Background: Platelet microparticles (PMP) are elevated in and associated with disease activity in a number of diseases including cancer, neurological conditions, autoimmune diseases, and infectious diseases. This finding raises the possibility of a removal of these microparticles as a therapeutic strategy. The Hemopurifier is an experimental device consisting of a plasma separator and an affinity resin containing Galanthus nivalis agglutinin (GNA) affinity resin that has previously been shown to remove extracellular vesicles in vitro and in vivo. In this proof on concept study, we sought to determine ex vivo removal of platelet-derived microparticles from healthy human plasma by the Hemopurifier. Methods: Two hundred milliliters of thawed healthy human plasma were circulated over the Hemopurifier device at a rate of 100 mL/minute. Plasma samples were taken at time points equivalent to a 4-, 6- and 8-hour clinical Hemopurifier session in a healthy adult. Microparticles were isolated from these timepoints and analyzed for treatment concentration changes. Platelet microparticle counts were determined by binding to antiCD41. Results: The Hemopurifier removed 98.5% of platelet microparticles during the equivalent of a 4-hour clinical session. Conclusions: We demonstrated that an extracorporeal device with a GNA affinity resin removes platelet microparticles from normal healthy plasma. Next steps would be demonstration of the removal of PMPs from plasma by the Hemopurifier in different disease states and characterization of the cargo within removed PMPs.

There is no proven therapy for Long COVID, a post-acute illness characterized by a myriad of diverse symptoms including fatigue, dyspnea, and brain fog following SARS-CoV-2 infection. Extracellular vesicles (EVs) have been implicated in Long COVID pathogenesis by promoting viral and inflammatory signaling with their molecular cargo. In this study, we investigated whether EV abundance and glycome characteristics are altered in plasma from people with Long COVID and whether they can be targeted for removal using a glycan-binding affinity resin. Large (100−500 nm) and small (40−200 nm) EVs were isolated from plasma of participants in the post-acute phase of COVID-19 and analyzed by nanoparticle flow cytometry to measure concentration and glycan characteristics. Plasma of those with Long COVID contained elevated levels of both large and small EVs, and mannose-positive large EVs were significantly increased in comparison to recovered controls (p < 0.05). EV capture assays using Galanthus nivalis agglutinin (GNA) affinity resin demonstrated small EV removal positively correlated with mannose-positive EV abundance (r = 0.341, p < 0.05). NanoString analyses identified seven EV-associated miRNAs significantly depleted by GNA affinity resin treatment of plasma. PROGENy pathway inference of validated miRNA-mRNA interactions suggests these reductions may lead to a downregulation of JAK-STAT signaling and upregulation of Estrogen, VEGF, and PI3K pathways, resulting in a favorable rebalancing of immune and tissue-repair networks. These findings reveal specific glycome EV-miRNA cargo signatures in Long COVID and the potential clinical benefits of a lectin capture therapeutic strategy to remove these pathogenic vesicles and their inflammatory cargo.

Kidney transplantation is considered the benchmark treatment for end-stage kidney disease patients, yet the scarcity of suitable kidneys poses a significant hindrance for patients and healthcare providers. One approach is to extend the criteria for the use of kidneys from deceased brain death and deceased circulatory death donors. Use of these organs, especially from these extended criteria donors, is associated with ischemia reperfusion injury and resultant delayed graft function as well as increased rates of allograft rejection. To lessen these complications as well as increase the time of organ viability assessment, machine perfusion has been evaluated on recovered kidneys. In this study we examined the immunogenic molecular content of perfusates from discarded organs that had undergone Controlled Oxygenated Rewarming (COR). Perfusates were analyzed for extracellular vesicles (EVs), DNA (Deoxyribonucleic acid), and microRNAs. These perfusates were then pumped over a plasma separator containing a lectin affinity resin. Following treatment, a significant diminution in extracellular vesicles, dsDNA (double-stranded DNA) associated with EVs, and microRNAs (miRNA) were observed. Specifically, in three out of the four renal perfusates analyzed there was significant removal of small EVs (<200 nm) and vesicles loaded with dsDNA (p < 0.05). Notably, depletion of larger EVs (100-500 nm) was found to be significant in all treated perfusates (p < 0.01). NanoString analysis of miRNA found 5 species potentially involved in renal dysfunction (hsa-let 7a-5p, hsa-miR-148b-3p, hsa-miR-148a-3p, hsa-miR-29b-3pb and hsa-miR-99a5p) to be significantly depleted in treated renal perfusates (p ≤ 0.05). These results support a future study incorporating this treatment method into a dynamic machine perfusion circuit to explore if reduction of these mediators is associated with improved function of retrieved kidneys.

Background: Immunotherapy with anti-PD-1 inhibitors has revolutionized the treatment of many solid tumors, however only 30-40% of patients will have a lasting clinical response. Tumor-derived extracellular vesicles (EVs) have been implicated in the spread of solid tumors and resistance to these agents. A lectin-affinity plasmapheresis device called the Hemopurifier (HP) has been developed and shown to remove extracellular vesicle in vitro and in patients. We hypothesize that the treatment of patients who are not improving on anti-PD-1 therapies will be safe, decrease EV concentrations, and improve anti-tumor T cell activity. Methods: This safety, feasibility and dose finding study is designed in a 3+3 safety study design with three treatment cohorts. Participants who are determined to be not responding to anti-PD-1 monotherapy will be assigned to receive either one, two, or three (HP) treatments over a one-week period prior to their next scheduled dose of anti-PD-1 antibody. Advancement from one cohort to the next will be determined by a DSMB. Data collection will include adverse events, safety labs, EV concentrations and T cell measurements, repeat imaging and survival status. The primary outcome of the study will be the safety of the HP in this population with additional endpoints to include the kinetics of EV removal and rebound following HP treatment, in addition to the effects on T cell numbers and activity, Ethics and dissemination: The clinical protocol and amendment to the study protocol have been approved by the Ethics Committees and Research Governance Offices at three participating clinical sites in Australia. The Therapeutic Good Authority (TGA) has been notified. The clinical trial is listed on the Australia New Zealand Clinical Trials Registry. Informed Consent is obtained from all participants prior to any protocol procedures being performed. Australia New Zealand Registration number ACTRN12624000732583

There is no proven therapy for Long COVID, a post-acute condition characterized by persistent symptoms following SARS-CoV-2 infection. Extracellular vesicles (EVs) are emerging as mediators of disease pathogenesis through their molecular cargo. We investigated whether EV glycosylation is altered in Long COVID plasma and whether these vesicles can be selectively targeted using a glycan-binding affinity resin. Large (100–500 nm) and small (40–200 nm) EVs were isolated from post-acute COVID-19 plasma and analyzed by nanoparticle flow cytometry to assess surface glycosylation. Small EV capture assays were performed using Galanthus nivalis agglutinin (GNA) affinity resin. Plasma miRNA profiles before and after GNA treatment were evaluated using NanoString nCounter analysis, and potential downstream pathway effects were computationally inferred using validated miRNA–mRNA interactions and PROGENy. Mannose-positive large EVs were significantly increased in Long COVID compared to recovered controls (p < 0.05). GNA-mediated small EV capture correlated with mannose-positive EV abundance (r = 0.341, p < 0.05), and seven miRNAs were significantly reduced following treatment. Computational pathway analysis suggested modulation of key signaling pathways, including JAK-STAT, Estrogen, VEGF, and PI3K. These findings suggest a glycan-associated EV signature in Long COVID and support further investigation of lectin-based capture as a potential strategy to target vesicle-associated molecular cargo.

Kidney transplantation is considered the benchmark treatment for end-stage kidney disease patients. Nevertheless, the demand for donor kidneys is continuously on the rise and the scarcity of suitable kidneys poses a significant hindrance for patients and healthcare providers. One approach is to extend the criteria for the use of kidneys from deceased brain death and deceased circulatory death donors. Use of these organs especially from these extended criteria donors is associated with ischemia reperfusion injury (IRI) and resultant delayed graft function (DGF) as well as increased rates of allograft rejection. One approach to try to lessen these complications as well as increase the time the assessment of organ viability is the use of machine perfusion on recovered kidneys. In this study we obtained perfusates from discarded organs that had undergone Controlled Oxygenated Rewarming. Perfusates were analyzed for extracellular vesicles (EVs), dsDNA associated with EVs and microRNAs. These perfusates were then pumped over the Aethlon Hemopurifier, a plasma separator with an affinity resin containing the lectin Galanthus nivalis agglutinin (GNA). Following treatment with the Hemopurifier, a diminution in extracellular vesicles, dsDNA associated with EVs and microRNAs was observed. These results support a future study of the Aethlon Hemopurifier as part of a machine perfusion circuit to explore if the device decreases these mediators in a dynamic circuit and is associated with improved function of retrieved kidneys.