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: Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

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: We reviewed magnetic resonance (MR) staging examinations of 98 patients with malignant lymphoma who failed other therapy and were under evaluation for bone marrow transplantation. MR scan results were compared with blind posterior iliac crest aspirations and biopsies. Images of vertebral, pelvic, and femoral marrow were obtained using a standard T1-weighted, short repetition time (TR), short time to echo (TE) (TR700/TE22), spin-echo (T1-SE) method in 92 patients and short TI inversion recovery (STIR) technique (TR1,500/TE36/TI100) in all. On standard T1-SE sequence, normal marrow is bright due to the predominance of marrow fat, and tumor is dark. With STIR images, water containing tumor has a very high signal intensity in a dark (fat suppressed) background. Thirteen patients had positive MR scans and marrow biopsies, whereas 49 had negative MR scans and biopsies. Of 36 discordant MR/histology results, 10 had positive biopsies and negative MR exams; eight of these had microscopic infiltration (less than or equal to 5%) with tumor. MR detected marrow tumor either in the crests or elsewhere in 25 of 75 (33%) patients with negative study biopsies. We could confirm marrow involvement in 15 of these 25 (60%) by clinical methods. Therefore, up to one third of the patients evaluated with routine biopsies may have occult marrow tumor detectable by MR exam. In patients with negative marrow biopsies, especially those with Hodgkin's disease or intermediate to high-grade non-Hodgkin's lymphomas, MR scans found focal lesions distant from the crests. Biopsy better detected lower grade microscopic involvement. We conclude that optimal marrow staging of lymphoma patients incorporates both biopsy and MR imaging.

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: The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).

Description: Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.