Aging of red blood cells and impaired erythropoiesis following prolonged administration of dichloromethylene diphosphonate containing liposomes in rats

OBJECTIVES: To investigate whether macrophage-depleted rats may serve as a model for studying red blood cell (RBC) aging. METHODS: Rats were macrophage-depleted by 4 weekly injections of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-CL). The macrophage content of spleens and bone marrows (BMs) was investigated by immunohistochemistry and light microscopy and by flow cytometry, respectively, after staining with macrophage-specific monoclonal antibodies. In addition, the ultrastructure of residual BM macrophages and their ability to phagocytose zymosan was studied. BM was also studied for apoptosis (by the TUNEL reaction) and for erythroid progenitor cell content. Furthermore, RBC indices, morphology, life span (by 51Cr labeling) and aging features (MCV, density, 4.1a/4.1b membrane protein ratio, anti-spectrin IgG binding, microvesiculation) were investigated. Serum TNF-alpha, iron, total iron-binding capacity (TIBC) and ferritin were also determined. RESULTS: Prolonged treatment with Cl2MDP-CL caused an almost complete depletion of macrophages in the spleen and a 58% reduction of those in the BM; the residual BM macrophages were activated as judged by their ultrastructure and phagocytic capacity in vitro. These alterations were accompanied by an increase in RBC life span and age-related RBC changes, as well as by mild anemia associated with a reduced reticulocyte count, reduced BM erythroid progenitors, increased numbers of apoptotic cells in the BM, low serum iron, high TIBC and increased serum TNF-alpha levels. CONCLUSIONS: Rats subjected to prolonged macrophage depletion showed an increased prevalence of senescent RBC in the circulation due to their impaired clearance by macrophages. Hence, these animals provide a model system in which mechanisms of RBC aging can be delineated. They also showed impaired erythropoiesis, presumably related to a reduction in BM macrophages and increased production of proinflammatory cytokines by residual activated marrow macrophages and other cells.     

Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP)

In order to explore the effect on bone marrow macrophages of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP), mice were injected intravenously with a preparation of such liposomes at a dose known to deplete spleen and liver macrophages. Two days later, the macrophages in the marrow of the femoral bones were quantified by flow cytometry using a macrophage-specific monoclonal antibody (F4/80), and their ultrastructure and phagocytic activity towards zymosan particles was assessed. To determine the effect on erythropoiesis of liposome-encapsulated Cl2MDP-induced changes in bone marrow macrophages, red blood cell parameters and the formation of erythroid burst-forming unit (BFU-E)-derived colonies in vitro were evaluated. In mice injected with liposome-encapsulated Cl2MDP, there was a 54% and 67% decrease in the total number of bone marrow macrophages as compared to uninjected controls and mice treated with empty liposomes, respectively. Moreover, residual macrophages showed an abnormal ultrastructure, with reduced numbers of crystalloid inclusions and increased numbers of large myelin figures. However, the phagocytic activity of these cells was unimpaired or slightly enhanced. In mice injected with liposome-encapsulated Cl2MDP there was an approximately 60% decrease in the percentage and total number of circulating reticulocytes and a 54% reduction in the BFU-E number, demonstrating deregulation of erythropoiesis under conditions of macrophage loss and impairment. The results suggest that mice treated with liposome-encapsulated Cl2MDP are a model for studying the role of macrophages in erythropoiesis.     

Effects of Poloxamer 188 on red blood cell membrane properties in sickle cell anaemia

Vaso‐occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in‐vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy.     

基质金属蛋白酶在老龄鼠基底节区血管的表达

目的　观察老龄鼠易卒中区基底节血管壁明胶酶A和明胶酶B的表达 ,以探讨基质金属蛋白酶在易卒中区血管壁损伤中的作用。方法　应用免疫组织化学方法分析基底节区血管壁明胶酶A和明胶酶B的表达 ,对比研究老龄鼠与 3个月龄自发性高血压大鼠及青年组大鼠的脑血管壁基质金属蛋白酶表达的差异性。结果　老龄鼠基底节区血管壁明胶酶A和明胶酶B表达明显 ,同样自发性高血压大鼠表达也高 ,而青年组大鼠表达极少。结论　年龄因素和高血压均是脑卒中的危险因素之一 ,而基质金属蛋白酶在二者的高表达可能是血管壁病变的重要原因。     

Clinical utility of the new Beckman-Coulter parameter red blood cell size factor in the study of erithropoiesis

CHr has been used as a diagnostic tool, together with biochemical markers, to distinguish IDA from ACD, and is incorporated to NKF-K/DOQI guidelines for the monitoring of rHuEPO therapy. The measurement of CHr has been restricted to the analysers of a single manufacturer, Siemens. Red blood cell size factor (RSf) is a new parameter provided by Beckman-Coulter which joins together the volume of erythrocytes and the volume of reticulocytes Rsf = square root (MCV x MRV). The aims of the study were to establish the values of RSf in normal population and in different types of anemia to investigate its clinical usefulness in the study of erythropoiesis and its correlation with CHr. Samples from 449 patients (learning group) were run sequentially on both LH 750 (Beckman-Coulter) and Advia 2120 (Siemens) analysers. Good correlation between CHr and RSf was observed, r(2) = 0.85. Receiver operating characteristic curve analysis for RSf and the diagnosis of restricted erythropoiesis. [table: see text] The diagnostic usefulness of RSf was evaluated on a validation group which included 220 consecutive patients with anemia. This study shows a very good level of agreement between RSf and CHr. Both are suitable parameters for the study of erythropoiesis.     

Clinical significance of red blood cell distribution width in the elderly: a potential indicator of bone marrow stimulation by erythropoietin

Summary To investigate the clinical significance of red blood cell distribution width (RDW) and haemoglobin distribution width (HDW) in the elderly and their relationships with erythropoietin (EPO) secretion, we measured red cells parameters using a Technicon HI system and serum EPO using a radioimmunoassay in 247 elderly subjects (normal: n= 150; preanaemic iron deficiency: n= 24; iron deficiency anaemia: n= 8; senile anaemia: n= 65). RDW was slightly higher in the elderly subjects with preanaemic iron deficiency (14.1 ± 1.1%) than in the normal elderly subjects (13.5±0.7%). It was highest in iron deficiency anaemia (16.1 ± 1.3%), while the increase in senile anaemia was limited (13.9 ± 1.2%). The HDW increased only in iron deficiency anaemia. There was a strong positive relationship between EPO and RDW in iron deficiency anaemia (r= 0.817, P<0.01). Moreover, this correlation was also found in preanaemic iron deficiency (r= 0.456, P < 0.05), but not in senile anaemia, suggesting that bone marrow hypoactivity may partly play a role in the pathogenesis of senile anaemia. All the eight subjects with iron deficiency anaemia had a RDW ≥ 14.9% (mean + 2SD of normal subjects), while 55 (85%) of the 65 with senile anaemia had a RDW < 14.9%. Both the RDW and EPO levels of six anaemic subjects with high RDW values (≥ 14.9%) after oral iron therapy for 56–78 days decreased significantly. Our results suggest that RDW is useful to distinguish iron deficiency anaemia from senile anaemia, and may be a potential parameter of bone marrow stimulation by EPO.     

中枢交感神经活性抑制对老年大鼠心肌细胞收缩功能的影响

目的探讨中枢交感神经活性抑制对老年大鼠心功能、单个心室肌细胞收缩幅度和细胞存活率的影响。方法选择月龄>18个月的SD大鼠30只,随机分为老年可乐定组和老年对照组,每组15只,另选4~6月龄的SD大鼠18只为成年组,老年可乐定组大鼠以可乐定腹腔注射2周,其他2组用生理盐水腹腔注射2周,进行血流动力学参数测定,酶分离大鼠心肌细胞,用含不同浓度钙离子或异丙肾上腺素(ISO)的KH液对心肌细胞进行表面灌流,观察心肌细胞的收缩幅度,计算其存活率。结果与成年组大鼠比较,老年对照组左心室压力最大上升和下降速率(±dp/dtmax)降低,左心室舒张末压(LVEDP)升高,且心肌细胞在不同浓度钙离子或ISO刺激下收缩幅度较成年组降低(P<0.05)。老年可乐定组大鼠±dp/dtmax改善,LVEDP降低,心肌细胞在同等条件刺激下收缩幅度较老年对照组增加(P<0.05);3组大鼠间细胞存活率无统计学差异(P>0.05)。结论抑制中枢交感神经紧张性可改善老年大鼠心功能,增加心肌细胞的收缩幅度。     

Cadmium-induced changes of antioxidant and metabolic status in red blood cells of rats: in vivo effects

Abstract: Chronic exposure of adult rats to dietary untake of cadmium (15 mg CdCl₂/day/kg for 30 days) leads to development of anemia and thrombocytosis. Anemia is characterized by significant reticulocytosis (13.1 ± 1.0%), anysocytosis, poikilocytosis, iron deficiency and marked alterations of antioxidant and metabolic status of red blood cells. Activities of SOD, catalase, GPx and GR were significantly increased in red blood cells of cadmium-treated rats. In treated animals cadmium induced an increase of red cell reduced and oxidized glutathione with no changes of GSSG/GSH ratio. However, significant reduction of lipid peroxidation was found. Plasma levels of tocopherol and ascorbate, as well as activity of glutathione-S-transferase, were all significantly increased in cadmium-treated rats. The energy metabolism of red blood cells was deeply altered in cadmium-treated rats. The levels of ATP, ADP, AMP and TAN were significantly increased while ATP/ADP ratio and adenylate energy charge (AEC) were significantly reduced. The level of 2,3-BPG was somewhat lower, but 2,3-BPG/Hb ratio was considerably higher, in red blood cells of cadmium-treated rats.     

IgA肾病并发肉眼血尿相关急性肾损伤临床及病理分析

目的分析IgA肾病(IgAN)患者发生肉眼血尿相关急性肾损伤(AKI)的临床、病理特点及其转归,探讨其对预后的影响及相关因素。方法对2004年1月至2007年8月在北京大学人民医院肾内科确诊为IgAN且同时伴发肉眼血尿相关AKI患者的临床、病理资料进行分析,并对这些患者进行随访。肾脏病变的程度根据KatafuchiIgAN评分标准进行评价。结果5例经临床和肾脏病理证实的IgAN患者发生肉眼血尿相关AKI,占同期确诊IgAN患者的1.3%,占发作肉眼血尿患者的13.5%。肾脏病理提示均存在大量红细胞管型伴不同程度的肾小管损伤。其中3例患者血清肌酐(Scr)在14d内完全恢复正常,1例患者Scr在1个月后明显好转,418d后恢复正常。另1例患者至最后一次随访(20个月)时Scr仍未恢复正常。未恢复者与恢复者相比,年龄较大,肉眼血尿持续时间较长,肾小球球性硬化积分和小管-间质病变积分较高。结论发作性肉眼血尿可以引起AKI的发生,且并非所有IgAN患者发生的肉眼血尿相关AKI都能完全恢复。影响肉眼血尿相关AKI患者预后的因素可能有年龄、肉眼血尿持续时间、小管-间质损伤程度以及硬化肾小球比例。     

Prevention of lymphocytic thyroiditis and insulitis in diabetes-prone BB rats by the depletion of macrophages

Summary Diabetes-prone biobreeding (BB) rats often develop lymphocytic thyroiditis. Intraperitoneal administration of silica to young BB rats (40-days-old) nearly completely prevented the development of lymphocytic thyroiditis as well as insulitis. Since silica is known to be toxic to macrophages, these data suggest that the presentation of autoantigen(s) on the specific target cells such as thyroid and pancreatic B cells by antigen-presenting cells (e.g., macrophages) would be the initial step in the development of organ-specific autoimmune diseases in diabetes-prone BB rats.     

Expression of macrophage inflammatory protein-1α in Kupffer cells following liver ischemia or reperfusion injury in rats

AIM: To explore the expression of macrophage inflammatory protein-1α (MIP-1α) in Kupffer cells (KCs)following liver ischemia/reperfusion injury IRI in rats.METHODS: Forty male SD rats were divided randomly into five groups. A model of partial warm ischemia/reperfusion injury in the rat liver was established. KCs were isolated and incubated one hour, six hours, 12 h,and 24 h after the reperfusion. Tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β) in the supernatants were measured by ELISA. MIP-1α in KCs was detected by immunocytochemical and RT-PCR.RESULTS: No or few MIP-1α protein and mRNA were expressed in the KCs of the control group. Its expression in the IRI group had a significant increase after the reperfusion (P ＜ 0.05), which was contrary to the control group.CONCLUSION: The active behavior of the MIP-1α gene in KCs following liver ischemia/reperfusion injury is assumed to be one of the major causes for the hepatic ischemia/reperfusion injury.     

The effect of steroid pulse therapy on the development of acute pancreatitis induced by closed duodenal loop in rats

Background: Background: The aim of the present study was to evaluate the effect of a high dose of methylprednisolone (MP) on the development of acute pancreatitis (AP) in rats induced by closed duodenal loop (CDL). Methods: Pancreas web weight, volume of ascites, hematocrit, serum amylase activity, concentrations of interleukin (IL)-1β and IL-6, organ blood flow in both the pancreas and the kidney, and histological findings of the pancreas were studied 6 h after the induction of AP. Results: The intravenous administration of MP (30 mg/kg body weight) significantly reduced the increase in pancreas web weight, volume of ascites, hematocrit, serum amylase activity, concentrations of IL-1β and IL-6, histological edema, and necrosis observed in CDL pancreatitis. The administration of MP also apparently improved both the pancreatic and the renal blood flow. Conclusions: The present results suggest that these cytokines influence at least the progression of AP, and that the mechanism by which MP pulse therapy inhibits the development of AP partly involves the inhibition by MP of the release of the cytokines. Received: July 19, 2001 / Accepted: December 14, 2001     

Expression of macrophage inflammatory protein-1α in Kupffer cells following liver ischemia or reperfusion injury in rats

AIM: To explore the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) in Kupffer cells (KCs) following liver ischemia/reperfusion injury IRI in rats. METHODS: Forty male SD rats were divided randomly into five groups. A model of partial warm ischemia/reperfusion injury in the rat liver was established. KCs were isolated and incubated one hour, six hours, 12 h, and 24 h after the reperfusion. Tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the supernatants were measured by ELISA. MIP-1alpha in KCs was detected by immunocytochemical and RT-PCR. RESULTS: No or few MIP-1alpha protein and mRNA were expressed in the KCs of the control group. Its expression in the IRI group had a significant increase after the reperfusion (P < 0.05), which was contrary to the control group. CONCLUSION: The active behavior of the MIP-1alpha gene in KCs following liver ischemia/reperfusion injury is assumed to be one of the major causes for the hepatic ischemia/reperfusion injury.     

高压氧对人脐带华通胶间充质干细胞移植心肌梗死大鼠炎性因子的作用

目的探讨高压氧对移植干细胞在梗死心肌组织局部的炎性状况。方法选择SD大鼠65只,随机分为假手术组5只,心肌梗死组15只,干细胞移植组15只,高压氧组15只,高压氧+干细胞移植组(联合组)15只。建立心肌梗死大鼠模型,模型建立后第1天进行高压氧治疗30d。心肌梗死后第7天进行人脐带华通胶间充质干细胞移植,分别于心肌梗死前、心肌梗死后1、7、14、28d采集血清,ELISA法检测血清TNF-a、白细胞介素1β(IL-1β)、IL-6水平,采用EdU染色法,标记人脐带华通胶间充质干细胞,免疫荧光法检测移植细胞存活及增殖率。结果除外联合组14d时IL-6,联合组3、14、28d时TNF-a、IL-6和IL-1β较心肌梗死组、高压氧组、干细胞移植组明显降低(P<0.05);与干细胞移植组比较,联合组干细胞移植后7、21d心肌组织局部干细胞数量及增殖率明显增高(80.6%vs 55.0%,79.1%vs 34.3%,P<0.05)。结论高压氧能够显著降低梗死心肌组织中的炎性因子水平,有效改善移植干细胞生存的微环境,提高移植干细胞在受损心肌局部的生存及增殖率。     

Autoimmunity and normal immune functions in aged humans

The high frequency of ANA, A-LDL and RF in advanced age suggests that AABs are present in the majority of aged subjects. CIC incidence determined by three methods is far below AAB incidence; only the Clq solubility test suggests an increased CIC incidence in aged as compared to young subjects. Simultaneous occurrence of AABs of different specificities or CIC determined by two or three methods is rare and both AAB and CIC levels are usually low. AAB prevalence in CIC-positive individuals seems to depend on the specificity of the AAB. CIC positivity is associated with relatively low Clq concentrations; however, usually not with Clq concentrations below the normal range. Neither ANA nor CIC positivity seems to correlate with DNA synthetic response to PHA, but ANA positivity may be associated with low responses to allogeneic cells. ANA positivity and, to a lesser extent, CIC positivity seems to be connected with enhanced killer cell activity. The concept of some AABs and CIC as autoregulatory factors of the humoral immune system compensating for the thymus-dependent regulation in old age is stressed.     

Successful collection of peripheral blood progenitor cells in patients with acute myeloid leukaemia following early consolidation therapy with granulocyte colony-stimulating factor-supported high-dose cytarabine and mitoxantrone

We evaluated the feasibility of collecting peripheral blood progenitor cells (PBPC) in patients with acute myeloid leukaemia (AML) following two cycles of induction chemotherapy with idarubicin, cytarabine and etoposide (ICE), and one cycle of consolidation therapy with high-dose cytarabine and mitoxantrone (HAM). Thirty-six patients of the multicentre treatment trial AML HD93 were enrolled in this study, and a sufficient number of PBPC was harvested in 30 (83%). Individual peak concentrations of CD34⁺ cells in the blood varied (range 13.1–291.5/μl; median 20.0/μl). To reach the target quantity of 2.5 × 10⁶ CD34⁺ cells/kg, between one and six (median two) leukaphereses (LP) were performed. The LP products contained between 0.2 × 10⁶ and 18.9 × 10⁶ CD34⁺cells/kg (median 1.2 × 10⁶/kg). Multivariate analysis showed that the white blood cell count prior to HAM and the time interval from the start of HAM therapy to reach an unsupported platelet count > 20 × 10⁹/l were predictive for the peak value of CD34⁺ cells in the blood during the G-CSF stimulated haematological recovery. In 16 patients an intraindividual comparison was made between bone marrow (BM) and PBPC grafts. Compared to BM grafts, PBPC grafts contained 14-fold more MNC, 5-fold more CD34⁺ cells and 36-fold more CFU-GM. A CD34⁺ subset analysis showed that blood-derived CD34⁺ cells had a more immature phenotype as indicated by a lower mean fluorescence intensity for HLA-DR and CD38. In addition, the proportion of CD34⁺/Thy-1⁺ cells tended to be greater in the PBPC grafts. The data indicate that sufficient PBPC can be collected in the majority of patients with AML following intensive double induction and first consolidation therapy with high-dose cytarabine and mitoxantrone.     

Diuretics and carbohydrate metabolism: The effects of furosemide and amiloride on blood glucose,plasma insulin and cations in the rat

Summary The effects of two unrelated diuretics, furosemide and amiloride on blood glucose, plasma insulin and glucose tolerance in the conscious rat are reported. Furosemide (1 mg/kg or 2 mg/kg) given intravenously caused an immediate but highly transient 23% and 53% fall in plasma insulin followed by a rise in blood glucose. The hyperglycaemic effect alone occurred in mild streptozotocin diabetic animals. Furosemide given with or 30 min before intravenous glucose (0.5 G/kg) caused glucose intolerance with diminished insulin response. None of these effects were observed in adrenalectomised animals. Amiloride (1, 5 or 10 mg/kg) given I.V. alone immediately increased plasma insulin (up to 213 U/ml) without affecting blood glucose. This effect was attenuated by mild streptozotocin diabetes. Amiloride given with or 30 min before glucose increased the insulin area without affecting glucose disappearance. Chronic administration enhanced glucose disappearance with increased plasma insulin response, and caused hyperkalaemia. Chronic furosemide administration had no effects. Possible mechanisms for these effects are discussed.Presented in part at the 7th Annual meeting of the European Association for the study of diabetes, Southampton, 1971.Wellcome Junior Research Fellow.