Skewed T cell receptor repertoire of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein-Barr virus-infected B cells in clonal restriction.

The proliferation of Vdelta1(+) gammadelta T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vdelta1(+) T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vdelta1(+) T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vdelta1(+) T cells in half the patients. A skew was also detected in certain healthy donors, and the Vdelta1(+) T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV-lymphoblastoid cell lines (LCL), and the Vdelta1(+) T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vdelta1(+) T cells from healthy EBV-seropositive individuals. These results suggest that human Vdelta1(+) T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease.

Up regulated expression of tumour necrosis factor {alpha} converting enzyme in peripheral monocytes of patients with early systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems. OBJECTIVE: To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor alpha (TNFalpha) converting enzyme (TACE). METHODS: PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS). RESULTS: In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3alpha (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14(+) monocytes both in patients with SSc and controls. TACE expression on CD14(+) cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016). CONCLUSIONS: Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFalpha and other immunoregulatory molecules.

Stem cell factor prevents Fas-mediated apoptosis of human erythroid precursor cells with Src-family kinase dependency.

The Fas ligand (Fas-L) expressed on mature erythroblasts may induce apoptosis of more immature erythroid cells that express Fas, whereas stem cell factor (SCF) may prevent Fas-mediated cell death in hematopoietic progenitor cells. The manner in which SCF prevents Fas-mediated cell death still is unclear. Given the essential role of SCF and the potentially important involvement of the Fas/Fas-L system in the development of erythrocytes, we studied mechanisms related to SCF prevention of Fas-mediated apoptosis. We used primary cultured human erythroid colony-forming cells (ECFC) derived from CD34+ cells and enriched glycophorin A positive (GPA+) c-kit+ cells in ECFC. Apoptosis of ECFC was induced by an Fas-L mimetic monoclonal antibody CH11. DNA fragmentation and the activation of caspase-3 and caspase-8 were measured using commercially available kits. Characterization of expanded cells was performed using multiparameter flow cytometry. Lyn kinase activity was measured by enolase kinase assays. SCF inhibited the CH11-induced DNA fragmentation of ECFC as well as enriched GPA+ c-kit+ cells in ECFC, but not those of GPA+ c-kit- cells. SCF also inhibited the activation of caspase-3 and caspase-8, without downregulation of the surface expression of Fas, suggesting that SCF prevents apoptosis through uncoupling of Fas ligation from subsequent caspase activation. PP2, a specific inhibitor of Src-family kinases, antagonized the effects of SCF in preventing Fas-mediated apoptosis. We propose that SCF prevents Fas-mediated apoptosis of erythroid progenitor cells in a manner dependent on the activity of Src-family tyrosine kinases. We also identified active Lyn in erythroid cells. These data suggest the presence of a novel Src-family-dependent function of SCF in the development of erythrocytes.

Signal transduction in primary cultured human erythroid cells.

Development of erythrocytes is a complex process governed by multiple cytokines. Colony assays have revealed the physiologic importance of these cytokines, although biochemical studies of highly purified human colony-forming unit-erythroid (CFU-E) generated in vitro from CD34+ cells have only recently begun. Studies from our groups and others suggested that signal transduction in primary erythroid cells differs considerably from that in cell lines or primary cells from other species. In this review, we summarize results of these studies with emphasis on possible implications for hematotherapy.

Effects of beta2-glycoprotein I and monoclonal anticardiolipin antibodies on extrinsic fibrinolysis.

Antiphospholipid antibodies (aPLs) are associated with an increased incidence of thrombosis, but the mechanisms responsible for thrombosis are unclear. The present study investigated the effect of both beta2-glycoprotein I (beta2-GPI) and aPLs on the activity of extrinsic fibrinolysis. The remaining tissue-plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, plasminogen activator inhibitor (PAI) -1 was measured by a chromogenic assay using synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. Without PAI-1, beta2-GPI did not affect t-PA activity. When 14.3 ng/ml PAI-1 was added to 3.6 U/ml t-PA, the remaining t-PA activity was increased from 48.9% to 60.4% by the addition of beta2-GPI (190 microg/ml). The effect of beta2-GPI did not require phospholipids. The beta2-GPI seems to protect t-PA activity from the inhibition by PAI-1. When monoclonal anticardiolipin antibodies (aCLs), EY1C8, and EY2C9, which were established from a patient with antiphospholipid syndrome, were further added to the mixture with a diluted phospholipid (Platelin) to investigate the influence of aPL, the remaining t-PA activity decreased to 50.1 and 80.7%. Monoclonal aCLs appeared to inhibit the effect of beta2-GPI, that is, these monoclonals inhibited the fibrinolytic activity by an elevation in PAI-1 activity. These results suggest the possibility that the impairment of fibrinolytic activity by aCLs is one of reasons for the increased incidence in thrombosis in patients with aCLs.

Heterogeneous behavior of anti-beta2-glycoprotein I antibodies on various commercially available enzyme immunoassay plates coated with beta2-glycoprotein I.

OBJECTIVE: To evaluate commercially available enzyme immunoassay (EIA) plates for the measurement of anti-beta2-glycoprotein I autoantibody (anti-beta2-GPI). METHODS: Sera from 10 patients with the antiphospholipid syndrome, and 3 monoclonal anti-beta2-GPI antibodies established from patients with antiphospholipid syndrome, were assayed for binding to solid phase beta2-GPI on 20 commercially available plates. RESULTS: Several commercially available EIA plates were found to be of potential value for the measurement of anti-beta2-GPI autoantibody. Some plates were unsuitable for anti-beta2-GPI detection, possibly due to less beta2-GPI on the plates, or to differences in the nature of conformational changes of beta2-GPI induced by the plates. CONCLUSION: Differences among EIA plates need to be considered when measuring beta2-GPI antibodies.

Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34(+) cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 micromol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V-binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

The putative mechanism of thrombosis in antiphospholipid syndrome: impairment of the protein C and the fibrinolytic systems by monoclonal anticardiolipin antibodies.

The mechanism of thrombosis in patients with antiphospholipid syndrome is not clear. To investigate it, we examined the effect of monoclonal anticardiolipin (aCL) antibodies and beta2-glycoprotein I (beta2-GPI), which is required for formation of the aCL epitopes, on activated protein C (APC) and on fibrinolytic activity. First, APC activities were measured in the presence and absence of beta2-GPI or gamma M immunoglobulin (IgM) monoclonal aCLs (EY1C8 and EY2C9), or both, established from peripheral blood lymphocytes obtained from a patient with aCL. beta2-GPI exhibited a procoagulant activity by inhibiting APC activity as well as an anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL, and then only in the presence of beta2-GPI. The remaining tissue plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, and plasminogen activator inhibitor (PAI)-1 was measured by a chromogenic assay using the synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. beta2-GPI protected t-PA activity from inhibition by PAI-1. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. Thrombosis in patients with aCL can be explained in part by both the inhibition of APC anticoagulant activity and the impairment of fibrinolytic activity by aCL.

The influence of beta2-glycoprotein I on tissue factor activity.

The beta2-glycoprotein I (beta2-GPI) is known for its procoagulant as well as anticoagulant properties. The influence of beta2-GPI on tissue factor (TF) activity was investigated by chromogenic assays for both factor Xa generation and factor VIIa activity. When 1.36 mg/mL beta2-GPI was added to phospholipids prior to the addition of TF and factor VIIa, the TF activity was moderately inhibited by beta2-GPI in a concentration-dependent manner. Inhibition of TF activity by beta2-GPI required incubation for at least 20 min. When beta2-GPI was added to the mixture of phospholipids and TF followed by the addition of factor VIIa, the TF activity was little affected by the addition of beta2-GPI. The TF activity was inhibited when beta2-GPI was added after forming a complex with TF and factor VIIa. The results suggest that beta2-GPI inhibits both the reconstitution of TF with phospholipids and factor Xa generation by the TF factor-VIIa complex but does not affect the formation of a TF factor-VIIa complex.

Efficacy of delayed granulocyte colony-stimulating factor after full dose CHOP therapy in non-Hodgkin's lymphoma: a pilot study for a leukocyte count oriented regimen.

Bone marrow toxicity is a great challenge for physicians treating patients with non-Hodgkin's lymphoma (NHL) and prescribed chemotherapy. Granulocyte colony-stimulating factor (G-CSF) prevents myelotoxicity, but the optimal timing and scheduling of G-CSF administration has not been ascertained. We investigated leukocyte count oriented G-CSF administration schedules, as related to full dose administration of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) chemotherapy, with shortened intervals. Thirty-eight Japanese patients with NHL were included in this study. The standard CHOP combination was administered in six cycles. Patients were given G-CSF in a dose of 2 micrograms/kg/day, subcutaneously starting the day when total leukocytes were < 3,000/microliters. When leukocyte count remained at > 3,000/microliters, G-CSF was started 10 days following CHOP. Treatment with G-CSF was discontinued after the leucocyte count reached > 10,000/microliters, and CHOP was started the next day (CHOP-G treatment; CHOP-G). Doses were not modified in any patient. Patients over 70 years of age received 2/3 of the standard dosage. In the first cycle of CHOP, the day of initiation of G-CSF was 9.6 days following CHOP in the first cycle and 7.7 to 8.5 days during 2 to 6 cycles. The mean duration of G-CSF injection was 7.4 days with a range from 6.8 to 8.0 days, in each CHOP cycle. The mean intervals of CHOP-G was 14.7 days during six consecutive courses, and there was no prolongation of the intervals, even in later cycles. In 23 patients who received all six cycles of CHOP-G, the overall response rate was 91.3% (73.9% complete remission; CR and 17.4% partial remission; PR). In 32 patients with intermediate grade NHL, the overall response rate was 84.4% (65.5% CR and 18.8% PR). Thus, the full dose CHOP with G-CSF, based on the leukocyte count oriented schedule, can be achieved with shortened intervals, an approach which will increase the quality of life (QOL) for the patients by reducing the days of treatment as well as the cost of G-CSF.

Purification of erythroid progenitor cells and characterization of erythropoietin receptors.

Highly purified murine and human erythroid progenitor cells at the colony-forming unit-erythroid (CFU-E) stage of development were prepared and recombinant human erythropoietin (rEp) was radioiodinated with retention of full biological activity. Specific binding of 125I-rEp to the murine cells revealed 950 receptors on the cell surface. Three hundred had a Kd of 0.09 nM while the remaining receptors had a Kd of 0.57 nM. The human erythroid progenitor cells also had two classes of receptors with a similar number per cell and similar distribution. The high affinity receptors had a Kd of 0.15 nM while the remaining receptors had a Kd of 0.37 nM. 125I-rEp was rapidly internalized into the cells at 37 C and metabolized to iodotyrosine. When cross-linking of 125I-rEp to the murine erythroid progenitor cells was performed with disuccinimidyl suberate, two labelled bands of 100 and 85 kDa were demonstrated. The radioactivity of both bands was reduced when binding was performed in the presence of excessive unlabelled rEp indicating a specific interaction of 125I-rEp with the receptor.