Biointeraction of Erythrocyte Ghost Membranes with Gold Nanoparticles Fluorescents

The application of new technologies for treatments against different diseases is increasingly innovative and effective. In the case of nanomedicine, the combination of nanoparticles with biological membranes consists of a “camouflage” technique, which improves biological interaction and minimizes the secondary effects caused by these remedies. In this work, gold nanoparticles synthesized by chemical reduction (Turkevich ≈13 nm) were conjugated with fluorescein isothiocyanate to amplify their optical properties. Fluorescent nanoparticles were deposited onto the surface of hemoglobin-free erythrocytes. Ghost erythrocytes were obtained from red blood cells by density gradient separation in a hypotonic medium and characterized with fluorescence, optical, and electron microscopy; the average size of erythrocyte ghosts was 9 µm. Results show that the functional groups of sodium citrate (COO-) and fluorophore (-N=C=S) adhere by electrostatic attraction to the surface of the hemoglobin-free erythrocyte membrane, forming the membrane–particle–fluorophore. These interactions can contribute to imaging applications, by increasing the sensitivity of measurement caused by surface plasmon resonance and fluorescence, in the context of biological membranes.

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer’s disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs “mitovesicles.” We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.

A modified method for isolating mouse islets of an adequate quality, quantity, and purity

Mouse islets are widely used in diabetes research. Thus an adequate quality, quantity, and purity of islets are needed for high-quality investigations. We performed a combination of filtration and density gradient separation and optimized many steps in the islet isolation procedure, including perfusion, digestion, and purification. Our results show that an increased quality, quantity, and purity of isolated islets can be achieved using these modifications. Moreover, this method can guarantee maximal recovery and purity of the isolated islets and is easy to perform with practice.

Evaluation of Isolation Methods for Circulating Tumor Cells (CTCs)

Background: Detection of CTCs is a poor prognostic factor for many cancer types; however, their very low frequency represents an obstacle for their detection. The objective of the current study was to compare the performance of commonly used methods for CTCs isolation. Methods: The evaluated methods using spiking experiments of MCF7, SKBR3 and MDA MB-231 breast cancer cell lines were (i) ficoll density gradient separation (DGS), (ii) red blood cell lysis (Erythrolysis) isolation, (iii) positive immunomagnetic selection (EpCAM Dynal beads), (iv) two different negative immunomagnetic separation systems (Dynal vs Miltenyi CD45 beads) as well as (v) the Cell Search platform and (vi) the ISET system. Results: The recovery rates of Erythrolysis and DGS were 39% and 24%, respectively. Magnetic isolations are ranked from the worse to the best recovery rate as follows:, Myltenyi-anti-CD45 microbeads (24%); Dynal-anti-EpCAM beads (75%); Dynabeads-anti-CD45 (97%). CTCs isolation from blood samples using the CellSearch and ISET systems revealed that the recovery rate for Cell Search and ISET was 52% and 95%, respectively. Conclusions: Dynal-anti-CD45 beads have the best recovery rate compared to other magnetic methods. Furthermore the recovery rate of ISET was higher compared to Cell Search, especially for the more aggressive MDA-MB 231 cell line.

Red Blood Cell Depletion for Pediatric Major ABO Mismatch BMT: Evaluation of the Risk of Hemolysis and Comparison of Two Techniques

Introduction: Major ABO mismatch occurs when the recipient has preformed isoagglutinins against the donor. Since unmanipulated bone marrow transplant (BMT) grafts contain donor red blood cells (RBCs), major ABO mismatch BMT infusions can cause hemolytic reactions that lead to renal dysfunction. To prevent this complication, RBC depletion of the graft can be performed by various techniques. Strong evidence-based recommendations regarding RBC depletion are lacking. In particular, it is unclear what residual volume of incompatible RBCs in the graft is safe, especially for pediatric patients. Methods: We conducted a retrospective cohort study of pediatric patients who had major ABO mismatch BMT at a single center. No patients had pre-BMT plasmapheresis to reduce isoagglutinin titers. RBC depletion was performed on the BMT grafts with either hydroxyethyl starch (HES) sedimentation or Ficoll density gradient separation. All patients received similar supportive care around the BM infusion that included hyperhydration and premedication with acetaminophen, diphenhydramine, and hydrocortisone. Serum creatinine the day after the BM infusion was compared to creatinine the day of the BM infusion. Since 2006 all patients had urine screened for blood post-BMT. Results: Sixty-three patients were identified who received a major ABO mismatch BMT between 9/2004 and 6/2015 (39 had RBC depletion with HES, 24 with Ficoll). Compared to Ficoll RBC depletion, patients who received BM grafts that had RBC depletion with HES received significantly more incompatible RBCs (Table 1). Hemoglobinuria was significantly more common in HES patients, but evidence of post-BM infusion renal impairment was not (Table 1). All patients had donor engraftment with a similar time to neutrophil engraftment for both groups (Table 1). Considering only the HES patients, 8/8 (100%) patients with >25% rise in creatinine had hemoglobinuria compared to 8/21(38%) patients with ≤25% rise in creatinine (p=0.003). Also among just the HES patients, the median amount of incompatible RBCs infused was not significantly different between patients with (0.74 ml/kg) and without (0.62 ml/kg) hemoglobinuria (p=0.42), or between patients with (0.56 ml/kg) and without (0.62 ml/kg) a >25% rise in creatinine (p=0.86). Table 1: Comparison of patients who had RBC depletion with HES vs. Ficoll Table 1.HES n=39Ficoll n=24p-valueMedian volume of RBCs/patient weight 0.62 ml/kg0.04 ml/kg<0.0001Number with urine positive for blood* 16/29 (55%)1/20 (5%)0.0003Number with >25% rise in creatinine 8 (21%)5 (21%)1.0Number with >50% rise in creatinine 4 (10%)0 (0%)0.29Median day of neutrophil engraftment 20210.11 *considering only patients transplanted after 2006. Conclusion: Our study is the first to analyze pediatric major ABO mismatch BMT infusion hemolysis after two different RBC depletion techniques: HES and Ficoll. Our results suggest that RBC depletion with Ficoll achieves less residual RBCs in the BM graft and likely less resulting hemoglobinuria. However, after both techniques hemolysis-induced renal impairment is rare. The volume of residual incompatible RBCs in the infused BM graft does not appear to strongly determine if clinical hemolysis occurs. The volume that is safe likely depends on other variables that influence the risk of clinical hemolysis. Disclosures No relevant conflicts of interest to declare.