Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors.

We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.     

Clinical trials of intrasplenic arterial infusion of interleukin-2 (IS-IL-2) to patients with advanced cancer

We tried a infusion of interleukin-2 (IL-2) of a relatively low dose via an intrasplenic arterial catheter connected to a chronometric infusion (IS-IL-2). Eighteen patients of colorectal cancer with metastases to the liver or lung or of unresectable hepatoma received a 24 hour continuous infusion with low dose recombinant of IL-2 (mainly 8 × 10⁵ JRU/day) for 25–40 days. All patients tolerated this protocol of the therapy and the main toxic effects were fever and general fatigue. Such serious toxicity as previously reported by high dose IL-2 therapy was not observed. Data of hepatic and renal functions were normal. IS-IL-2 therapy induced a high incidence of eosinophilia (12/18) and thrombocythemia (12/18). Peripheral natural killer (NK) and LAK activities were augmented in all patients and total white blood cell counts were increased during IS-IL-2 therapy. An increase in IL-2 receptor expression of peripheral blood mononuclear cells and significant rises in numbers of Leull (CD16)+, OKMl(CD11)+ and OKIal(HLA-DR)+ were observed. Of 18 patients 12 were evaluable for their response to therapy. Partial response (PR) was observed in one unresectable hepatoma and 11 demonstrated no change (NC) or progressive disease (PD). Six patients were not evaluable because of additional therapy (3 cases) or decreasing tumor cell markers having no measurable lesions (3 cases). Three patients of colorectal cancer from an unresectable group were presumed to have micrometastases to the liver as suggested by an elevated serum CEA level. After receiving IS-IL-2 therapy they demonstrated a decrease in the serum CEA level for more than 3 years after treatment. We conclude that continuous IS-IL-2 administration can result in an increase of their therapeutic efficacy of IL-2 administration and in a decrease its toxicity.     

Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood

The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.     

Functional inhibition by cyclosporin A of the lymphocyte receptor for the AIDS virus (HIV)

The Human Immunodeficiency Virus (HIV) displays a selective tropism for cells expressing the CD4 molecule which, by itself, represents at least part of the specific receptor for this virus. However, modification of the activation state of each individual cell seems critical not only for virus replication but also for its binding and subsequent penetration into its target. We demonstrate here that Cyclosporin-A (CSA), a drug which inhibits IL-2 dependent T-lymphocyte proliferation and differentiation and which is known for its immunosuppressive activity, can prevent subsequent virus binding to cells otherwise susceptible to HIV. Normal T-lymphocytes were preincubated in vitro with CSA at concentrations that were in the same range than those reached in the serum of treated patients. This resulted in the complete disappearance of HIV receptors (HIV-R), as assessed by the direct measure of specific binding of fluoresceinated HIV (HIV-FITC), and in the subsequent inhibition of HIV replication in cultured cells. Moreover CSA pretreatment of IL-2 independent transformed cells derived from the CEM line, before their infection, strongly inhibited HIV adsorption as well as further virus replication. These results provide a new experimental basis for the potential application of CSA in the treatment of HIV-related diseases.     

Enhancement of interleukin-2 release in rats by treatment with bleomycin and Adriamycin in vivo

Summary Spleen cells from rats previously injected with bleomycin (10 mg/kg) or Adriamycin (1 mg/kg) were able to release higher levels of interleukin-2 (IL-2) than cells from untreated animals. The difference in IL-2 release was detected after the cells were exposed to a suboptimal dose of concanavalin A (0.5 g/ml) for 24 h. By cytofluorimetry, these drugs did not change the proportion of W3/25⁺ (helper) or OX-8⁺ (suppressor) T-cell subsets. In contrast, the immunosuppressive drug cyclophosphamide inhibited the IL-2 release from spleen cells under the same conditions. It is suggested that some anti-cancer antibiotics may be able to enhance the release of IL-2 while other cytotoxic drugs with more immunosuppressive potential could inhibit the release of this mediator.     

Bone marrow transplantation for leukemia and aplastic anemia: management of ABO incompatibility.

Between February 1971 and October 1980, 34 patients with leukemia or aplastic anemia received bone marrow transplants from HLA-identical siblings whose lymphocytes did not react in a mixed leukocyte culture. The donors of 10 patients were ABO-incompatible, and for five pairs the ABO incompatibility was major. Plasma exchanges followed by a red blood cell exchange transfusion reduced the anti-A titres to 1:4 or less in these patients. The ABO incompatibility had no adverse effect on the results of marrow transplantation. Twenty-two patients, including 16 of the 20 who received their transplant after Jan. 1, 1980, are still living. Seven of the 15 patients with acute leukemia have survived 89 to 466 days, and 4 of the 6 with chronic myelogenous leukemia (CML) have survived 117 to 545 days. Of the 13 patients with aplastic anemia, 11 are alive up to 8 years after transplantation. Marrow transplantation, when possible, is the treatment of choice for young patients with acute leukemia in remission and for patients with aplastic anemia. Marrow transplantation may also prove to be effective in patients with CML.     

Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.     

Interleukin-2 activates human peripheral blood eosinophils

The effect of interleukin (IL)-2 on eosinophil survival and mediator release was investigated in vitro. Human peripheral blood eosinophils were isolated and purified from mildly atopic donors and cultured on albumin-coated wells with different concentrations of IL-2, interferon (IFN)-gamma, and granulocyte-macrophage colony stimulating factor (GM-CSF) and their viability was evaluated after 4 days in culture. Eosinophils were cultured with IL-2 (1000 u/ml), IFN-gamma (1000 u/ml), or GM-CSF (10 ng/ml) for 18 h, or with platelet activating factor (PAF) (10(-6) M) for 20 min, and the release of eosinophil peroxidase (EPO) and IL-6 was measured. Nedocromil sodium (10(-5) M) was added with each of the above cytokines to study the inhibitory effect of this drug on EPO release. A significant increase of EPO release was induced by IL-2, IFN-gamma, and GM-CSF after 18 h in culture. IL-2 as well as IFN-gamma induced a significant IL-6 release from eosinophils. Nedocromil sodium significantly inhibited EPO release from eosinophils induced by IL-2 or PAF. These results show that IL-2 can activate peripheral blood eosinophils to release granule mediators (EPO) and cytokines (IL-6). Taken together with the presence of IL-2 receptors on eosinophils, we conclude that IL-2 is an important mediator in allergic inflammation and a possible target for pharmacological modulation.     

Effect of virus-modified tumor cell extracts,autologous mononuclear cell infusions and interleukin-2 on oncolytic activity of effector cells of patients with advanced ovarian cancer

Summary We studied the biological responses of six ovarian cancer patients after intraperitoneal (i.p.) injections of virus-modified tumor cell extracts (VMTE) and autologous peripheral blood mononuclear cells, collected by leukapheresis after two injections of VMTE. VMTE was prepared from allogeneic ovarian cell lines, OV2774 and CaOV3, modified by influenza virus, A/PR8/34. A dose of 9 mg VMTE was given i.p. in total of 2–4 injections, and (1–9) × 10⁸ autologous mononuclear cells were infused i.p., 24 h after the second VMTE injection, and 24 h and 72 h after the third VMTE injection. Both peripheral blood (PB) and peritoneal cavity (PC) effector cell cytotoxicity was significantly enhanced against the K562 cell line in the majority of patients, 24–48 h after the second and third VMTE injections. This was accompanied by a dramatic influx of neutrophils into PC (57-550-fold), increase in absolute numbers of lymphocytes, (including large granular lymphocytes) and monocytes, and resulted also in a significant decrease in the number of ascitic tumor cells (98% reduction). The infusion of autologous mononuclear cells did not appear to influence either cytotoxicity or cell infiltration of the peritoneal cavity. We also investigated the in vitro effect of recombinant interleukin-2 (IL-2) on effector cells from PB and PC from patients before and after VMTE treatment. Cytotoxicity of both of these compartments was significantly potentiated after culture with IL-2. In three out of five VMTE-treated patients, PC cytotoxicity was significantly higher after activation with IL-2 than that of patients before VMTE treatment. These data suggest that VMTE induces regional cellular immunity, which could be further potentiated by culture of PC effector cells with IL-2. Thus, combination of VMTE and IL-2 after regional administration could represent the effective therapy for patients with advanced ovarian cancer.     

NK and K cell activity in children with leukemias and aplastic anemias before and after allogeneic bone marrow transplantation

Mononuclear leukocytes from the peripheral blood and bone-marrow of children affected with aplastic anemia and leukemia were investigated for K-cell activity (antibody-dependent cellular cytotoxicity) and NK-cell activity before and after allogenous bone-marrow transplantation. 51Cr liberation test against murine Graffi erythroblast leukemic cells covered with xenoantibodies and K-562 cells were used for identification. Strongly lowered NK- and K-cell activities could be found in aplastic anemia prior to bone-marrow transplantation. However, NK-cell activity was only lowered significantly in leukemic patients with indication of bone-marrow transplantation. K-cell and NK-cell activities normalised after bone-marrow transplantation. K-cell and NK-cell activities could be observed to be reconstituted very early after bone-marrow transplantation.     

Lymphokine release, suppressor cell generation, cell surface markers, and cytotoxic activity in cancer patients receiving natural interleukin-2

We monitored patients treated for 5 days with continuous infusion of increasing doses (3 to 6 x 10(6) U/d) of natural interleukin-2 (IL-2). CD16+, CD25+, and CD56+ cells increased after treatment. Plasma tumor necrosis factor-alpha (TNF-alpha) levels, but not interferon-gamma (IFN-gamma) levels, increased during IL-2 treatment, but spontaneous and IL-2-stimulated TNF-alpha secretion in vitro remained abnormally low. However, mitogen-stimulated TNF-alpha release was normal. Mitogen-stimulated, but not IL-2-stimulated, IFN-gamma release was strongly depressed. Low spontaneous and IL-2-stimulated cytotoxicity on K562 or Daudi increased after treatment. Low suppressor cell generation also normalized after treatment. This appears to be the first reported study of immunologic monitoring of cancer patients treated with natural rather than recombinant IL-2.     

Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils. Inhibition of mediator release without affecting PI hydrolysis or Ca2+ fluxes.

Cyclosporin A (CSA) inhibits IgE receptor-mediated exocytosis from rat basophilic leukemia (RBL) cells and human peripheral blood basophils in a dose-dependent manner over the therapeutic range of CSA concentrations achieved in vivo. Half-maximal inhibition was observed at 0.2 micrograms/ml CSA. The effect of CSA on several biochemical parameters involved in receptor-mediated activation of RBL cells was examined. Maximum inhibition of secretion occurred when CSA was added 5 min before activation, and inhibition was nearly maximum when the drug was added 2 min before the cells were triggered. The same results were observed when RBL cells were stimulated with A23187, a calcium ionophore. These results suggest a mechanism other than inhibition of protein synthesis is involved. Inhibition by CSA of release by either secretagogue persisted, even if CSA was removed from the buffer before the cells were triggered. No inhibition was observed of either receptor-mediated phosphatidylinositol hydrolysis, 45Ca2+ uptake, or the rise in the intracellular concentration of free Ca2+ under the same conditions that produced greater than 80% inhibition of serotonin release. These results demonstrate that the early events in signal transduction are not affected, and suggest that the intracellular target for CSA participates in a later stage of exocytosis. Furthermore, the data suggest that CSA suppresses cells other than T lymphocytes and predict that patients on CSA therapy may have altered response to allergens.     

Interleukin-2 induces chemotactic deficiency in patients with onco hematologic malignancies and autologous bone marrow transplantation.

Unusual gram positive bacteremia has been reported in non granulopenic patients receiving recombinant human interleukin-2 (IL-2) suggesting a beneficial effect of anti gram positive prophylaxis in such patients. We report here studies on granulocyte functions examined during the course of high dose IL-2 therapy (16 to 24 million IU/m2/days for 11 to 18 days) administered during a period of 35 days in 14 patients including 4 solid tumors, 5 chronic myeloid leukemias, 4 recipients of autologous bone marrow transplant (ABMT) and 1 recipient of syngeneic bone marrow transplant. Neutrophils functions were studied before IL-2 administration (d 0), after the first cycle (d 8) and after the third cycle (d 36). Nylon fiber adherence, superoxide production, random migration, phagocytosis, nitroblue tetrazolium reduction, lysozyme and elastase release were not impaired significantly throughout therapy. However N-Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) stimulated chemotaxis of granulocytes, normal before therapy, was significantly impaired as early at d 8 and severely inhibited at d 36 (p less than 0.001). Three septicemia, one corynebacteria parvum septicemia and two gram-negative septicemia despite normal neutrophil counts and oxacillin or Penicillin G plus Pefloxacin prophylaxis, occurred among the 14 patients studied. Although neutrophil functions were not more depressed in transplanted patients than in the other non transplanted patients, special attention should be paid to such patients in whom delayed immune reconstitution could increase the risk of sepsis.     

Serum levels of interleukin-2 in cancer patients: preliminary considerations

In order to investigate the production of interleukin-2 (IL-2) in human neoplasms, we determined IL-2 and soluble IL-2 receptors (sIL-2R) in serum from 18 patients with lymphoma and 28 patients with solid tumors, with (15 cases) or without (13 cases) metastases. As controls, 58 healthy subjects were evaluated. Low levels of IL-2 were not observed in patients with lymphoma or limited solid tumor but abnormally low concentrations of IL-2 were seen in 4/15 metastatic solid tumors, associated with abnormally high values of sIL-2R. This preliminary study confirms in vivo the reduced IL-2 production in metastatic solid neoplasms, previously documented in vitro.     

Therapy with interleukin-2 induces the systemic release of phospholipase-A2

Therapy with interleukin-2 (IL-2) induces remissions in some forms of cancer. This treatment however, is accompanied by side-effects which, in part, may be mediated by the formation of eicosanoids and plateletactivating factor. We investigated the systemic release of phospholipase A₂ (PLA₂), a rate-limiting enzyme in the formation of these lipid mediators, in patients receiving IL-2. In a pilot study of 4 patients we observed an increase in PLA₂ activity in serial plasma samples obtained during the first day after a bolus infusion of IL-2, which increase closely correlated with that of antigen levels of secretory phospholipase A₂ (sPLA₂) as measured by enzyme-linked immunosorbent assay (r=0.92;P<0.001). In 20 patients, receiving 12×10⁶–18×10⁶ IU IL-2/m², we then investigated the course of antigenic levels of sPLA₂ in relation to those of the cytokines tumour necrosis factor (TNF) and interleukin-6 (IL-6) (both cytokines may induce sPLA₂ in vivo). From 4 h on, sPLA₂ levels significantly increased, reaching a peak 24 h after the IL-2 infusion. Subsequent IL-2 infusions even induced a further increase of sPLA₂. This increase of sPLA₂ was presumably not due to a direct effect of IL-2 on, for example, hepatocytes, since this cytokine, in contrast to IL-1, IL-6, TNF and interferon , was not able to induce the synthesis of sPLA₂ by Hep G2 cells in vitro. Consistent with this, plasma levels of TNF and IL-6 in the patients rose, reaching peak levels before a zenith of sPLA₂ occurred, i.e at 2 h and 4 h after the start of the IL-2 infusion respectively. sPLA₂ levels significantly correlated with the development of the side-effects increase in body weight (r=0.49;P<0.0001) and decrease in mean arterial blood pressure (r=0.40;P<0.0001). Moreover, maximum sPLA₂ levels induced by IL-2 were higher in patients who had progressive disease after therapy than in patients who had stable disease or a partial response.     

The pharmacologic regulation of interleukin-1 production: the role of prostaglandins

The role of prostaglandins in the regulation of lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) production by murine C3H/HeN resident peritoneal macrophages was studied. IL-1 production was initially studied in the presence of piroxicam and indomethacin, both inhibitors of prostaglandin biosynthesis. IL-1 was assayed using the IL-1-dependent proliferative response of C3H/HeJ thymocytes. LPS stimulation resulted in 15 to 20 ng/ml of prostaglandin E2 (PGE2) produced in the first hour of culture. IL-1-containing supernatants from drug-treated macrophages at dilutions of up to 1:32 resulted in enhanced thymocyte proliferation compared to control, non-drug-treated cultures and contained less than 2 ng/ml of PGE2. Similar enhancement of proliferation could be obtained by incubating non-drug-treated supernatants with monoclonal anti-PGE2 but not anti-thromboxane B2 (TxB2) antibody. Further dilutions of the drug-treated supernatants gave thymocyte proliferation responses which were indistinguishable from control cultures and, correspondingly, had identical values for IL-1 production. The absence of an effect on IL-1 production was confirmed by quantitation of intracellular IL-1 alpha using goat anti-IL-1 alpha antibody and by quantitation of supernatant IL-1 receptor competition assay. Exogenous PGE2, in the concentration range produced in macrophage supernatants (10-20 ng/ml), directly inhibited IL-1-stimulated thymocyte proliferation. Finally, when macrophages were stimulated with LPS for 24 hr in the presence of added PGE2, thymocyte proliferation was inhibited at the lowest supernatant dilutions, but as the IL-1-containing supernatants were diluted out, the assay curves were indistinguishable from non-PGE2-treated control. Thus, in this system, PGE2 has no effect on IL-1 synthesis, but rather has a direct inhibitory effect on thymocyte proliferation. Nonsteroidal anti-inflammatory drugs are not stimulating IL-1 production but are, in fact, relieving inhibition of the thymocyte IL-1 assay caused by the presence of prostaglandins.     

Radiotherapy-based conditioning is effective immunosuppression for patients undergoing transplantation with T-cell depleted stem cell grafts for severe aplasia

BACKGROUND: We studied the outcome of individuals with aplastic anemia conditioned with a radiation-containing regimen followed by an infusion of stem cell grafts that had been depleted of lymphocytes with CAMPATH-1H (antiCD52; humanised). METHODS: The conditioning regime consisted of fractionated (f) TBI 8 Gy followed by f total nodal irradiation (TNI) 6 Gy. In addition, patients received CY 60 mg/kg on 2 consecutive days. Cytokine-mobilized peripheral blood grafts from HLA-identical siblings were T-cell depleted with CAMPATH-1H 'in the bag'. CsA was commenced on day -1 and continued until day +90. RESULTS: Seventeen heavily transfused patients with aplastic anemia, median age 18 years (range 14-56 years), were studied. The median time from diagnosis to transplantation was 172 days (range 34-443 days). The median CD34(+) cell number infused was 3.47 x 10(6)/kg (range 1.03-18.4 x 10(6)/kg). All patients engrafted. Recovery was fast and patients reached 0.5 x 10(9)/L polymorphs by median day 11 (range 9-17 days). Toxicity from the conditioning included grade 4 hematologic toxicity in all patients. Another major toxicity was gastrointestinal mucosal damage, which exceeded grade 2 in two instances. One patient developed thrombotic thrombocytopenic purpura, which responded to substitution of CsA with tacrolimus and plasmapheresis. Another patient, who had normal blood counts, died of infection on day 241. Chimerism studies at 6 months post-transplantation confirmed the donor origin of hematopoiesis in all seven patients tested. None of the patients developed acute or chronic GvHD. There was no delayed graft failure and 94% of patients had survived disease free at a median of 1303.5 days (range 216-2615 days) from graft infusion. DISCUSSION: In this cohort of multiply transfused patients, the radiation-containing schedules described in this study led to universal engraftment with limited toxicity despite T-cell depletion. No patient developed GvHD or late graft failure. Lower doses of radiation-containing conditioning should be explored further.     

Gene Expression Profiling Identifies HOXB4 as a Direct Downstream Target of GATA-2 in Human CD34+ Hematopoietic Cells

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.     

不同麻醉方法对胃肠道肿瘤手术病人细胞免疫功能的影响

目的:探讨不同麻醉方法对胃肠道肿瘤患者围术期外周血T细胞亚群以及白细胞介素-2(IL-2)的影响.方法:选择胃肠道肿瘤手术患者40例,随机分为两组.采用单纯全麻者为对照组,采用全麻复合硬膜外麻醉者为治疗组.分别于麻醉前、麻醉后不同时间点抽取静脉血,流式细胞术测定CD4+T及CD8+T细胞亚群数量,ELISA法测定血清IL-2的浓度.结果:麻醉后两组患者的CD4+T细胞、CD4+/CD8+比值和血清IL-2浓度均有所下降,与麻醉前比较差异有统计学意义(P＜0.05),治疗组患者的CD4+T细胞及IL-2下降程度不如对照组患者明显,且恢复较快,两组间差异有统计学意义(P＜0.05).结论:胃肠道肿瘤患者在麻醉手术后其细胞免疫功能受到不同程度的抑制,但复合麻醉较单纯全麻的免疫抑制效应低且恢复较快.     

IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2

The proliferative activity of thymocytes cultured with IL-2 and submitogenic concentrations of PHA is increased by 3- to 10-fold in the presence of IL-4. In contrast, IL-4 alone is unable to induce proliferative activity in thymocyte cultures and its synergistic activity is only apparent to concentrations of IL-2 above 1 U/ml. The costimulatory activity of IL-4 is abrogated by the monoclonal anti-IL-4 antibody 11B11. Furthermore, potentiation of the IL-2-mediated thymocyte proliferation is not seen with IL-1, IL-3, IFN-gamma, and granulocyte-macrophage CSF. Thymocytes are at least as responsive to IL-4 as B cells and the IL-4 costimulatory activity in fractionated thymocytes appears to be restricted mainly to the Lyt-2+/L3T4- population. In contrast, purified resting mature T cells do not respond to IL-4 plus IL-2, although they did proliferate in response to IL-4 in combination with PMA. These findings indicate that thymocytes and mature T cells are responsive to the costimulatory activity of IL-4 under quite different conditions, and that IL-4 may play an important role in thymocyte maturation in the thymus.