Evidence of a Phospholipid Binding Species within Human Fibrinogen Preparations

Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent K_d=5 μM, B_max=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3–6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143–210 in the alpha chain, and positions 59–77 and 101–139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.     

Kinetics of peripheral blood mononuclear cell mobilization with chemotherapy and/or granulocyte-colony-stimulating factor: implications for yield of hematopoietic progenitor cell collections

BACKGROUND: Peripheral blood progenitor cells (PBPCs) are commonly collected and used to reconstitute hematopoiesis after high-dose chemotherapy. However, strategies for optimal collection and assessment of leukapheresis components are not standardized. STUDY DESIGN and METHODS: Hematopoietic progenitor cell assays were performed on 369 leukapheresis components collected from 95 patients who had received doxorubicin-based chemotherapy and/or granulocyte-colony-stimulating factor (G-CSF). Precollection patient hematologic values, leukapheresis collection values, component hematopoietic progenitor cell assays, and patient outcome measures were summarized. The kinetics of mononuclear cell (MNC) and PBPC mobilization were assessed among four patient groups. RESULTS: Patient group was a significant predictor of the peripheral blood MNC count on the day of collection (p<0.0001), and that value was a significant predictor of granulocyte-macrophage– colony-forming unit (CFU-GM) yield (p<0.0001). This relationship between the peripheral blood MNC count on the day of collection and CFU- GM yield differed according to patient group (p<0.0001). CFU-GM made up a larger fraction of peripheral blood MNCs collected from patients who received chemotherapy plus G-CSF than collected from those who received G-CSF alone. Moreover, the peripheral blood MNC count and the corresponding CFU-GM yield increased significantly on consecutive days of collection in patient groups receiving chemotherapy and G-CSF but were unchanged or decreased in patients receiving G-CSF alone. CONCLUSION: The relationship between peripheral blood MNC count and leukapheresis component CFU-GM yield differed significantly between patients who received chemotherapy and G-CSF and those who received G- CSF alone for the mobilization of PBPCs. Patient peripheral blood MNC count and component CFU-GM yield are useful for both assessing and suggesting revisions to PBPC mobilization and collection strategies.     

预激态补骨脂素抑制 K562细胞增殖的实验研究

本研究的目的是观察预激态补骨脂素对K562细胞增殖的影响，为补骨脂素的临床应用提供实验依据。取预激态补骨脂素和晚激态补骨脂素处理的细胞，在培养后检测台盼蓝拒染细胞数和白血病细胞集落，并对它们在台盼蓝拒染细胞抑制率(TBIR)、细胞增殖抑制率(CPIR)和集落形成抑制率(CFIR)方面的差异进行比较。结果表明：预激态补骨脂素对K562细胞增殖有抑制作用；随着预激态补骨脂素浓度的增加，其抑制作用也随之增强；预激态补骨脂素与晚激态补骨脂素的TBIR、CPIR、CFIR各值比的差异不显著；为使预激态补骨脂素要充分发挥对K562细胞的抑制作用，补骨脂素的紫外线照射时间应在10分钟以上；与K562细胞作用时间也应大于12小时；抑制作用会因预激态补骨脂素预激后搁置时间的延长而下降，在6小时内作用最强。结论：预激态补骨脂素和晚激态补骨脂素对K562细胞的增殖均表现出抑制作用，有望作为临床的抗肿瘤用药。     

In vitro binding of L-triiodothyronine to receptors in rat liver nuclei. Kinectics of binding, extraction properties, and lack of requirement for cytosol proteins.

Isolated hepatic nuclei from euthyroid rats were incubated with tracer (125I)L-triiodothyronine (T3) and increasing doses of nonradioactive T3 for 30 min at 37degrees C. The T3 bound specifically to nuclear sites increased with increasing T3 doses to a plateau, which represented the nuclear binding capacity, M. Addition of 1 mM KCN, NaF, dinitrophenol, oriodoacetate did not affect nuclear binding, indicating that active metabolism was not required. Kinetic studies showed that the nuclear sites were equilibrated with T3 within 30 min of incubation (one-half maximal binding at 3 min) and that the rate of release of T3 in vitro (0.058 min-1) was the same for endogenous T3 or for T3 bound to nuclei in vitro. Nuclear T3 resisted extraction with 0.14 M NaC1 buffered at pH 7.5, but both endogenous hormone and T3 bound in vitro were readily extracted by 0.4 M KC1 at pH 8.0. The elution profiles of endogenous and in vitro-bound T3 from Sephadex G-100 columns showed a common protein peak with a molecular weight of 60-65,000, assuming a globular protein. Scatchard analysis of in vitro displacement studies showed a single class of binding sites. Mean M equals 0.23 times 10-9 M or 0.85 ng T3 for nuclei isolated from 1 g of liver. Mean M closely corresponded to that anticipated from reported in vivo studies. The apparent association constant Ka for the nuclear sites, 5.55 times 108 M-1, was lower than in studies in vivo, probably attributable to the different ionic milieu of nuclei in the incubation buffer and in the intact cell. Thus, the identity of the nuclear T3 binding sites studied in vitro to those reported for endogenous hormone is demonstrated by similar binding capacities, release rates, analogue binding affinities (previously reported), and localization to chromatin nonhistone proteins of comparable molecular weight. The role of cytosol protein in nuclear binding was assessed by comparing binding parameters for extensively washed nuclei and nuclei incubated either with contaminating or added cytosol. No difference in Ka or M was found. Moreover, it was unlikely that specific cytosol proteins were already present in nuclei and functioned during incubation as a shuttle for T3, since Ka and M for nuclei obtained from athyreotic rats were similar to Ka and M for nuclei from euthyroid animals. Thus, an initial interaction between T3 and specific cytosol proteins does not appear to be a prerequisite for translocation of T3 to nuclear sites.     

Donor screening for antibody to hepatitis B core antigen and hepatitis B virus infection in transfusion recipients

BACKGROUND: Testing for antibody to hepatitis B core antigen (anti-HBc) as a surrogate for hepatitis C viremia is no longer needed for blood donor screening. Currently, the important question is how much its use supplements hepatitis B surface antigen (HBsAg) donor screening in preventing transfusion-transmitted hepatitis B virus (HBV) infection. STUDY DESIGN AND METHODS: In a study conducted in the 1970s, 64 blood donors were associated with 15 cases of HBV (1.0%) in 1533 transfusion recipients. Sera from 61 donors at donation and 29 follow-up visits were available for present-day assays for HBsAg, HBV DNA, anti-HBc, and antibody to HBsAg (anti-HBs). RESULTS: HBsAg was found in four previously negative blood donors; HBV DNA was limited to three of these four. Anti-HBc was detected in six HBsAg-negative donors. Two other donors were negative in all assays at donation, but positive for anti- HBc and anti-HBs 2 to 4 months later. The remaining donors were negative for all HBV markers, which left five recipient cases unexplained. No HBV transmission was observed when anti-HBs sample-to- negative control values were > or = 10. CONCLUSION: Some 33 to 50 percent of cases of hepatitis B that could be transmitted by transfusion of blood from HBsAg-negative donors are prevented by anti- HBc screening. Anti-HBc-positive donors unequivocally positive for anti- HBs should be considered noninfectious for HBV and should be allowed to donate. Anti-HBc screening of paid plasmapheresis donors, supplemented by anti-HBs testing, would reduce the amount of HBV to be processed by virus inactivation and increase the content of anti-HBs in plasma pools.     

Percutaneous umbilical blood sampling and umbilical vein transfusions. Rapid serologic differentiation of fetal blood from maternal blood

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.     

Donor levels of serum alanine aminotransferase activity and antibody to hepatitis B core antigen associated with recipient hepatitis C and non- B, non-C outcomes

BACKGROUND: Hepatitis virus(es) that are neither hepatitis B (HBV) nor hepatitis C (HCV) (non-B, non-C [NBNC]) may be transmitted by transfusion. The present study assessed donor values for alanine aminotransferase (ALT) and antibody to hepatitis B core antigen (anti- HBc) for their association with HCV and NBNC hepatitis outcomes among allogeneic blood recipients. STUDY DESIGN AND METHODS: Data on blood donors and recipients enrolled in the Transfusion- Transmitted Viruses Study in four United States cities from 1974 through 1980 were supplemented by anti-HBc testing of donors and anti-HCV evaluation of recipients. Two statistical approaches estimated the value of these indirect tests in detecting donors associated with HCV seroconversion and NBNC hepatitis in recipients. RESULTS: For HCV cases, donor ALT alone (at > or = 60 IU/L) had a sensitivity and a specificity of 30 and 96 percent, respectively, and anti-HBc alone (at > or = 60% inhibition) had a sensitivity and specificity of 53 and 86 percent, respectively. The two markers combined had a sensitivity and a specificity of 69 and 83 percent. For NBNC hepatitis cases, each measure had low sensitivity (20%) that was not improved by using both (28%) [corrected]. CONCLUSION: The indirect tests proved to be equal in sensitivity to the first-generation anti-HCV tests. The positive predictive power of these indirect tests in the 1980s was sufficient to affect HCV incidence in studies during that period. Improved anti-HCV assays, however, replaced the need for indirect tests. The sensitivity of indirect tests for NBNC hepatitis contributed little.     

Humidification during high-frequency oscillation ventilation is affected by ventilator circuit and ventilatory setting

Background:  High-frequency oscillation ventilation (HFOV) is an accepted ventilatory mode for acute respiratory failure in neonates. As conventional mechanical ventilation, inspiratory gas humidification is essential. However, humidification during HFOV has not been clarified. In this bench study, we evaluated humidification during HFOV in the open circumstance of ICU. Our hypothesis is that humidification during HFOV is affected by circuit design and ventilatory settings.
Methods/Materials:  We connected a ventilator with HFOV mode to a neonatal lung model that was placed in an infant incubator set at 37°C. We set a heated humidifier (Fisher & Paykel) to obtain 37°C at the chamber outlet and 40°C at the distal temperature probe. We measured absolute humidity and temperature at the Y-piece using a rapid-response hygrometer. We evaluated two types of ventilator circuit: a circuit with inner heating wire and another with embedded heating element. In addition, we evaluated three lengths of the inspiratory limb, three stroke volumes, three frequencies, and three mean airway pressures.
Results:  The circuit with embedded heating element provided significantly higher absolute humidity and temperature than one with inner heating wire. As an extended tube lacking a heating wire was shorter, absolute humidity and temperature became higher. In the circuit with inner heating wire, absolute humidity and temperature increased as stroke volume increased.
Conclusion:  Humidification during HFOV is affected by circuit design and ventilatory settings.