Furosemide inhibits regenerative cortical spreading depression in anaesthetized cats

Ionic perturbations occur during cortical spreading depression (SD), a phenomenon implicated in migraine pathophysiology. We studied the effect of 0.2,2 and 20 mg kg⁻¹ iv ( n =4) furosemide on cortical direct current (d.c.) potential, cerebrovascular laser Doppler flux (rCBF_LDF), artery diameter and NO concentration in the parietal cortex of the anaesthetized cat during repetitive SD. In vehicle treated animals ( n =4), SD activity was sustained for 50 1.8 min. However, duration of SD activity was significantly reduced when compared to vehicle to 39 6.6 ( n =4), 3.1 8.3 ( n =4) and 27.3 11.3 min ( n =4), at 0.2, 2 and 20 mg kg⁻¹ iv furosemide respectively. It is hypothesized that the mechanism of inhibition of SD d.c. activity by furosemide may be through alterations in cortica ion buffering capacity or inhibition of cell swelling in neurones or glia. These mechanisms may represent potential novel drug targets in future migraine therapy.     

A prospective, randomized study of the use of platelet concentrates irradiated with ultraviolet-B light in patients with hematologic malignancy

BACKGROUND: Irradiation of platelet concentrates (PCs) with ultraviolet- B (UVB) light inactivates the contaminating white cells and might be an alternative to filtration for the prevention of alloimmunization to HLA antigens and subsequent refractoriness to further platelet transfusions in multiply transfused patients with bone marrow failure. STUDY DESIGN AND METHODS: Patients with hematologic malignancy, mainly acute myeloid leukemia, were prospectively assigned in a random manner to receive either UVB-irradiated or control, nonirradiated PCs. All patients were given red cells that were white cell reduced by filtration. Transfusion efficacy and alloimmunization were assessed by means of corrected count increments, requirement for red cells and PCs, and measurement of lymphocyte-reactive antibodies. RESULTS: UVB-irradiated PCs had a clinical efficacy similar to controls as judged by corrected count increments at 1 to 6 and 12 to 24 hours and by the median requirement for red cell and platelet transfusions. Alloimmunization determined by measurements of lymphocyte-reactive antibodies using both conventional and antiglobulin-augmented lymphocytotoxicity techniques was not abolished in recipients of UVB-irradiated PCs (4/30, 13%) but was less than that in controls (5/20, 25%; p = NS). The mean number of platelet transfusion episodes prior to the occurrence of alloimmunization was greater in the control group (27 vs. 10; p = 0.017). CONCLUSION: In this trial, UVB irradiation did not diminish the clinical efficacy of platelet transfusions. There was a small but nonsignificant reduction alloimmunization, but no difference in refractoriness of the two groups was observed. Larger prospective randomized studies are required to confirm these findings and to compare UVB irradiation with white cell reduction.     

Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies

Antineutrophil cytoplasmic autoantibodies (ANCA) have been described in sera of patients with several forms of systemic vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. The two main targets of ANCA in vasculitis are proteinase 3 (PR3) and myeloperoxidase (MPO). ANCA are capable of activating neutrophils primed by tumor necrosis factor-alpha (TNF-alpha) in vitro, which may be relevant for the induction of the vascular inflammation observed in vivo. Recently, it has been suggested that engagement of Fc gamma receptor IIa (Fc gamma RIIa) on the neutrophils is involved in the activation by ANCA. In the present study, we show that activation of the neutrophil respiratory burst by anti-PR3 and anti-MPO is strongly enhanced after TNF priming and lost on removal of the Fc parts of the antibodies. Similar results were obtained when the neutrophils were activated with antibodies against known membrane antigens without major changes in the expression of the target antigens. The TNF-induced enhancement of the neutrophil activation was not observed when adherence of the cells was prevented by continuous stirring of the suspension or by the addition of CD18 antibodies before TNF exposure. Hence, our results indicate that engagement of both Fc gamma RIIa and beta 2 integrins is instrumental in neutrophil activation induced by ANCA.     

A functional comparison of CD34 + CD38- cells in cord blood and bone marrow

We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as > 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.     

An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal

To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light- density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.     

Oral iloprost in Raynaud's phenomenon secondary to systemic sclerosis: a multicentre, placebo-controlled, dose-comparison study

OBJECTIVE: To identify the optimal dose of oral iloprost on the basis of efficacy and tolerability in patients with Raynaud's phenomenon secondary to systemic sclerosis. DESIGN: Multicentre, randomized, parallel-group comparison of two different doses of oral iloprost and placebo. SETTING: European university hospitals. PATIENTS: A total of 103 patients with Raynaud's phenomenon secondary to systemic sclerosis. INTERVENTION: Patients received one of three treatments for 6 weeks: placebo, oral iloprost 50 microg or oral iloprost 100 microg. Each treatment was taken twice daily, giving total daily doses of iloprost of 100 and 200 microg. MEASUREMENTS: The frequency, total daily duration and severity of Raynaud's attacks were recorded in a specially designed patient diary; physician's global assessment and adverse events were recorded at visits to the clinic. Analysis was performed on an intention-to-treat population. RESULTS: A total of 103 patients were recruited, 89 completed the assessments throughout the treatment period and 82 completed an additional 6 weeks of follow-up after treatment. Thirty-five patients received placebo, 33 received iloprost 50 microg and 35 received iloprost 100 microg. The mean percentage reductions in the frequency, total daily duration and severity of Raynaud's attacks were numerically greater in the iloprost groups at the end of treatment and at the end of follow-up. At the end of treatment (6 weeks), there were significant treatment differences in the total daily duration of attacks (P = 0.03), but not in the severity (P = 0.07) or the frequency of attacks (P = 0.37). At the end of follow-up (12 weeks), there were significant treatment differences in the total daily duration of attacks (P = 0.001) and in the severity of attacks (P = 0.007), but not in the frequency of attacks (P = 0.07). Percentages of patients improved at the end of treatment as assessed by the physician were 44% placebo, 57% iloprost 50 microg and 64% iloprost 100 microg (not significant). Side-effects were reported by 80% of patients on placebo, 85% on oral iloprost 50 microg and 97% on oral iloprost 100 microg. Premature discontinuations of treatment in each group were 9, 30 and 51%, respectively, with 6, 27 and 51% being due to adverse events. CONCLUSION: The results on the daily duration of Raynaud's attacks suggest that both 50 and 100 microg oral iloprost twice daily may be effective in the treatment of Raynaud's phenomenon secondary to systemic sclerosis. The 50 microg iloprost dose was better tolerated in this patient group.      

A distinct profile of six soluble adhesion molecules (ICAM-1, ICAM-3, VCAM-1, E-selectin, L-selectin and P-selectin) in rheumatoid arthritis

Soluble forms of ICAM-1, VCAM-1, E-selectin, L-selectin, P-selectin and, more recently, ICAM-3 are known to exist in human serum and have elevated levels in numerous diseases. Previous studies have demonstrated that in rheumatoid arthritis (RA) the levels of circulating sICAM-1 and sE-selectin are elevated relative to healthy controls. We have compared the serum profiles of these six soluble adhesion molecules in patients with RA (n = 22) to those seen in healthy controls (n = 10) using sandwich ELISA. In the patients, there were significant elevations of serum sICAM-1 (P < 0.0001), sICAM-3 (P = 0.0327), sVCAM-1 (P = 0.0025), sL-selectin (P = 0.0194) and sP-selectin (P = 0.0025), but not E-selectin (P = 0.0672). However, only sP- selectin was found to correlate with disease activity in the patients (r = 0.461, P < 0.05). Thus, there is a distinct profile of soluble adhesion molecules in RA of which only sP-selectin correlates with disease activity.      

Marrow transplantation for acute lymphoblastic leukemia: factors affecting relapse and survival

Transplant outcome was analyzed in 690 recipients of bone marrow transplants (BMTs) for acute lymphoblastic leukemia (ALL) in first (n = 299) or second remission (n = 391). Actuarial 5-year leukemia-free survival was 42% +/- 9% (95% confidence interval) and 26% +/- 6%, respectively; relapse rates were 29% +/- 9% and 52% +/- 8%, respectively. Five-year leukemia-free survival was 56% +/- 18% in children and 39% +/- 10% in adults (P less than .02) transplanted in first remission. In first-remission adults, non-T-cell phenotype, male to female donor-recipient sex-match and graft-v-host disease (GVHD) were associated with decreased leukemia-free survival; inclusion of corticosteroids in the regimen to prevent GVHD was associated with increased leukemia-free survival. Variables associated with decreased leukemia-free survival after second-remission transplants were age greater than or equal to 16 years and relapse occurring while on therapy. Variables associated with increased probability of relapse were similar for first- and second-remission transplants and included GVHD prophylaxis without methotrexate and absence of GVHD. In first- remission transplants, leukocyte count greater than or equal to 50 x 10(9)/L at diagnosis was also associated with increased relapse; in second remission, relapse while receiving chemotherapy was also associated with increased posttransplant relapse. These data emphasize the importance of both disease- and transplant-related variables in predicting outcome after BMT. They may be used to explain differences between studies, design future trials, and identify persons most likely to benefit from BMT.     

Limiting-dilution analysis of the effects of colony-stimulating factors, phytohemagglutinin, and hydrocortisone on hematopoietic progenitor cell growth

The effects of colony-stimulating factors (CSFs), phytohemagglutinin (PHA), and hydrocortisone on the growth of human bone marrow hematopoietic progenitor cells (granulocyte-macrophage; GM) were analyzed in a limiting-dilution assay (LDA). Both low-density bone marrow cells separated by discontinuous Percoll gradients and a T cell- depleted and progenitor-enriched cell fraction obtained by the combination of counterflow elutriation centrifugation and Percoll gradients were examined in LDA. GCT (monocytoid cell line-conditioned medium containing GM-CSF), human placenta-conditioned medium, bladder carcinoma cell line 5637-conditioned medium (containing GM- and G-CSF), and recombinant CSF (G-CSF) directly induced proliferation of progenitors with single-hit kinetics. In some instances, however, PHA- stimulated lymphocyte-conditioned medium (containing G- and GM-CSF) showed deviation from single-hit kinetics, which demonstrated the presence of factor(s) suppressive to progenitor growth. In a T cell- depleted, progenitor-enriched fraction, PHA alone was found to suppress progenitor growth at a level as low as 100 ng/mL. The addition of hydrocortisone (10(-6) mol/L) increased the progenitor frequency but suppressed progenitor growth at 10(-4) mol/L. LDA appears to be a valuable method for exploring mechanisms of factors regulating hematopoietic cell growth.     

The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca(2+)-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium- induced phospholipid scrambling

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca(2+)- induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca(2+)-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca(2+)-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca(2+)-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD- phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca(2+)-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.