hazard models were constructed to evaluate plasma EBV as a prognostic factor for failure-free survival (FFS). FFS was estimated by the Kaplan-Meier method. Results: Pretreatment EBV(+) plasma was associated with treatment failure with a hazard ratio of 2.1 (95% CI 1.2-3.6, p=0.01) after adjusting for International Prognostic Score, treatment arm, and histology. Of the EBV(+) patients with follow-up specimens (n=45), patients with EBV(+) plasma beyond 1 month of therapy (n=9) had inferior FFS compared to those who cleared their plasma of EBV (n=36), (3-year FFS 44% versus 69%, respectively; log rank p=0.03). Conclusions: HL patients with EBV(+) plasma at baseline have inferior FFS compared to others. Among patients with EBV(+) plasma at baseline, those in whom plasma EBV persists or reappears after initiation of therapy have inferior FFS. Such patients may benefit from experimental or intensified therapies.

Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences

Sequence analysis of the ∼20 kb 5′-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a–1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining ∼6·2 kb of the GAV genome downstream of ORF1a–1b to a 3′-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3′-coterminal subgenomic (sg) mRNAs of ∼6 kb and ∼5·5 kb. Primer extension and 5′-RACE analyses showed that the sgmRNAs initiate at the same 5′-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5′-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26·2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5′-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.