Microrheological characteristics of reticulocyte in vivo

Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticulocytes, which synchronously grow, were got in vivo. The measurements of deformation index, orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of reticulocytes turning into red blood cells in vivo. There were obvious changes in the micro- rheological characteristics of reticulocytes in the course of turning into erythrocytes. The present study is significant in clinic for studying erythrocytes' microrheological characteristics when there are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes microrheological characteristics. It makes up a deficiency in the study on microrheological characteristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.     

Microrheological characteristics of reticulocyte in vivo

Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticu-locytes, which synchronously grow, were got in vivo. The measurements of deformation index, orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of reticulocytes turning into red blood cells in vivo. There were obvious changes in the micro-rheological characteristics of reticulocytes in the course of turning into erythrocytes. The present study is significant in clinic for studying erythrocytes' microrheological characteristics when there are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes microrheological characteristics. It makes up a deficiency in the study on microrheological characteristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.     

60Co辐射对网织红细胞微观流变学特性的影响

采用 ⁶⁰Co大剂量全身均匀急性辐射的方法,造成一种辐射贫血的动物模型.以便在几天内连续研究 ⁶⁰Co辐射对新生的网织红细胞及红细胞流变学特性的影响.采用一种在低粘切变流场中能将红细胞变形指数DI分解为取向指数（DI）_or和小变形指数（DI）_d的新型激光衍射法,对网织红细胞及红细胞的变形指数、取向指数、综合变形指数（IDI）等血液流变学特性参数进行测量,发现在 ⁶⁰Co大剂量辐射后,新生的网织红细胞及红细胞流变学特性存在明显异常.将这种 ⁶⁰Co辐射造成的贫血模型与文宗曜等提出的用抗体诱导的大量同步化的球形红细胞贫血模型相比较,后者更具有明显的优点.同时为研究辐射对血液流变特性的影响及正确地挑选红细胞衰老模型提供了理论与实验的基础.     

苯肼对红细胞在体衰老过程中微观流变特性的影响

在Brunara等人用苯肼使动物造成急性溶血性贫血的方法基础上,建立一种由急性溶血性贫血后,而诱发家兔幼红细胞增多的非正常生理状态的红细胞在体衰老模型,继而研究新生红细胞从产生到死亡死亡过程,即衰老过程的流变学特性的变化规律。通过对新生红细胞的压积、变形、取向及与之相应的全血的粘度、血沉等指标的连续60多天的监测,发现红细胞在衰老过程中的微观流变学特性确实有明显改变。红细胞在体衰老过程中微观流变特性逐渐变差。     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Real‐time in vivo imaging of fungal migration to the central nervous system

Recent technical advances have afforded valuable new insights into the pathogenesis of fungal infections in the central nervous system (CNS), which continue to cause devastating complications, particularly in immunocompromised individuals. To cause CNS mycosis, organisms such as Cryptococcus neoformans become blood borne and progress through a series of pathogenic checkpoints that culminate in fungal replication in the brain. Critical steps include fungal arrest in the vasculature of the brain, interaction and signalling of the fungal and endothelial cells leading to transmigration with subsequent parenchymal invasion and fungal replication in the CNS. Previous studies that made use of in vitro and ex vivo approaches contributed greatly to our understanding of brain invasion by fungi. However, the knowledge gained from previous studies relied on in vitro models that did not account for vascular haemodynamics. For this reason, more refined approaches that model blood flow and vascular anatomy are required, andultimately studying fungal invasion and dissemination in vivo. Indeed, in vivo imaging (also known as intravital imaging) has emerged as a valuable technique to probe host–pathogen interactions. In this review, with a focus on C. neoformans, we will provide an overview of the applications of the prior techniques and recent advances, their strengths and limitations in characterizing the migration of fungi into the brain, and unanswered questions that may provide new directions for research.     

Labeling of cynomolgus monkey bone marrow-derived mesenchymal stem cells for cell tracking by multimodality imaging

Recently, transplantation of allogeneic and autologous cells has been used for regenerative medicine. A critical issue is monitoring migration and homing of transplanted cells, as well as engraftment efficiency and functional capability in vivo. Monitoring of superparamagnetic iron oxide (SPIO) particles by magnetic resonance imaging (MRI) has been used in animal models and clinical settings to track labeled cells. A major limitation of MRI is that the signals do not show biological characteristics of transplanted cells in vivo. Bone marrow mesenchymal stem cells (MSCs) have been extensively investigated for their various therapeutic properties, and exhibit the potential to differentiate into cells of diverse lineages. In this study, cynomolgus monkey MSCs (cMSCs) were labeled with Molday ION Rhodamine-B™ (MIRB), a new SPIO agent, to investigate and characterize the biophysical and MRI properties of labeled cMSCs in vitro and in vivo. The results indicate that MIRB is biocompatible and useful for cMSCs labeling and cell tracking by multimodality imaging. Our method is helpful for detection of transplanted stem cells in vivo, which is required for understanding mechanisms of cell therapy.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> assays for osteoclast apoptosis

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.     

红细胞成熟脱核机制的研究

面对解决血液来源匮乏的问题,开发新的血源已经成为当前医疗中的迫切需要,其中一种重要的手段就是体外产生功能性的红细胞,而脱核是关键的一步．虽然目前的研究表明已经可以在体外条件下产生成熟的脱核红细胞,但是诱导分化的效率相对比较低．不仅如此,我们对红细胞脱核机制研究的并不是很清楚．本文对于目前研究比较成熟的三种脱核理论,包括细胞凋亡学说、不对称分裂学说、膜泡运输理论,以及脱核过程中重要的蛋白质、microRNA进行了阐述,并对体外制备血细胞的前景进行了展望．     

Studies of metabolic control using NMR and molecular genetics

The techniques of NMR spectroscopy and molecular genetics have provided new and powerful approaches to studying the control and organisation of cellular metabolism in vivo. We review here our recent applications of these methodologies to the study of energy metabolism in yeast and mammalian cells. © 1997 John Wiley & Sons, Ltd.     

The power and the promise of organoid models for cancer precision medicine with next-generation functional diagnostics and pharmaceutical exploitation

As organ-specific three-dimensional cell clusters derived from cancer tissue or cancer-specific stem cells, cancer-derived organoids are organized in the same manner of the cell sorting and spatial lineage restriction in vivo, making them ideal for simulating the characteristics of cancer and the heterogeneity of cancer cells in vivo. Besides the applications as a new in vitro model to study the physiological characteristics of normal tissues and organs, organoids are also used for in vivo cancer cell characterization, anti-cancer drug screening, and precision medicine. However, organoid cultures are not without limitations, i.e., the lack of nerves, blood vessels, and immune cells. As a result, organoids could not fully replicate the characteristics of organs but partially simulate the disease process. This review attempts to provide insights into the organoid models for cancer precision medicine.     

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34⁺ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34⁺ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.     

Wound healing effect of collagen-hyaluronic acid implanted in partially injured anterior cruciate ligament of dog

The purpose of this study was to evaluate the cell compatibility of collagen-hyaluronan (HA) substrate in vitro by assessing ACL cell cultures. Additionally, the use of collagen-HA substrate as a dressing material for partial ACL defects as well as its effect on collagen synthesis and angiogenesis were evaluated in vivo. The initial attachment and proliferation of dog ACL cells on silk matrix covered with collagen-HA substrate (SMCH) was greater than that observed on silk matrix alone. Silk matrix and SMCHs were implanted as dressing materials into partial ACL defects located at the knees of dogs, and they were harvested six weeks after implantation. A histological evaluation of the collagen-HA substrates revealed the presence of monocytes and the absence of giant cells in all cases. MT staining of the SMCH-grafted group showed a higher level of granulation tissue formation consisting of fibroblasts and collagen fibers compared to the silk matrix-grafted group. In addition, CD31 staining revealed that the SMCH-grafted area showed more blood vessel formation than the silk matrix-grafted area. These results suggest that the collagen-HA substrate was cell-compatible in vitro and enhanced collagen synthesis and new blood vessel formation in vivo.     

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase la can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during ‘in vivo’ reticulocytes maturation. This new hexokinase micro-heterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.     

Deterioration and disappearance of mitochondria during reticulocyte maturation

This study correlates the decreasing aerobic metabolism with the increased deterioration of mitochondria in maturing reticulocytes. The respiration of reticulocytes, separated into four age groups according to their density, was determined manometrically. Their Krebs tricarboxylic acid cycle activity was determined using [2-¹⁴C] acetate as a substrate for [¹⁴C]CO₂ formation. The ability of reticulocytes of various age groups to couple the phosphorylation to oxidation processes was estimated by studying the effect of 2,4-dinitrophenol as an uncoupler.     

Hematopoietic Stem/Progenitor Cell Sources to Generate Reticulocytes for Plasmodium vivax Culture

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1–2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34⁺-enriched populations of PB and BM, CD34⁺-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34⁺-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.     

Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs

Background aimsMesenchymal stromal cells (MSC) possess multilineage differentiation potential and characteristics of self-renewal. It has been reported that MSC can acquire characteristics of cells in the periodontal ligament (PDL) in vitro. Moreover, the transplantation of MSC has been shown to be a promising strategy for treating periodontal defects. However, little is known about the fate of MSC in periodontal tissue in vivo. The aim of this study was to trace the paths of MSC after transplantation into periodontal tissues in vivo.MethodsMSC labeled with bromodeoxyuridine (BrdU) were transplanted into periodontal defects of beagle dogs. Six weeks after surgery, the animals were killed and decalcified specimens were prepared. Migration and differentiation of MSC were detected by single/double immunohistochemistry and a combination of immunohistochemistry and in situ hybridization.ResultsBrdU-labeled MSC were observed distributing into periodontal tissue that included alveolar bone, PDL, cementum and blood vessels and expressing surface markers typical of osteoblasts and fibroblasts.ConclusionsCumulatively, our data suggest that MSC migrate throughout periodontal tissue and differentiate into osteoblasts and fibroblasts after transplantation into periodontal defects at 6 weeks in vivo, and have the potential to regenerate periodontal tissue.     

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study

BackgroundA very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival.Methods and findingsWe examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach.In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted.ConclusionsImmature CD71⁺ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Dr. Anstey and co-authors found that P. vivax-infected immature reticulocytes and erythrocytes accumulate in the same splenic compartments, suggesting existence of a cryptic endosplenic lifecycle in chronic P. vivax infection that maximizes survival and replication in the spleen.     

小鼠网织红细胞微观流变学特性的研究

按Bishop方法,在小鼠血液里诱导生成大量网织红细胞,然后提取网织红细胞,对其电泳率、渗透脆性、膜的流动性、细胞的变形能力和取向性进行了系统研究。研究结果表明网织红细胞在转变为成熟红细胞的短短时间内,其微观流变学特性发生了明显的变化：电泳率变小、渗透脆性变好、膜的流动性变大、细胞的变形能力变强、取向性变好,最终发育成具有全面功能的成熟红细胞。     

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in Pig spleens

Summary Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a ³H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar.The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G₂, about 1.2 to 1.3 hours for M; about 17 to 16.5 hours for S and about 1.5 to 1.7 hours for G₁. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.The expert technical assistance of Mrs. A. Fischer is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 112.