Introduction: leukocyte-reduced blood components.
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Blood Component Transfusion↗
Biomedical subjects
T A Lane
Publications and source records attributed to T A Lane.
Phagocytosis of a fluorescently labeled perflubron emulsion by a human monocyte cell line.
We hypothesized that fluorocarbon-based lipid emulsions are phagocytosed by monocytes and that many of the in vivo side effects related to the infusion of these particulate emulsions are due to release of cytokines by these monocytes. To clarify whether these emulsions are actually phagocytosed we attempted to measure by flow cytometry the apparent uptake of a fluorescently labeled high-concentration (90%, w/v) perflubron (perfluorooctyl bromide [PFOB]) emulsion by a differentiated human monocyte cell line. A fluorescent chromophore (Zynaxis Cell Science) was used to label the egg yolk phospholipid in a perflubron emulsion. This phospholipid label was used to track the perflubron emulsion during overnight incubation with the human monocyte (THP-1) cell line which had been differentiated, by exposure to PMA, into macrophage-like cells. Our results indicate that after 24 hours of incubation with the labeled perflubron emulsion, 64.9% (+/- 11.0) of differentiated THP-1 cells had cell-associated emulsion (ingested and/or membrane bound) whereas 24.4 (+/- 6.8%) of the control cells had cell-associated emulsion. We speculate that this technique may be a useful method to track the intravascular persistence and extravascular distribution of such emulsions, and that the degree of uptake of the emulsion by macrophages in this assay may correlate with its in vivo half life.
Cell Line↗
Leukocyte reduction of cellular blood components. Effectiveness, benefits, quality control, and costs.
Cellular blood components contain passenger donor leukocytes. Transfusion of passenger leukocytes may be associated with alloimmunization to leukocyte antigens, febrile transfusion reactions, refractoriness to platelet transfusion, severe pulmonary dysfunction, graft-vs-host disease, the transmission of infectious diseases, and immune modulation. Advanced leukocyte-reduction filters enable the removal of up to 99.9% of leukocytes from cellular blood components. Clinical trials suggest that the use of leukocyte-reduction filters may prevent or diminish the probability of febrile transfusion reactions, alloimmunization, and cytomegalovirus infection, but controversy exists regarding the effectiveness of leukocyte reduction in preventing immune modulation. There is no evidence that available techniques will prevent graft-vs-host disease. Cost-benefit analyses support the use of leukocyte-reduction filters for well-defined indications. Standards for leukocyte reduction of red blood cells have been defined, but issues regarding the quality control of leukocyte-reduced blood components require additional study.
Cost-Benefit Analysis↗
Leukocyte depletion of cellular blood components.
Clinical studies have indicated that the use of leukocyte-reduced cellular blood components produced in the laboratory may prevent febrile reactions and delay or prevent alloimmunization to HLA antigens and refractoriness to platelet transfusion. Additional investigations regarding the effects of the use of leukocyte-reduced blood components were reported during the past year. A recent study in patients with hematologic malignancy that employed the commonly used bedside leukocyte-reduction filters failed to confirm a decrease in the rate of alloimmunization, except in a subgroup of patients with acute myelogenous leukemia. Another major multicenter trial confirmed the effectiveness of leukocyte-reduced blood components in the prevention of cytomegalovirus infection. The effect of allogeneic leukocytes in transfused blood on immune function in patients undergoing colorectal surgery continues to receive attention. Whereas one study failed to demonstrate an adverse effect of standard blood components on disease recurrence or survival, a second study demonstrated a marginally significant decrease in infectious complications in patients who received only leukocyte-reduced blood. Increasingly efficient leukocyte-reduction filters have been developed for cellular blood components, many of which are best suited for laboratory filtration of unstored blood. Laboratory studies indicate that prestorage leukocyte-reduction of cellular blood components does not impair erythrocyte or platelet function and will not increase the incidence of microbial contamination of blood. New methods that employ flow cytometry should enable improved quality control of blood components rendered leukocyte-reduced by the newer, more efficient filters. Finally, a cost-benefit analysis suggests that the appropriate use of leukocyte-reduction filters for acute leukemia patients may reduce the cost of health care to these patients.
Blood Component Transfusion↗
Hepatitis B in a prenatal population.
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California↗
Current trends: evolving concepts in transfusion medicine. The need for standardization of cryoprecipitate-derived fibrin adhesive.
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Blood Transfusion↗
Effect of high concentration perflubron emulsion on platelet function.
Perfluorocarbon (PFC) and lipid emulsions (eg. Fluosol, Intralipid,) containing phospholipid have been reported to modify platelet function after intravenous infusion. Platelet activation might be responsible for the generation of mediators responsible for PFC-induced side effects. In view of this, we investigated the effect of a highly concentrated perfluorocarbon emulsion, containing 90% (w/v) perfluorooctylbromide (perflubron,;PFOB), on porcine and human platelet activation and function. We measured both (1) stimulated ex-vivo porcine platelet aggregation pre and post infusion of either perflubron or control (vehicle only) emulsions and (2) stimulated in vitro human platelet calcium flux in the presence of perflubron emulsion or control emulsions. Platelet aggregation stimulated by collagen, ADP or arachadonic acid (AA) was inhibited in ex-vivo porcine platelets following infusion of perflubron emulsion at a dose of 3 ml/kg (0.2 ml/kg/min). Inhibition was dose-dependent and was decreased when the dose of perflubron emulsion was reduced to 1.0 or 0.3 ml/kg. Infusion of saline or "vehicle" emulsions had little or no effect on stimulated ex-vivo pig platelet aggregation. Fluosol infusion was associated with inconsistant inhibition of platelet aggregation. A23187- or AA-stimulated calcium flux of human platelets in vitro was inhibited in the presence of 1% (v/v) perflubron emulsion. Similar effects were seen with Fluosol, or Intralipid. This inhibition was agonist dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Leukocyte reduction in blood component therapy.
PURPOSE: To review methods of preventing or minimizing the adverse effects associated with the transfusion of passenger leukocytes present in cellular blood components and to define groups of patients who are at risk for adverse effects. DATA SOURCES: English-language articles on transfusion medicine. STUDY SELECTION: Original reports describing the pathogenesis of leukocyte-induced adverse effects in transfusion recipients and the influence of leukocyte-reduced blood components on these effects. DATA EXTRACTION: Evaluation of the diagnosis, transfusion history, and treatment of the study patients; the methods and results of leukocyte reduction; and specific outcomes, including development of alloimmunization to leukocytes, febrile reactions to transfusion, and platelet refractoriness. DATA SYNTHESIS: Passenger leukocytes are the chief cause of alloimmunization to human leukocyte antigen (HLA) and leukocyte-specific antigens in transfusion recipients. Alloimmunization may result in febrile transfusion reactions, platelet refractoriness, and acute lung injury. Leukocytes are also the vector for transfusion-associated cytomegalovirus infection. Technologic advances in the leukocyte reduction of cellular blood components have made it possible to reduce the number of leukocytes to fewer than 10(7) per transfusion. Findings suggest that the use of leukocyte-reduced cellular blood components may minimize or prevent recurrent febrile reactions and alloimmunization to leukocyte antigens. Cytomegalovirus may not be transmitted by blood components containing fewer than 10(7) leukocytes. CONCLUSIONS: Leukocyte reduction in red blood cell and platelet transfusions using third-generation filters is indicated for selected patients who are likely to receive long-term transfusion support, to prevent recurrent febrile reactions and to prevent or delay alloimmunization to leukocyte antigens. Leukocyte-depleted transfusions may also be indicated to delay or prevent refractoriness to platelet transfusion.
Blood Component Transfusion↗
Reducing the risk for transfusion-transmitted cytomegalovirus infection.
OBJECTIVE: To define the groups of patients at risk for transfusion-transmitted cytomegalovirus infection and to define the methods to reduce this risk. DATA SOURCES: English-language publications on transfusion medicine. STUDY SELECTION AND DATA EXTRACTION: Studies were selected that described cytomegalovirus infection in transfusion-dependent patients. Special attention was paid to reports that included observations about the prevalence and clinical manifestations of cytomegalovirus infection and recommendations for the prevention of infection. DATA SYNTHESIS: Some patients with impaired immune responses who have never been exposed to cytomegalovirus are at risk for transfusion-transmitted cytomegalovirus infection. This infection, which is associated with substantial morbidity and mortality, can be avoided by additional screening of blood donors or by special processing of components for transfusion. CONCLUSIONS: Transfusion products that are unlikely to transmit cytomegalovirus infection can be prepared by filtration to remove leukocytes or can be obtained by selecting donors who are seronegative for antibodies to cytomegalovirus. These products are indicated for certain groups of immunosuppressed patients, including pregnant women who are cytomegalovirus seronegative, premature infants of low birth weight who are born to cytomegalovirus-seronegative mothers, cytomegalovirus-seronegative recipients of allogeneic bone marrow transplants from cytomegalovirus-seronegative donors, and cytomegalovirus-seronegative patients with the acquired immunodeficiency syndrome (AIDS).
Cytomegalovirus Infections↗
Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.
Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.
Animals↗
The effect of peritoneal macrophage-derived factor(s) on ovarian progesterone secretion and LH receptors: the role of calcium.
Macrophages and their secretory products, cytokines, play an integral role in many reproductive processes. In this study we examined the effect of conditioned media from cultured human peritoneal macrophages on progesterone production by granulosa cells and the role of calcium in this process. Macrophages were pretreated with various concentrations of a calcium channel blocker (verapamil) or a calcium ionophore (A23187). Macrophage-conditioned media (MCM) or cell-free media that contained calcium channel modifiers were added at three dose levels to cultured porcine granulosa cells. Progesterone production and LH receptor content were determined. Macrophage-conditioned media alone elevated basal progesterone production, but significantly attenuated granulosa cell LH receptor content. These effects were neither potentiated nor suppressed by pretreating macrophages with verapamil. However, production of the LH receptor lowering factor(s) appeared to be suppressed by calcium ionophore. We conclude that (1) one or more factors produced by macrophages have a net stimulatory effect on basal progesterone production and these factor(s) may not be calcium-dependent and (2) macrophage-derived secretory products reduce granulosa cell LH receptor content. The production of these factor(s) may be calcium-dependent.
Animals↗
Hypoxia induces endothelial cells to increase their adherence for neutrophils: role of PAF.
We investigated the interactions of polymorphonuclear neutrophils (PMN) and endothelial cells in myocardial ischemia using a hypoxia model. We exposed porcine aortic (PAEC) and porcine coronary microvessel (PCMEC) endothelial cells to 2% O2 for 2 h (PO2 = 53 mmHg) and measured the adherence of unstimulated neutrophils (PMN) to both control and hypoxia-conditioned endothelial cell monolayers. Hypoxia conditioning increased PMN adherence to PAEC and PCMEC by 51 and 101%, respectively, above control levels. The increase in PMN adhesion to PAEC was associated with a threefold increase in endothelial cell-associated platelet-activating factor (PAF) compared with control PAEC. The conditioned media from PAEC exposed to hypoxia also contained sixfold more PAF than control conditioned media, and it activated PMN to become adherent to untreated PAEC. The hypoxia-induced PAEC adhesion response was inhibited by preincubating PMN with the specific PAF receptor antagonist, L-659,989. We conclude that PAF is produced by cultured endothelial cells in response to hypoxia and that PAF generation is chiefly responsible for the increased adherence properties of hypoxia-conditioned endothelial cells. This response may play a major role in regulating PMN margination during myocardial ischemia.
Animals↗
Effect of a high concentration perfluorocarbon emulsion on platelet function.
Perfluorocarbon (PFC) and lipid emulsions (eg. Fluosol, Intralipid,) containing phospholipid have been reported to modify platelet function after intravenous infusion. Platelet activation might be responsible for the generation of mediators responsible for PFC-induced side effects. In view of this, we investigated the effect of a highly concentrated perfluorocarbon emulsion containing 90% (w/v) perfluorooctylbromide (perflubron; PFOB), on porcine and human platelet activation and function. We measured both: a) stimulated ex-vivo porcine platelet aggregation pre and post infusion of either perflubron or control (vehicle only) emulsions, and b) stimulated in vitro human platelet calcium flux in the presence of perflubron emulsion or control emulsions. Platelet aggregation stimulated by collagen, ADP or arachidonic acid (AA) was inhibited in ex-vivo porcine platelets following infusion of perflubron emulsion at a dose of 3 ml/kg (0.2 ml/kg/min). Inhibition was dose dependent and was decreased when the dose of perflubron emulsion was reduced to 1.0 or 0.3 ml/kg. Infusion of saline or "vehicle" emulsions had little or no effect on stimulated ex-vivo pig platelet aggregation. Fluosol, infusion was associated with inconsistent inhibition of platelet aggregation. A23187- or AA-stimulated calcium flux of human platelets in vitro was inhibited in the presence of 1% (v/v) perflubron emulsion. Similar effects were seen with Fluosol, or Intralipid. This inhibition was agonist dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Variation in blood component irradiation practice: implications for prevention of transfusion-associated graft-versus-host disease.
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Blood Cells↗
Heterologous down-modulation of luteinizing hormone receptors by prolactin: a flow cytometry study.
Although the binding, internalization, and regulation of LH, PRL, and their respective receptors have been extensively studied, it is not known whether the receptors are coordinately regulated. Using double labeling experiments, we have previously shown that receptor-bound LH and PRL can be colocalized in identical endosomes of granulosa cells. We hypothesize that high levels of PRL may induce a heterologous down-modulation of LH receptors, consequently reducing ovarian responsiveness to further gonadotropin stimulation. In this study we used a novel procedure to enrich endosomes containing internalized PRL and to determine whether unoccupied LH receptors were cointernalized in granulosa cells. Porcine granulosa cells were obtained from medium-sized (3-5 mm) follicles and cultured for 4 days in the presence of FSH. Fluorescein isothiocyanate-labeled PRL (FITC-PRL) was used as a ligand to induce internalization of PRL receptors and as a marker to label endosomes. Granulosa cells were incubated with FITC-PRL at either 4 or 37 C for various times. At the end of the incubation, cells were trypsinized to remove surface receptors and then homogenized. The postnuclear fraction containing endosomes and other subcellular organelles was sorted using a FACStar Plus cell sorter. Results from 14 separate sorting experiments showed that 1) FITC-PRL-treated cells exhibited a sorting pattern distinct from that of FITC-BSA-treated or untreated cells; 2) excess unlabeled PRL partially shifted the sorting profile to one similar to that in controls; 3) the differences in sorting profiles were not due to free FITC; and 4) using this method, it was possible to isolate FITC-PRL-containing endosomes that were virtually devoid of other contaminating subcellular particles. Fluorescently positive (FITC-PRL-containing) organelles were collected and assayed for LH receptors using [125I]hCG as a tracer. When the cells were incubated with FITC-PRL at 37 C for 3 h, the number of available LH receptors (as determined by [125I]hCG binding) was 37% higher in particles containing FITC-PRL than in those devoid of FITC-PRL. If the cells were allowed to preincubate with FITC-PRL at 4 C for 10-16 h before raising the temperature to 37 C, the number of available LH receptors in FITC-PRL-containing endosomes was about 7-fold higher than that in FITC-negative endosomes. Results from this study suggest that PRL not only induces internalization of its own receptor, but also causes down-modulation of unoccupied LH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Protein kinase C inhibitors block the enhanced expression of intercellular adhesion molecule-1 on endothelial cells activated by interleukin-1, lipopolysaccharide and tumor necrosis factor.
Interleukin 1 (IL-1), bacterial lipopolysaccharide (LPS) and tumor necrosis factor (TNF alpha) enhance the adherence properties of endothelial cells (EC) for neutrophils (PMN). This is mediated in part by the up-regulation of Intercellular Adhesion Molecule 1 (ICAM-1) on EC. Phorbol esters, which activate protein kinase c (PKC) and enhance the adherence properties of EC for PMN also up-regulate the ICAM-1 expression on EC. We investigated the effect of PKC inhibitors on ICAM-1 expression of human umbilical vein EC (HUVEC). Staurosporine (STS) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented inflammatory mediator-dependent stimulation of both ICAM-1 expression and PMN adherence by HUVEC (ID50 for STS = 2.7-2.9 microM; for H-7 = 7.6-8.8 microM). Inhibition was dose and time-dependent and was not due to HUVEC toxicity. The STS analog K252a and the H-7 analog W-7 were less potent inhibitors of ICAM-1 up-regulation and adherence promotion. Prolonged exposure of HUVEC to phorbol myristate acetate down-regulated PKC activity and inhibited subsequent ICAM-1 up-regulation by this agent and by IL-1. We conclude that inflammatory mediator induced stimulation of HUVEC expression of ICAM-1 and promotion of adherence properties are mediated in part by activation of PKC.
Cell Adhesion↗
Granulocyte storage.
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Blood Component Transfusion↗
Effects of cytokines on porcine granulosa cell steroidogenesis in vitro.
Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.
Animals↗