Hemorheological disorders in children with thermal trauma

Цель исследования - изучение динамики изменения реологических свойств крови и их корреляции с параметрами эритроцитов в процессе лечения детей после термической травмы. Методика. Исследование проведено на 32 детях в возрасте от 3 до 17 лет с ожогами площадью от 10 до 70% поверхности тела в период лечения, начиная с 3-х суток после термической травмы и до полного закрытия ожоговых ран и выписки пациента из стационара. Изучали состояние цитоскелета эритроцитов методом термоиндукции, их агрегацию и дезагрегацию с помощью модифицированного нами реоскопа Н. Schmid-Schönbein и соавт. [1973], их деформируемость и морфологию агрегатов эритроцитов, а также рассчитывали эритроцитарные индексы. Результаты. Установлено, что после ожога происходит резкое усиление агрегации эритроцитов и прочности агрегатов, но значительно снижается их деформируемость. Нарушение реологических свойств крови сопровождает все стадии ожоговой болезни и сохраняется при выписке больных из стационара. Нарушения гемореологии коррелируют с изменениями концентрации фибриногена, состоянием цитоскелета эритроцитов и их физическими свойствами - размером эритроцитов и содержанием в них гемоглобина. Заключение. Стойкие и выраженные нарушения реологических свойств крови у детей при ожоговой болезни могут являться важнейшей причиной редукции снабжения кислородом жизненно важных органов и развития их недостаточности. Простым, хотя и предварительным, методом оценки состояния гемореологии может служить измерение эритроцитарных индексов. Aim. To study hemorheological changes and their correlation with the size of red blood cells (RBCs) during the treatment of thermal injury in children. Methods. The study included 32 children ages 3 to 17 with burns affecting 10% to 70% of the total body area during the treatment period between the 3rd day after the burn and complete wound closure. Aggregation and disaggregation of red blood cells was analyzed using a H. Schmid-Schönbein et al. [1973] rheoscope in our modification. In addition, erythrocyte deformability, morphology of erythrocyte aggregates, and the state of erythrocyte cytoskeleton were studied using thermoinduction, and erythrocyte indices were calculated. Results. After a burn injury, erythrocyte aggregation and the strength of erythrocyte aggregates were increased whereas their deformability was decreased. Disorders of blood rheology accompanied all stages of burn disease and remained at the patient discharge. Hemorheological disorders correlated with changes in fibrinogen concentrations and with changes in the erythrocyte cytoskeleton, size, and the amount of contained hemoglobin. Conclusion. The stable and pronounced disorders of blood rheological properties in children with burn disease may be an essential cause for reduction of oxygen supply to vital organs and their failure. The measurement of erythrocyte indices may represent a simple though preliminary method for assessing hemorheology.

A Dynamic Motility Mechanism for Reticulocyte Enucleation and Maturation

Terminal erythropoiesis involves differentiation of erythroblasts to enucleated reticulocytes, which further remove major organelles to become mature red blood cells. Several pieces of evidence indicate that the extrusion of the nucleus out of the orthochromatic erythroblasts is a fairly rapid process that lasts approximately 10 minutes. However, the detachment of the extruded nucleus from the nascent reticulocyte takes longer time, and possibly requires the aid of phagocyte to anchor the nucleus. The mechanism involved in this process is poorly understood. Previous studies have demonstrated that mDia2 is critical for erythroblast enucleation. Loss of mDia2 in mice lead to compromised cytokinesis in orthochromatic erythroblasts and decrease in enucleation. To further reveal the mechanism of the defect in enucleation in mDia2 deficient erythroid cells, we first monitored the enucleating erythroblasts using real-time microscopy. The wild type enucleating mouse erythroblast showed a dynamic motility to detach the nucleus, which can be facilitated by the macrophages. The mDia2 knockout erythroblasts retained the capacity to extrude the nucleus, even in the bi-nucleated cells. However, the nascent reticulocytes were rigid and lacked the dynamic movement to expel the nuclei. Since mDia2 is critical for linear actin polymerization, we next performed a Western blot assay of RBC ghost and found that actin and many other critical RBC cytoskeleton proteins were significantly decreased in mDia2 knockout mice (Fig 1A). Transmission electron microscope (TEM) analysis of RBC cytoskeleton revealed that spectrin filament connecting two junctional complexes was longer in mDia2 knockout RBCs (Fig 1B), which explains their rigidity and inefficient movement to detach the extruded nuclei. Further studies using stochastic optical reconstruction microscopy (STORM) revealed disorganized distribution of the major components of erythrocyte cytoskeleton, including actin (Fig 1C) and β-spectrin, in mDia2 knockout reticulocytes. The dynamic motility of the nascent reticulocytes is also required for the remodeling and removal of proteins to further mature into RBCs. We found an increase in proteins such as CD71 and CD44, as well as DNA/RNAs, in mDia2 knockout reticulocytes. Mechanistic studies revealed that mDia2 functions on vesicle trafficking through its interaction with Chmp5, one of the critical components of the ESCRT-III complex involved in vesicle formation and trafficking. This interaction stabilized Chmp5 since its level was dramatically reduced in mDia2 knockout erythroblasts and reticulocytes (Fig 1D-E). Our study reveals a critical role of the dynamic motility of the nascent reticulocyte in enucleation and maturation. Abnormal membrane cytoskeleton in mDia2 null mice compromises these processes. In one hand, mDia2 mediated actin polymerization ensures the flexibility of the erythrocyte cytoskeleton. On the other hand, mDia2 also involves vesicle biogenesis and discharging through the regulation of ESCRT complexes during reticulocyte maturation. Our study thus sheds lights on the understanding of reticulocyte maturation mechanisms. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse inPlasmodium falciparumegress

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellularPlasmodium falciparumparasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10–30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.