Hurler and Hunter syndromes: mutual correction of the defect in cultured fibroblasts.

The biochemical defect of cultuired skin fibroblasts from Hurler or Hunter patients (faulty degradation of sulfated mucopolysaccharide, resulting in excessive intracellular accumulation) may be corrected if cells of these two genotypes are mixed with each other or with normal cells. The effect is mediated by substances released into the medium.

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

Endogenous ADP prevents PGE1-induced tyrosine dephosphorylation of focal adhesion kinase in thrombin-activated platelets.

Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.

Measuring platelet aggregation with microplate reader. A new technical approach to platelet aggregation studies.

Platelet aggregation measurements were done with the use of a commercially available microtiter plate reader with specific modification of the mode of agitation of the samples. Satisfactory aggregation curves were obtained with use of an external horizontal agitator, with an amplitude of 1.3 mm and minimum frequency of 1,360 cycles/minute. With the use of the 96 available wells in the microtiter plates, all test and control platelet samples, with replicates, were observed simultaneously and the output data obtained within 10-15 minutes. The technique was validated by demonstrating the similarity of dose-response curves, obtained with a standard aggregometer and with the microtiter technique, of platelets stimulated by adenosine diphosphate, thrombin, and arachidonic acid.

Aberrant morphology of platelets stored in five day containers.

Platelets for transfusion, stored in either of two new types of container (PL-732 and CLX), demonstrated unusual morphological alterations after 2 or 3 days of storage. The atypical forms observed included crescents, elongated tubular forms and rings. Development of these forms was not seen if the permeability of the container was inhibited and the pH kept below 6.7. The variables which differentiate these new containers from those previously used, and which appear to be related to the changes described, are pH, pO2 and presence of a leachable plasticizer. The functional behaviour of these platelets, as assessed by serotonin uptake and resistance to hypotonic shock, was not different from that of control platelets.

Ouabain affects platelet reactivity as measured in vitro.

Ouabain, a digitalis glycoside and an inhibitor of the Mg2+-dependent Na+-K+ ATPase, was used to probe the role of intracellular Na+ levels in the regulation of platelet reactivity. Platelets preincubated with 10 to 150 microM ouabain exhibited a potentiated aggregation response to collagen (14.4 to 180 micrograms/mL), ADP (4 to 12 microM) and thrombin (0.03 to 0.10 unit/mL). Ouabain markedly decreased the time interval between addition of collagen and the onset of shape change. At submaximal concentrations of collagen, thrombin and ADP, preincubation with ouabain increased the rate and amplitude of the aggregation response. Irreversible aggregation was achieved in ouabain-treated platelets by using concentrations of ADP which induced only reversible aggregation in the absence of ouabain. In addition, chelation of extracellular calcium with EGTA or EDTA (2 mM) failed to block reactivity to collagen, ADP or thrombin in ouabain-treated platelets. These results suggest that ouabain induces a "preactivation state" in platelets, perhaps via modulation of intracellular Na+ levels.

Red blood cell substitutes: evolution of approaches to demonstrating efficacy.

There has been a striking advancement in our understanding of red cell substitutes over the past decade. Throughout this period, regulatory oversight influenced many aspects of product development. The approach to demonstrating efficacy was an important example. I will review some events of the decade so that we may consider the question, "If we had it to do over again, could we do any better?" At the start of the decade, there were sufficient data on the mechanism of hemoglobin-induced renal toxicity to reach consensus among investigators that the content of alpha beta-dimers in any hemoglobin product must be strictly limited. An array of additional, unexpected reactions were seen in early clinical studies, reactions that led to several conclusions: hemoglobin is a pharmacologically active material; more basic studies are needed; and it would all be so much easier if communications between investigators were open and unhindered. Meaningful studies of efficacy could not be approached until the safety issues had been solved or until the community was convinced that they were not serious. The initial approach was based on the meager experience gained with previous products, e.g., Fluosol. That experience led to policies which required that, for example, if the product was to be used as a red cell substitute, it must be compared with red cells, and if it was to be used as an oxygen carrier, it must be shown to support organ function. Endpoints of clinical trials were based on delivery of a clear clinical benefit to the patient--surrogate endpoints could be used only if their relationship to clinical benefit had been demonstrated.

Econazole inhibits thapsigargin-induced platelet calcium influx by mechanisms other than cytochrome P-450 inhibition.

Cytochrome P-450 has been suggested as a mediator of the signal between depleted platelet calcium stores and an increase in plasma membrane permeability to calcium which follows depletion of the stores. This hypothesis is based on the observations that inhibitors of cytochrome P-450, such as the imidazole antifungal agents, also inhibit influx of a calcium surrogate (manganese) into calcium-depleted platelets. We tested the effects of econazole and of a cytochrome P-450 inhibitor, carbon monoxide (CO), on thapsigargin (TG)-induced platelet 45Ca2+ influx. TG specifically depletes internal calcium stores and activates store-regulated calcium influx. Econazole blocked 45Ca2+ influx when it was added before TG (IC50 11 microM). Econazole at a concentration (20 microM) that inhibited 83% of TG-induced calcium influx was not inhibitory to TG-induced calcium efflux from 45Ca(2+)-loaded platelets, and did not affect calcium fluxes in resting platelets. This econazole concentration was also inhibitory to calcium influx even when it was added after the stores had been calcium-depleted by EGTA and TG for 15 min and the signal to increase calcium influx had already been generated. Inhibition of cytochrome P-450 with CO bubbled through platelet suspensions did not change calcium influx in resting cells and potentiated TG-induced calcium influx (160% of control calcium accumulation at 20 min). This effect appeared to be concentration-dependent, such that a 5 min exposure to CO produced a greater influx potentiation than a 3 min exposure. These observations indicate that (1) cytochrome P-450 does not mediate store-regulated calcium influx, and (2) econazole probably inhibits store-regulated calcium influx by an alternative mechanism, such as interaction with plasma membrane calcium channels.

Dimethyl sulfoxide: effects on function of fresh platelets and on the viability of platelets in storage.

Dimethyl sulfoxide (DMSO) is used as a cryoprotective agent when platelets are frozen. We examined the effect of DMSO (0.1 to 10%) on platelet aggregation, release, and prostaglandin synthesis (as indicated by malondialdehyde formation) in response to thrombin, collagen, arachidonic acid and calcium ionophore. Inhibition was observed at the lowest levels of DMSO, varied with the type of stimulus, and was reversed by washing the platelets. Inhibition of aggregation, release, and malondialdehyde formation were dose-dependent with thrombin or collagen. DMSO did not inhibit malondialdehyde formation stimulated by arachidonic acid, nor did it consistently inhibit any function stimulated by calcium ionophore. When platelets were stored as platelet-rich plasma at 20 to 24 degrees C for 48 hours, with and without 5 percent DMSO, and subsequently washed, the platelets stored with DMSO were more reactive in vitro. These results indicate that platelet function inhibition by DMSO not only is reversible, but protects the platelets during storage. The factor limiting the use of DMSO in platelet storage is potential systemic toxicity, not its effects on platelets.

Oxidation reactions of human, opossum (Didelphis virginiana) and spot (Leiostomus xanthurus) hemoglobins: a search for a correlation with some structural-functional properties.

1. Relative to human HbA, opossum (Didelphis virginiana) hemoglobin was found to be more susceptible to autoxidation. While the initial rate of autoxidation of spot (Leiostomus xanthurus) hemoglobin is close to that of HbA, complete oxidation occurs in 50 hr. 2. Direct addition of hydrogen peroxide (H2O2) induced oxidation of hemoglobins in a definite order: spot Hb > HbA > opossum Hb. Excess H2O2 led to heme degradation and precipitation that occurred much faster for spot Hb than the case with other proteins. 3. Exposure of hemoglobins to a continuous flux of H2O2, generated by the glucose/glucose oxidase system, induced the formation of heterogeneous protein-associated oxidation products. 4. Differential reactivity among these hemoglobins under the same or different oxidative conditions, with respect to methemoglobin formation and stability of the ferric form, may reflect the differences in the local heme environment of these proteins.

Heparin-induced thrombocytopenia: confirmation of diagnosis with in vitro methods.

Profound thrombocytopenia developed in a patient during treatment with heparin for venous thrombosis. The platelet count increased toward normal when heparin administration was stopped, but fell abruptly when the drug was again given. Platelet aggregation occurred when heparin was added to the patient's platelet-rich plasma, or to normal platelets plus the patient's serum. This serum also effected release of 3H-serotonin from normal platelets. This pattern of aggregation was clearly different from that occasionally caused by heparin in a control population. The data is consistent with an effect of heparin on platelets, possibly mediated by on immune mechanism.

The defect in Hurler and Hunter syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation.

Cultured fibroblasts, derived from patients with the Hurler and Hunter syndromes, show defective degradation of sulfated mucopolysaccharide. The aberrant metabolism of Hurler cells can be corrected by secretions of fibroblasts of genotype other than Hurler, and similarly, the defect of Hunter cells can be corrected by secretions of fibroblasts of genotype other than Hunter. The active factors in these secretions, which are heat labile and associated with macromolecules, accelerate the degradation of mucopolysaccharide.

Quantitative assessment of platelet morphology by light scattering: a potential method for the evaluation of platelets for transfusion.

Normal fresh platelets are discoid, and platelets that have been stored become spherical. Discoid morphology correlates with normal viability and imparts to a platelet suspension a "streaming" appearance when agitated. We developed a device that quantitates the percent of discoid cells in a suspension by means of laser light scattering at low angles. It performs this assay within 3 minutes on platelets contained in a plastic blood bag. The ability of the device to quantitate discoid platelets was demonstrated in the following ways: (1) comparison of percentage discs calculated from light-scattering data with visually observed optical streaming; (2) correlation of percentage discs from light scattering with the percentage determined by phase microscopy; and (3) observation of changes during room temperature storage. Platelet suspensions containing extreme (dendritic and balloon) forms were examined, and we conclude that these forms do not significantly affect the light-scattering assay. The optical measurements were also used to accurately approximate the total platelet counts of the suspensions. This device may have a role in the evaluation of platelets for transfusion.

Survey on the current use of leukapheresis and the collection of granulocyte concentrates.

This study was designed to define leukapheresis practice. A voluntary questionnaire on leukapheresis was sent to 280 FDA-registered blood-collecting establishments performing leukapheresis. Of the facilities questioned, 67.9 percent responded. The survey results indicate that most facilities use intermittent-flow blood cell separators, while 22.6 percent use more than one separation method. Establishments routinely use 6% hydroxyethyl starch (HES 450/0.70) and the majority using trisodium citrate as the anticoagulant. Forty-eight percent use corticosteroids, primarily dexamethasone, to pretreat the donor. The frequency of donation was not specified by 25.3 percent of the report. Forty-two percent chose an individual donation frequency of two times per week. A limit on the total number of donations allowed per donor was not specified by 78.4 percent of the facilities. Community blood banks (including regional centers) performed 55.6 percent of all leukocyte concentrate collections. The donor reaction incidence was of 3.64 percent. Hospitals, of all types, performed 37.8 percent of the collections. The adverse reaction rate ranged from 2.84 to 9.72 percent. Adverse reactions occurred in donors 54.9 times per 1000 procedures. Ninety-four percent of reported reactions were mild, whereas moderate and severe reactions accounted for 5.9 and 0.4 percent, respectively. Granulocyte yields varied by the type procedure and the use of corticosteroids as well as among facilities. The majority (56.3%) held leukocytes at 22 to 25 degrees C prior to transfusion, while most of the remainder stored at 4 degrees C.

Redox reactivity of modified hemoglobins with hydrogen peroxide and nitric oxide: toxicological implications.

The rapid unloading of oxygen to tissue and the prevention of subunit dissociation have been the main concerns in the search for an effective hemoglobin-based red cell substitute. The presence of redox active iron however, raises some questions about its potential to enter into reactions that mediate the formation of cytotoxic oxygen free radicals. We tested the propensity of modified hemoglobins to undergo oxidative damage by peroxide (H2O2). We found differences in their susceptibility to oxidative modification and in their ability to form the highly cytotoxic ferryl species. This protein-associated oxidant may be a physiologically important contributor to reperfusion injury. Another potential mechanism of toxicity involves the reaction of cell-free hemoglobin with endothelium derived nitric oxide (NO). Marked hypertensive responses in intact animals infused with some of these hemoglobins were reported. Cell-free hemoglobin has the potential to bind the endothelial generated NO yielding methemoglobin and nitrate, an extremely rapid reaction in vivo. We describe subsequent redox reactions between NO and methemoglobin which may further deplete NO as a biological transducer, leading to greater effects on the extent of endothelial-dependent responses. The consequences of a potential linkage between oxidative toxicity of cell-free hemoglobin and its interaction with NO is addressed.