An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.     

下坡运动对大鼠骨骼肌肌浆网功能的影响

目的：观测研究下坡(离心)运动对大鼠骨骼肌肌浆网Ca2+-ATP酶活性,Ca2+摄取与释放在量与时程上的影响。此外,测定离子载体的刺激作用,即测定在含与不含(Ca2+离子载体)A23187时Ca2+-ATP酶活性的比值,用以评定囊泡的完整性。方法：成年雄性SD大鼠随机分为对照与离心运动组, 离心运动的大鼠分别于运动后即刻, 4, 24, 48, 72 和144h后取样 (n=7). 离心运动方式采用90min持续跑台下坡运动(-16°;15m/min)。取大鼠红股肌制备组织匀浆, 测定肌浆网Ca2+-ATP酶活性,Ca2+摄取与释放。结果：与对照组[19.25±1.38 nmol ·min-1·(mg protein)-1]相比, 肌浆网Ca2+摄取分别于运动后即刻和4h下降了29% and 36% (P<0.05), 24h依然降低(P<0.05). 肌浆网Ca2+释放与对照组[31.06±2.36 nmol·min-1·(mg protein)-1] 相比,也分别于运动后即刻和4h下降了37% and 39% (P<0.05), 24h持续降低(P<0.05). 用含离子载体测定的肌浆网Ca2+-ATP酶活性运动后4h降低了31%(P<0.05), 并于运动后24h仍然降低 (P<0.05)。运动后, 含与不含A23187时测定的Ca2+-ATP酶活性的比值未见显著性改变, 表明该运动没有明显改变肌浆网膜的完整性。结论：一次性低强度，长时间下坡运动导致肌浆网功能长时间降低, 运动后恢复期两天尚未完全恢复, 亦可构成离心运动诱导的骨骼肌某些功能降低的基础。提示这些变化可能产生于离心收缩时肌节长度不匀一性所造成的张力应激。     

Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.     

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P- selectin-mediated binding of platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P- selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.     

The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony- forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma- IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.     

Intravenous gammaglobulin treatment of chronic idiopathic thrombocytopenic purpura

High-dose intravenous gammaglobulin (IVIgG) was given to 12 children and adults with chronic idiopathic thrombocytopenic purpura (ITP) to avoid splenectomy or because they either failed to respond to or required maintenance with high doses of steroids and/or immunosuppressives. The average platelet count increase to initial therapy was 239,500/microliters (range 23,000-790,000). A concomitant IgG Fc receptor blockade, measured by IgG-sensitized 51Cr-labeled autologous erythrocytes, was seen in 11 of 11 patients tested, both splenectomized and not splenectomized, lasting 3-4 wk. Six or more months after treatment, 2 children are in remission, 2 children and 2 adults are stable requiring no therapy with platelet counts of approximately 50,000 and 30,000, respectively, 3 children require maintenance IVIgG therapy at 2-10-wk intervals, and 1 child and 2 adults have become refractory to further IVIgG. Splenectomy was not performed in 4 children. Two adults were able to discontinue daily prednisone. The 3 patients who became unresponsive to Swiss Red Cross gamma-globulin (IgSRK) therapy did so in conjunction with a markedly elevated platelet-associated IgG and IgM. Serum IgM increased an average of 103 mg/dl after the IVIgG infusions. No significant side effects were seen.     

Delayed activation of quiescent donor hematopoietic stem cells in the host marrow cavity by anti-host monoclonal antibody

To understand the mechanisms controlling hematopoietic engraftment in untreated, normal recipients, we investigated the fate of parental, donor hematopoietic stem cells after apparent graft failures in unconditioned F1 hybrid recipient mice. By administering an anti-host H- 2K monoclonal antibody, which targets host cells but spares the donor, we found that chimerism could be induced by delayed conditioning in animals with apparent graft failure. Engraftment kinetics in the host were followed by typing individual colony forming unit-- granulocyte/macrophage (CFU-GM) colonies for their origin and showed that parental cells, which were otherwise virtually absent, become promptly detectable within the marrow cavity after antibody administration. Marrow transfers to secondary hosts suggested that parental stem cells were present in the marrow of the untreated recipients. These findings establish that the elimination of all parental cells cannot account for the absence of peripheral blood chimerism in the unconditioned F1 hybrid recipient. Thus, viable and functional donor stem cells, which remain quiescent in the host marrow, can be activated by a selective conditioning regimen and can rescue an apparent graft failure. The selective activation in vivo of marked stem cells in an unirradiated microenvironment may be a useful system to study the regulation of cellular proliferation within the marrow cavity.     

Infusible platelet membrane microvesicles: a potential transfusion substitute for platelets

BACKGROUND: Several substitutes for intact, viable platelets have been used for transfusion, both to people and in animal models, with varied success. Infusible platelet membrane (IPM) is prepared from human platelets. IPM retains the glycoprotein (GP)lb receptor and has platelet factor 3 activity (procoagulant activity). However, factor V, serotonin, a cytoplasmic marker enzyme (purine nucleotide phosphorylase), GPIIb/IIIa complex, and HLA class I and II antigens are all absent in IPM. STUDY DESIGN AND METHODS: IPM is prepared from outdated platelets. The platelets were disrupted by freezing and thawing; they were washed and heated to inactivate possible viral contaminants, and then the sonicated membrane microvesicle fraction was separated and lyophilized. The hemostatic activity of IPM was measured by its ability to reduce the prolonged bleeding time in thrombocytopenic rabbits. RESULTS: Administration of IPM at a dose of 2 mg per kg results in a substantial reduction in the bleeding time. In a series of 23 experiments, a median preinjection bleeding time of 15 minutes was reduced to 6 minutes within 4 hours after IPM administration. Administration of IPM did show a mild enhancement in the thrombogenicity index, as measured in the Wessler rabbit model. This enhancement is, however, not significant, as a thrombogenicity index value of up to 0.6 is clinically acceptable. CONCLUSION: IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.     

体外制成复合软质再生颅骨修复材料填充犬颅骨缺损

目的:目前颅骨修补材料有很多种,但都为异源性无机骨替代物,并且应用该方法又要给患者再次行开颅手术,实验拟开展新型颅骨再生材料的研究。方法:实验于2006-05/11在解放军第一五七医院动物中心及中山大学附属第三医院动物实验室完成。①实验动物:30只犬随机分为实验组20只,对照组10只。②实验方法:应用纳米级羟基磷灰石为支架和成骨细胞培养,加入脱矿的犬类骨基质为载体的重组人类骨形成蛋白2,制成复合软质再生颅骨。实验组犬在右侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉的复合材料,左侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉和重组人类骨形成蛋白2的复合材料。对照组犬在右侧为单纯颅骨缺损,左侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉和重组人类骨形成蛋白2复合材料。实验过程中对动物处置符合动物伦理学标准。③实验评估:手术后1,2,3,6个月X射线片检查颅骨缺损修复情况,对再生的颅骨组织标本进行茜素红S染色,观察成骨能力及再生材料骨膜组织细胞体外培养情况。结果:实验动物均进入结果分析。术后1个月,成骨活跃,骨端新生骨小梁基本覆盖骨断端,缺损区可见较多新生骨小梁形成,骨端新生骨小梁向缺损区长入;术后2个月可见较多散在骨岛形成;术后3个月可见成熟骨,并有髓腔形成,缺损区大量新骨形成。而各对照组骨断端处有散在骨岛,或被增生的纤维结缔组织占据,可见大量纤维组织及毛细血管长入,植入的基质材料基本被吸收,无新骨生成。结论:应用纳米级羟基磷灰石为支架和成骨细胞培养,加入脱矿骨基质为载体的重组人类骨形成蛋白2,制成的复合软质再生颅骨能自身代谢并逐渐骨化,形成新的颅骨。