Function of miR-24 and miR-27 in Pediatric Patients With Idiopathic Nephrotic Syndrome

Background: The specific etiology and mechanism of idiopathic nephrotic syndrome (INS) in children remain unclear, so we investigated the pathogenesis of INS by measuring the effects two specific miRNAs on Th2 cells in children with this disease. Methods: Flow cytometry was used to measure the levels of Th2 cells and a cytometric bead array was used to measure the levels of IgE, interleukin (IL) -4, and IL-13. RT-PCR was used to measure the levels of miR-24 and miR-27 in CD4+TCD25− cells. PBMCs were isolated using Ficoll density gradient centrifugation, and transfected with different mimic or inhibitor miRNAs. RT-PCR was used to measure the expression of different RNAs, and flow cytometry was used to determine the percentages of Th2 cells. Results: Children with active INS had higher percentages of Th2 cells than healthy controls (P<0.05), but there was no significant difference for children in remission. The plasma levels of IgE, IL-4, and IL-13 were significantly increased in children with active INS (P<0.05). miR-24 and miR-27 were at lower levels in children with active non-atopic INS (P<0.05). Transfection experiments indicated that upregulation of each miRNA decreased the percentage of Th2 cells and the level of IL-4 (P<0.05), and down-regulation of each miRNA had the opposite effects (P<0.05). Conclusion: Children with active INS, with or without atopy, had higher levels of IgE, possibly related to their higher levels of IL-13 and IL-4 due to drift toward Th2 cells. miR-24 and miR-27 suppress the expression of Th2 cells and have a critical function in Th2 expression in INS.

Separation of X spermatozoa from cauda of epididymis prior to selection of female embryos in local goat

The objective of this study is the selection of X epididymal spermatozoa of local buck by discontinuous ficoll density gradient and using these spermatozoa for in vitro fertilization and identify the ratio of produced female embryos after identification by polymerase chain reaction (PCR). Epididymal spermatozoa were harvested from the cauda epididymis by slicing, and then spermatozoa were estimated and submitted to in vitro maturation and capacitation prior to separation of X from Y bearing spermatozoa and use for in vitro fertilization. Modified discontinuous ficoll density gradient method used for the separation of X and Y-bearing sperm by using 3 layers of discontinuous ficoll (60, 40, and 20%) or 4 layers of discontinuous ficoll (80, 72, 64, and 50%). The centrifugation applied at 200×g or 300×g. In the protocol of 3 layers of discontinuous ficoll (60, 40, and 20%) and centrifugation at 200×g and 300×g the results showed that the mean of the sperms at the 3rd was 79.014 ± 3.12 and 79.39 ± 2.11 respectively and this show the sperms was not completely separated. In the protocol of 4 layers of discontinuous ficoll (80, 72, 64, and 50%) and centrifugation at 200×g showed that the mean of the sperms at the 4th layer was (42.79 ± 1.38) as compared with the first three layers and with the protocol centrifugation at 300×g. The mean of the sperm lost showed (7.066 ± 0.56) and a little percent from centrifugation at 300×g showed 11.28 ± 1.55. Oocytes collected from ovaries which obtained from the slaughter house then matured oocyte was applied. The maturation rate for grade A and B used for IVF by spermatozoa after ficoll density gradient it was 67.60% and 52.74%, while the fertilization rate for the grade A and B was 70.68% and 78.26% respectively. The sex of embryos was determined by polymerase chain reaction (PCR) using specific primers for detection of SRY gene. The percentage of goat embryos obtained by IVF by sperms selected with ficoll density techniques after centrifugation at 200×g at different stage of development was 70.13%. The percentage of the male embryos was 16.67% while the female embryos was 83.33%. The results showed that the best method for selection of female embryos was by using 4 layers of discontinuous ficoll gradient method (80, 72, 64, and 50 %) with centrifugation at 200×g.

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.

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid lyse/fix protocol with a routine method

Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes are separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid lyse/fix method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples). Similar foci yields were scored for both protocols and reliable dose estimates were obtained for coded samples, with mean absolute differences from the actual doses of 0.26 and 0.27 Gy for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid lyse/fix protocol with a routine method

Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes are separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid lyse/fix method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples). Similar foci yields were scored for both protocols and reliable dose estimates were obtained for coded samples, with mean absolute differences from the actual doses of 0.26 and 0.27 Gy for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

VLA-4 Blockade by Low Molecular Weight Heparin: A Novel Therapeutic Approach in Sickle Cell Disease.

Abstract 2113 Background and Significance Vaso-occlusion in sickle cell disease (SCD) that leads to painful events and complications is caused in part by adhesion of sickled erythrocytes (SSRBCs) to components of the vascular wall and to circulating leukocytes (WBC). Very late activation antigen-4 (VLA-4) or α4β1 integrin expressed on RBCs and WBCs mediates SSRBC-WBC adhesion as well as SSRBCs adhesion to the endothelium via vascular cell adhesion molecule (VCAM-1). VLA-4 blockade has been used to treat pathologic inflammation in multiple sclerosis and Crohn's Disease by inhibiting the interaction between VLA-4 on the leukocyte and endothelial VCAM-1. Enoxaparin, a low molecular weight heparin (LMWH), has been recently shown to exhibit anti-adhesive properties via VLA-4 inhibition. The potential role of enoxaparin as an anti-adhesive therapy for SCD has not been explored. The objective of this study was to determine if enoxaparin could decrease VLA-mediated SS RBC adhesive interactions. Design and Methods Peripheral blood samples were obtained from pediatric patients (n=6) with homozygous SCD (5–18 years) at steady state. Normal control samples (n=3) were obtained from healthy volunteers. We evaluated the interaction between VLA-4 on isolated SSRBCs and immobilized VCAM-1 in a flow adhesion assay under physiologic flow conditions. RBCS were isolated from whole blood after ficoll density gradient separation. We observed adhesion of SSRBCs to immobilized VCAM-1 under three conditions: 1) unstimulated, 2) stimulated with Manganese (Mn2+) – an experimentally accepted method of integrin stimulation that puts VLA-4 in its active confirmation and 3) Mn2+ stimulated RBCs pretreated with enoxaparin. SSRBC-WBC aggregates were identified by classical flow cytometry. Whole blood samples were processed under three conditions: 1) unstimulated, 2) stimulated with Mn2+ and 3) stimulated with Mn2+ and pretreated with enoxaparin. Samples were separated on a ficoll density gradient to obtain an enriched WBC layer. Within this WBC layer, components of multicellular aggregates were identified using antibodies staining for WBCs (anti-CD45), or RBCs (anti-glycophorin A), and CD71 for reticulocytes. Results Mn2+ treatment enhanced VLA-4 adhesion of isolated SSRBCs to immobilized VCAM-1 in 5/6 patient samples. When Mn2+ stimulated samples were treated with enoxaparin, 4/6 patient samples showed a significant decrease in adhesion (p < 0.02). Mn2+ stimulation induced SS RBC-WBC aggregate formation in 3/4 patient samples tested. Both mature red blood cells and reticulocytes were involved in these aggregates. Aggregates were variable in size. All 4 patient samples treated with enoxaparin showed decreased aggregates. 3/4 patient samples showed aggregates decreased to levels at or below that observed in unstimulated samples. Conclusions Our data show that enoxaparin inhibits adhesion of pediatric SSRBCs to immobilized VCAM-1, presumably via blockade of activated VLA-4 on the SS RBC surface. Further, our data demonstrate that stimulation of VLA-4 can increase adhesive interactions of circulating SSRBCs and WBCs, and that these interactions can be disrupted by enoxaparin. The complications and sequelae in SCD begin in childhood warranting the need to find more treatment strategies for pediatric SCD. The fact that numerous adhesive interactions responsible for vaso-cclusive events in SCD are VLA-4-mediated makes enoxaparin an attractive therapeutic possibility to prevent and treat vaso-occlusive complications in SCD. Disclosures: No relevant conflicts of interest to declare.