ALBERT

Description: Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% ± 0.5% of total cell radioactivity) were raised relative to control values (2.0% ± 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% ± 17% of control values (n = 10,P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-α was, respectively, 65% ± 11% (n = 7, P = .03) and 57% ± 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% ± 9% and 25% ± 3% of control for GM-CSF and TNF-α, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.

Description: To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of β thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/β thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the /γ, βE/γ, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/β thalassemia, and other β thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

Description: Ten adult patients with human immunodeficiency virus (HIV)-associated malignancies (five with lymphoma and five with Kaposi's Sarcoma) were treated with a daily subcutaneous injection of interleukin-2 (IL-2) for 90 consecutive days in a phase I dose-escalation study. Seven patients had absolute CD4 counts below 200/mm3 at the time malignancy was diagnosed. Each lymphoma patient had obtained a complete or partial remission with standard chemotherapy before initiating IL-2. The daily dose of IL-2 did not change during the 90-day course of therapy. Seventeen courses of IL-2 therapy were completed at doses ranging from 0.4 x 10(6) U/m2/d to 1.2 x 10(6) U/m2/d without significant (grade III) toxicity. Two of two patients experienced grade III toxicity within 21 days of initiating IL-2 at a dose of 1.4 x 10(6) U/m2/d, but both patients subsequently completed 90 days of therapy at the maximum tolerated dose (MTD) of 1.2 x 10(6) U/m2/d. Although there were no significant increases or decreases in T-cell subsets at any dose level, there was an increase in absolute natural killer (NK) cell number at the three highest doses of IL-2 (mean percent increase 247; 95% confidence interval, 124 to 369) that was statistically significant (Wilcoxon one-sample signed rank test, P = .015). One patient developed an anti-IL-2 antibody titer that correlated with minimal NK cell expansion in vitro and in vivo. An increase in eosinophils was noted during 9 of 17 courses of IL-2 therapy without correlation to IL-2 dose, prior course of IL-2, or NK cell expansion. At the MTD, there was no consistent increase in the plasma HIV RNA level over time. Three of 10 patients had progressive disease while on study. During 50 months of IL-2 therapy, no patient was treated for an opportunistic infection. We conclude that daily low dose subcutaneous IL-2 can be self-administered safely with good compliance for prolonged periods of time to patients with HIV-associated malignancies, including those with profound immune deficiency. The majority of patients show selective expansion of innate immune effectors, ie, NK cells and/or eosinophils, in the absence of significant clinical toxicity or increased viral burden. These results suggest that low-dose IL-2 therapy should be studied further in phase II clinical trials for evidence of activity against malignancy and opportunistic infection in this patient population.

Description: The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X- CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

Description: Neutrophil elastase, proteinase-3, and azurocidin are primary components of neutrophil azurophilic granules and are encoded by closely linked genes (gene symbols ELA2, PRTN3, and AZU1, respectively) in a region of approximately 50 kb. These genes are coordinately expressed in a granulocyte-specific fashion, but the mechanisms defining this pattern of expression are unknown. To understand the role of chromatin organization in governing the expression of ELA2, PRTN3, and AZU1, we mapped this region of chromosome 19 and identified the adipsin (complement factor D) gene in proximity to the 3′ end of ELA2. We then examined the changes in chromatin structure at the locus which accompany myeloid cell differentiation and identified 17 DNase I hypersensitive sites (DHS 1 to 17) in U-937 cells, an early myelomonocytic cell line expressing high levels of neutrophil elastase. Chemically induced differentiation and concomitant downregulation of AZU1, PRTN3, and ELA2 transcription in U-937 cells is not accompanied by changes in the DHS-pattern. Mature neutrophils, however, do not carry any of these hypersensitive sites, indicating a large degree of chromatin remodeling at this locus accompanying terminal granulocytic differentiation. Sixteen of the 17 DHS identified in U-937 cells are also present in the HL-60 myelomonocytic cell line. Hematopoietic cell lines representing the early erythroid and lymphocyte lineages, and a nonhematopoietic cell line display a subset of the hypersensitive sites. The altered chromatin structure specific to cells that actively transcribe the AZU1-PRTN3-ELA2 genes suggests that chromatin reorganization is an important mechanism regulating the myeloid-specific transcription of this gene cluster.

Description: To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of β thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/β thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the /γ, βE/γ, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/β thalassemia, and other β thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

Description: Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% ± 0.5% of total cell radioactivity) were raised relative to control values (2.0% ± 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% ± 17% of control values (n = 10,P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-α was, respectively, 65% ± 11% (n = 7, P = .03) and 57% ± 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% ± 9% and 25% ± 3% of control for GM-CSF and TNF-α, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.

Description: Neutrophil elastase, proteinase-3, and azurocidin are primary components of neutrophil azurophilic granules and are encoded by closely linked genes (gene symbols ELA2, PRTN3, and AZU1, respectively) in a region of approximately 50 kb. These genes are coordinately expressed in a granulocyte-specific fashion, but the mechanisms defining this pattern of expression are unknown. To understand the role of chromatin organization in governing the expression of ELA2, PRTN3, and AZU1, we mapped this region of chromosome 19 and identified the adipsin (complement factor D) gene in proximity to the 3′ end of ELA2. We then examined the changes in chromatin structure at the locus which accompany myeloid cell differentiation and identified 17 DNase I hypersensitive sites (DHS 1 to 17) in U-937 cells, an early myelomonocytic cell line expressing high levels of neutrophil elastase. Chemically induced differentiation and concomitant downregulation of AZU1, PRTN3, and ELA2 transcription in U-937 cells is not accompanied by changes in the DHS-pattern. Mature neutrophils, however, do not carry any of these hypersensitive sites, indicating a large degree of chromatin remodeling at this locus accompanying terminal granulocytic differentiation. Sixteen of the 17 DHS identified in U-937 cells are also present in the HL-60 myelomonocytic cell line. Hematopoietic cell lines representing the early erythroid and lymphocyte lineages, and a nonhematopoietic cell line display a subset of the hypersensitive sites. The altered chromatin structure specific to cells that actively transcribe the AZU1-PRTN3-ELA2 genes suggests that chromatin reorganization is an important mechanism regulating the myeloid-specific transcription of this gene cluster.

Description: Interleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1β and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1β on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1–induced NF-κB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.

Description: The rapidity and duration of the response of non-transferrin-bound iron (NTBPI) to chelation therapy are largely unknown and have important implications for the design of optimal chelation regimens. Methodology was developed to measure simultaneously NTBPI, deferoxamine (DFO), and its major metabolite. NTBPI was present in all but 2 of 28 thalassaemia major (TM) patients who had received conventional subcutaneous DFO the previous night, suggesting a short duration of NTBPI clearance by DFO. The detailed kinetics of NTBPI were therefore studied in response to intravenous DFO at 50 mg/kg/27 h for 48 hours and compared in 17 regularly transfused TM and 8 untransfused thalassaemia intermedia (TI) patients to determine the influence of hypertransfusion and iron overload on NTBPI response. Before DFO infusion, NTBPI was present in all patients and was significantly higher in TI (4.52 +/- 0.53 mumol/L) than TM (2.92 +/- 0.03 mumol/L; P = .03). NTBPI values in TM correlated with transferrin saturation (r = .6, P = .03) but not with serum ferritin. Removal of NTBPI by intravenous DFO is in a biphasic manner. The initial rapid rate constant (alpha) was similar in TI (1.5 hour-1) and TM (1.6 hour-1), but the subsequent beta phase was slower (0.04 hour-1) in TI when compared with TM (0.4 hour-1, P = .002). Detectable NTBPI persisted during the beta phase, particularly in TI, despite an excess of plasma DFO also being present (steady state 8 mumol/L). On cessation of DFO infusion, NTBPI reappearance was rapid; the kinetics also being biphasic. The rapid initial rate constant (alpha = 2.5 hour- 1) lasted less than 30 minutes and was approximately equal to the summation of the initial rate constant for removal of DFO (1.8 hour-1) and its major metabolite (0.6 hour-1). This was followed by a slower return to pretreatment levels, usually between 6 and 12 hours, which was faster in TI than in TM. This marked NTBPI lability supports the use of continuous rather than intermittent DFO in high risk patients.