The Fcgamma receptor IIIA-158F allele is a major risk factor for the development of lupus nephritis among Caucasians but not non-Caucasians

OBJECTIVE: To determine whether inheritance of Fcgamma receptor (FcgammaR) alleles conferring lower affinity for IgG binding increases the risk of developing lupus nephritis. METHODS: We compared the frequency of low-affinity alleles of two FcgammaR polymorphisms (FcgammaRIIA and FcgammaRIIIA) among 235 patients with systemic lupus erythematosus (SLE) and proven nephritis (nephritis patients) and among 352 SLE patients with no evidence of renal disease (non-nephritis control subjects). The ethnic distribution of patients in the study was 49% Caucasian, 20% Hispanic, 17% Asian/Pacific Islander, 12% African American, and 2% from other ethnic groups. All patients were genotyped for the FcgammaRIIA-131R/H and FcgammaRIIIA-158V/F polymorphisms. We used contingency table analysis to compare allele and genotype distributions for nephritis patients and nonnephritis control subjects, including ethnic-specific strata. Multivariate logistic regression analyses included sex and disease duration as covariates. RESULTS: Univariate and multivariate analyses demonstrated a striking association between the low-affinity FcgammaRIIIA-158F allele and FF genotype and the risk of nephritis among Caucasians, but not among non-Caucasians (multivariate odds ratio [OR] 2.6 for Caucasians with FF genotype), (P = 0.0017). This association was even stronger among Caucasians with severe nephritis (OR 4.4, P < 0.0001). In contrast, inheritance of the low-affinity FcgammaRIIA-131R allele (and RR genotype) was not associated with an increased risk of lupus nephritis among any of the ethnic groups examined. CONCLUSION: The FcgammaRIIIA-158F allele is a major risk factor for the development of lupus nephritis among Caucasians, but not among non-Caucasians. These results suggest that ethnic variation is critical in defining the specific genetic factors underlying the pathogenesis of SLE, and they have important prognostic and therapeutic implications as well.     

Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells

CD34 is expressed on human and murine hematopoietic stem and progenitor cells and its clinical usefulness for isolation of stem/progenitor cells has been well established. Although expression of CD34 is regulated in a developmental stage-specific manner, the function of CD34 is not known. Recently we have shown that both a full-length and truncated form of CD34 protein is expressed by hematopoietic cells (Blood 84:691, 1994). To test whether failure to suppress either form of CD34 could affect terminal myeloid differentiation, we constitutively expressed these CD34 proteins in murine M1 myeloid leukemia cells, which can be terminally differentiated to macrophages by treatment with interleukin-6 of leukemia inhibitory factor. Surprisingly our results show that forced expression of the full-length but not the truncated form of CD34 impedes terminal differentiation by these agents. Because the difference between the two forms of CD34 protein resides in the length of their respective cytoplasmic tail domains, our findings strongly suggest that the cytoplasmic domain region of full-length CD34 is responsible for the observed maturation arrest phenotype. These findings suggest a potential negative regulatory role for full-length CD34 in hematopoietic cell differentiation and may explain, at least in part, the block in maturation observed in CD34+ acute myeloid leukemia.     

Restricted expression of a new acute myelogenous leukemia-associated antigen (NHL-30.5) on normal hemopoietic progenitor cells

We have previously reported the isolation of a monoclonal antibody (NHL- 30.5) that reacts with an antigen expressed on a substantial proportion of marrow and blood cells of most patients with newly diagnosed or relapsing acute myeloid leukemia. This antigen is also found on several cell lines derived from myeloid malignancies of human origin. It is not present on mature hemopoietic cells or on the majority of differentiating bone marrow cells. In order to determine whether the NHL-30.5 antigen may, nevertheless, be expressed on low-frequency primitive normal hemopoietic cells, not detected in standard antibody screening procedures, its expression was studied on clonogenic erythropoietic and granulopoietic cells. Light-density (less than 1.077 g/mL) suspensions of normal or chronic myelogenous leukemia bone marrow and peripheral blood cells were stained with NHL-30.5 and fluorescein isothiocyanate labeled second antibody and then sorted into two fractions using the fluorescence-activated cell sorter. The first contained the top 5% of cells with the highest fluorescence intensity. The remainder were collected in the second fraction. Colony assays of both fractions showed the first to be enriched in CFU-E, BFU-E, and CFU- C content (fourfold to 17-fold). The second fraction was correspondingly depleted of these progenitors. These findings reveal NHL-30.5 antigen expression to be a transient event during normal hemopoiesis that characterizes primitive hemopoietic cells on several pathways. Subsequent experiments showed that the presence of up to 10 micrograms/mL of purified NHL-30.5 antibody in colony assay cultures neither inhibited nor stimulated colony formation. Marrow fibroblasts (subcultured marrow adherent cells) were NHL-30.5 negative. Immunoprecipitation studies showed that the antigen detected by NHL- 30.5 is clearly distinct from that identified by My-10, another monoclonal antibody that has previously shown some similarities to NHL- 30.5. It thus appears that the NHL-30.5 antibody reacts with a new myeloid differentiation antigen of as yet unidentified function that is normally restricted in its expression to early stages of hemopoiesis.     

Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

Activation of apoptosis associated with enforced myc expression in myeloid progenitor cells is dominant to the suppression of apoptosis by interleukin-3 or erythropoietin

The inappropriate expression of c-myc in cells deprived of growth factors has recently been implicated in the activation of programmed cell death (apoptosis). The studies described here examine the ability of interleukin-3 (IL-3) or erythropoietin (Epo) to suppress apoptosis that occurs in association with enforced myc expression during cell cycle arrest of a murine IL-3-dependent myeloid progenitor cell line, 32D. G1 arrest was observed when culturing 32D cells to high density in medium supplemented with IL-3, or at subconfluent densities in medium supplemented with Epo. Under both conditions, endogenous c-myc expression was downregulated and viability was maintained. In clones of cells in which c-myc is constitutively expressed from a retroviral vector, enforced c-myc expression was associated with the activation of apoptosis at high cell densities. Similarly, enforced c-myc expression was deleterious to cell survival when these cells were cultured in Epo, as apoptosis was evident within 6 hours. The results support the concept that inappropriate c-myc expression activates apoptosis and that neither IL-3 nor Epo can suppress this program under these conditions.     

Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute lymphoblastic leukemia: a pilot prognostic factor analysis

Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B- cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized "good-risk" patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the "good-risk" children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for "good-risk" patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.     

Bone marrow transplantation for patients with Philadelphia chromosome- positive acute lymphoblastic leukemia

We report the treatment outcome of allogeneic bone marrow transplantation in ten patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Six patients are alive and well for 6 to 30 months (median 19 months) after transplantation. Four patients died with transplant related complications. In view of the poor prognosis associated with this disease, marrow ablation followed by allogeneic or syngeneic marrow grafting may be the preferred treatment modality if a suitable marrow donor is available.