Comparison of antileukemic immunity against U937 cells in atopic asthmatics versus healthy controls

To assess the antitumor effects in atopic asthmatics versus healthy adults, we designed this study using in vitro mononuclear cells (MNC) culture as an immunity model with human leukemic U937 cells as the target. MNCs were collected from asthmatic subjects and healthy controls. Conditioned media from the MNC cultures (MNC-CM) were collected after stimulation with various concentrations of phytohemagglutinin (PHA). We treated U937 cells with these MNC-CMs, then assayed their proliferation and differentiation after 5 days of culture. At lower PHA doses (1.25 microg/ml), as well as in absence of PHA, the asthmatic MNC-CMs inhibited U937 cells growth to a slightly greater extent than did the MNC-CMs from controls. In contrast, when higher doses of PHA were used (5, 10 microg/ml), this growth-inhibiting effect was dramatically reversed. The dual effect of MNC-CM in these two groups was also shown in U937 cell differentiation assay, assessed as follows: morphological change by Liu's staining, functional change by NBT reduction test and CD 14 expression by flow cytometric detection. We suggest that the antileukemic effects of MNCs from asthmatic patients result from a slightly immunopotentiated status. This immunity may be dramatically reversed, however, after marked activation of MNCs.     

Clonal analysis of intrahepatic T lymphocytes in chronic active hepatitis: Isolation of a T-cell line specific for hepatitis B core antigen from a patient with serological evidence of exposure to HBV

We have evaluated whether peripheral blood and hepatic lymphocytes from a patient with chronic active hepatitis (CAH) and antibodies to HBV in serum were specifically sensitized to HBV envelope antigens (HBsAg and pre-S Ag) or to HBcAg. No proliferation to HBV antigens was demonstrated upon stimulation of peripheral blood mononuclear cells either unfractionated or enriched in CD4+ (helper/inducer) T cells. Of 15 T-cell cloned lines (7 CD8+ and 8 CD4+) obtained by limiting dilution in the presence of PHA and recombinant IL2 from liver-infiltrating lymphocytes, one, designated H2, showed specific sensitization to HBcAg, whereas none demonstrated sensitization to viral envelope antigens. The H2 line displayed the CD8+ phenotype, suppressor activity on polyclonal immunoglobulin production and IL2-dependent, HBcAg-specific proliferation. These results suggest that in patients with CAH and serological evidence of previous exposure to HBV, it is possible to obtain lymphocytes specifically sensitized to HBcAg from liver biopsy.     

In vitro alpha-interferon treatment of peripheral blood mononuclear cells improves interleukin-2 activity in HBV-related chronic liver disease

To investigate mitogen-induced helper T cell activity in patients with HBV-related chronic liver disease (CLD), Interleukin-2 (IL-2) activity was assessed by an IL-2 bioassay using phytohaemagglutinin (PHA)-stimulated mononuclear cells (MNC). IL-2 activity was significantly reduced in patients with CLD (P less than 0.01), and was comparable to controls in those with minimal liver damage, indicating that decreased IL-2 activity is not due to the presence of HBV X MNC from 2 of the 3 patients treated with alpha-interferon (alpha-IFN) showed the highest IL-2 activity. In vitro preincubation of MNC with alpha-IFN before stimulation with PHA, led to a significant increase in IL-2 activity in all subjects (P less than 0.01). The improvement in IL-2 activity induced by alpha-IFN may be, in part, responsible for the therapeutic effect of this agent in HBV-related CLD.     

Cytokines and the chronic inflammation of rheumatic disease. III. Deficient interleukin-2 production in rheumatoid arthritis is not due to suppressor mechanisms

Despite the evidence for activated T cells in the joint in rheumatoid arthritis (RA), there is evidence of deficient lymphocyte proliferation to a variety of stimulants. We investigated the production of interleukin-2 (IL-2) after phytohemagglutinin (PHA) stimulation. We show that in the peripheral blood IL-2 production was similar in RA and controls (3.7 vs 3.0 U/ml, respectively). However, blood lymphocytes from patients with joint effusions produced significantly less IL-2 than from patients without effusions (1.8 vs 5.7 U/ml, respectively). The amount of IL-2 produced by synovial fluids (SF) cells was significantly less than that produced by the corresponding blood cells (1.0 vs 1.6 U/ml). Further experiments revealed that the decreased IL-2 production was not due to its removal by IL-2 receptor positive cells in the SF and cell mixing experiments did not reveal any suppressor influences.     

Anti-leukemic immunity against U937 cells in uremic patients

To examine anti-tumor immunity in uremic patients undergoing regular hemodialysis, we designed this study using in vitro mononuclear cell (MNC) cultures, with human leukemic U937 cells as the target. MNC were collected and cultured from uremic subjects and age- and gender-matched healthy controls. Conditioned media from the cultures (MNC-CM) were collected after stimulation with various concentrations of phytohemagglutinin (PHA). The proliferation-inhibiting and differentiation-inducing activities of the PHA-MNC-CM on U937 cells were evaluated. The growth inhibition activity of uremic patients' PHA-MNC-CM was lower than that of controls. The differentiation-inducing effects were evaluated by morphological scoring, superoxide production, and monocyte-associated antigen expression (CD14 and CD68). All three parameters demonstrated that the differentiation-inducing effect of MNC-CM increased with increasing doses of PHA. These effects, however, were significantly less in uremic patients compared to controls at higher doses of PHA.The levels of TNF-alpha and IFN-gamma in PHA-MNC-CM increased in a PHA dose-dependent manner and were much higher in the controls. We conclude that the capacity of MNC from uremic hemodialysis patients to produce anti-leukemic immunity is significantly lower than that of healthy controls.     

Enhanced complement-susceptibility and dysfunction of lymphocytes in paroxysmal nocturnal haemoglobinuria (PNH)

We investigated the complement-susceptibility of paroxysmal nocturnal haemoglobinuria (PNH) lymphocytes in relation to their dysfunction. When assessed by complement-mediated lysis induced by monoclonal antibodies (CD5 or CD20) and rabbit complement, the complement-susceptibility of lymphocytes from patients with PNH, both CD5+ (T cells) and CD20+ (B cells), was greater than that from controls (P less than 0.001). This susceptibility was further enhanced, both in normal controls (P less than 0.01) and in patients with PNH (P less than 0.001), when the activity of decay-accelerating factor (DAF) on the lymphocytes was blocked with anti-DAF monoclonal antibody. DAF amounts in mononuclear cells (MNC) from patients with PNH, measured by an enzyme-linked immunosorbent assay (ELISA), were lower than those of the controls (P less than 0.001). The expressions of DAF on T cells and on B cells from patients with PNH were significantly decreased (P less than 0.05 in T cells: P less than 0.01 in B cells). MNC from patients with PNH responded less to phytohaemagglutinin (PHA) and concanavalin A (Con A) than MNC from the controls (P less than 0.001). In contrast, the responses of PNH MNC to poke weed mitogen (PWM) or lipopolysaccharide (LPS) were not impaired. MNC from a normal donor preincubated with anti-DAF became less responsive to PHA or Con A. We conclude that PNH lymphocytes show enhanced complement-susceptibility and that they are involved in their own expression of DAF as well as in other types of peripheral blood cells. The results of the responses to various lectins suggest the dysfunction of T cells in PNH.     

霉酚酸对外周血T淋巴细胞活化抗原表达和增殖的影响

目的　探讨免疫抑制剂霉酚酸酯的活性代谢产物霉酚酸 (MPA)对外周血T淋巴细胞活化、增殖的影响。方法　分离外周血单个核细胞 ,以植物血凝素 (PHA)作为刺激剂 ,加或不加免疫抑制剂MPA和环孢素A(CSA) ,分组体外培养。以流式细胞仪检测 :T淋巴细胞表面标志CD3 ;T细胞活化抗原CD69/CD3 、CD2 5/CD3 、溴尿嘧啶脱氧核糖核苷 (BrdU)掺入率及细胞周期。结果　 (1)MPA、CSA均能显著抑制T淋巴细胞表面标志抗原CD3 的表达 ,96h时 ,MPA浓度 1组、CSA浓度 1组分别为(37 6 0± 7 89) %、(5 5 85± 13 6 4 ) % ,与对照组 (74 2 0± 7 5 1) %比较 ,差异有显著性 (P值分别为0 0 0 0、0 0 0 4 ) ;(2 )对预先活化 72h的T淋巴细胞 ,MPA能显著抑制CD3 的表达 ,继续培养 96h时 ,MPA组、CSA组分别为 (5 2 90± 7 35 ) %、(6 5 0 5± 10 82 ) % ,与对照组 (78 80± 5 0 9) %比较 ,差异有显著性(P值分别为 0 0 4 9、0 188) ;(3)MPA、CSA均不影响 2 4h时T淋巴细胞CD69的表达 ,MPA组、CSA组分别为 (5 5 0 3± 13 98) %、(38 30± 17 38) % ,与对照组 (5 0 11± 19 2 8) %比较 ,差异无显著性 (P >0 0 0 5 ) ;(4)MPA、CSA均能抑制T细胞表面CD2 5的表达 ,72h时 ,MPA浓度 1组、CSA浓度 1组分别为(37 15± 7 15 ) %、(6 2     

Limiting dilution analysis of IL-2 producing cells: I. Studies of normal human peripheral blood.

BACKGROUND. After marrow transplantation, the interaction of helper T lymphocytes from the donor with the patient alloantigens leads to cellular activation and release of IL-2 as initial events of the graft versus host reaction. A method for assessing the size of the pool containing allospecific helper T cells capable of producing IL-2 could be applied in the selection of better donors for marrow transplantation. MATERIAL AND METHODS. PBMC are added to replicate sets of wells each containing various amounts of EBV-LCL cells and PHA. After culture for some days the supernatant is removed from each well and added to IL-2 dependent CTLL-2 line. The proliferation of the CTLL-2 line is assessed by pulse labeling with 3H-thimidine. The precursor frequency of cell capable of producing IL-2 per ml/blood is estimated from the minimum X2 regression of the function of non-responding wells plotted as logarithmic function of the number of PBMC added per well. RESULTS. Approximately 30-40% of PBMC are found to produce IL-2 under the following conditions in culture: the optimal PHA concentration is 1.25 micrograms/ml, the optimal number of stimulator EBV-LCL cells is 1 x 10(3) and 3 days of culture are required. CONCLUSION. Here we report a rapid and quantitative technique of limiting dilution analysis that can estimate the frequency of peripheral blood mononuclear cells capable of secreting interleukin-2 following interaction with specific alloantigen.     

Phospholipase A2 activity associated with synovial fluid cells

High activity of phospholipase A2 (PLA2) has been detected in synovial fluids (SF) in inflammatory arthritides. Since the source(s) of SF PLA2 has not been identified, we tested PLA2 content in SF cells obtained from 11 SF. Cell sonicates were prepared at 5 X 10(6) cells/ml. In the supernatants of the sonicated SF cells (n = 11), PLA2 activity ranged from 38-755 U/ml, mean 368 +/- 243 (SD) U/ml, compared to 5-64 U/ml, mean 31 +/- 15 (SD) U/ml in sonicates of normal peripheral blood PMN (n = 5) (p less than 0.0001). Spontaneous release of PLA2 from unstimulated SF cells ranged from 26-365 U/ml, mean 131 +/- 144 (SD) U/ml (n = 5), whereas spontaneous release from peripheral blood PMN was negligible. Neither 10(-8) M FMLP nor 5 X 10(-6) M dexamethasone altered extracellular PLA2 release. To assess whether PLA2 adsorbs passively to cell membranes through hydrophobic interaction, normal peripheral PMN were incubated in crude SF (n = 7) with PLA2 ranging from 4,000-24,300 U/ml, or with purified human SF PLA2 or Naja naja PLA2. We found that soluble PLA2 adsorbed to PMN membranes in a concentration dependent fashion. PLA2 activity in sonicates of PMN incubated in crude SF ranged from 185-358 U/ml compared to controls of 21-64 U/ml. Sonicates of PMN incubated with purified human SF PLA2 (5,000-30,000 U/ml) showed progressive concentration dependent increase in PLA2 from 7 +/- 4 to 212 +/- 11 (SD) U/ml (p less than 0.001). PMN incubated with Naja naja PLA2 also showed marked increase in the content of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-transferrin dependent 59Fe uptake in phytohemagglutinin-stimulated human peripheral lymphocytes.

Human peripheral lymphocytes stimulated by phytohemagglutinin (PHA) have been used to demonstrate the characteristics of iron uptake from non-transferrin iron donors. When incubated with 59Fe(II)-ascorbate or 59Fe(III)-nitrilotriacetate (Fe-NTA), stimulated lymphocytes (2 micrograms/ml PHA) showed a tenfold increase in uptake of 59Fe as compared with the resting cells. The uptake of 59Fe from these iron donors was time and concentration dependent, showed saturation kinetics, and was not influenced by the addition of a tenfold excess of unlabeled Fe(III)2-transferrin (Fe-Tf). The amount of 59Fe accumulated from 59Fe-NTA was about one-third of that from 59Fe2-Tf, whereas the uptake of 59Fe from 59Fe-ascorbate was about tenfold higher. When stimulated with varying doses of PHA, lymphocytes showed maximal uptake of 59Fe from 59Fe2-Tf at the concentration of PHA (1 microgram/ml) optimal for 3H-thymidine incorporation. Lymphocytes stimulated with supraoptimal concentrations (5-20 micrograms/ml) of PHA showed slightly less iron uptake from 59Fe-Tf and had a longer transferrin cycle time, as compared with the cells under optimal stimulation. In contrast, the uptake of 59Fe from non-transferrin 59Fe donors in cells stimulated with 5-20 micrograms/ml PHA was greater than that in cells grown with 1 or 2 micrograms/ml PHA. It is suggested that lymphocytes take up iron from non-transferrin iron donors by processes different from the iron uptake pathway used by transferrin.     

Production of B-cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B-cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.     

Further characterization of interleukin-2 production by lymphocytes of patients with systemic lupus erythematosus

To further characterize the mechanisms responsible for defective interleukin-2 (IL-2) production in patients with systemic lupus erythematosus (SLE), we studied the effect of irradiation on the capacity of lymphocytes to produce this lymphokine when stimulated with phytohemagglutinin (PHA), or with a combination of PHA and a phorbol myristic acid ester (PMA). Irradiation increased PHA induced IL-2 production in patients with SLE and normal controls, and reached normal levels in 10 of 16 patients with SLE. This effect was due to inactivation of CD8+ suppressor cells. When PMA was used as a costimulant, maximal enhancement of IL-2 production was observed in both groups, but values in SLE remained significantly lower than in normals. These differences were not overcome by irradiation, raising the possibility that SLE suppressor cells act upon a site proximal to protein kinase C. Our studies have confirmed that active endogenous suppression may be responsible for most of the defective PHA induced IL-2 production in SLE and that this suppression is radiosensitive.     

Fluorinated 4-quinolones induce hyperproduction of interleukin 2.

The fluorinated 4-quinolones are a "new" group of antibiotics with a broad antibacterial spectrum. They are already widely used in clinical practice. Previous studies have shown that these drugs increase the uptake of [3H]thymidine into DNA of mitogen-stimulated lymphocytes but inhibit cell growth and immunoglobulin secretion. This study shows that the 4-quinolones strongly (up to 100 times) increase the recovery of interleukin 2 (IL-2) in culture supernatants of phytohemagglutinin (PHA)-stimulated normal human lymphocytes and also prolong the kinetics of IL-2 production. The effect was significant at clinically achievable concentrations (5 micrograms/ml). In addition to hyperproduction of IL-2, the level of RNA hybridizing with a human IL-2 cDNA probe was also intensely elevated (16-32 times) in PHA-stimulated lymphocytes cultured with ciprofloxacin (80 micrograms/ml). The mechanism responsible for 4-quinolone-mediated effects on T cells is at present unclear, but evidence is presented that suggests the effect is not exerted at the level of protein kinase C activation. Ciprofloxacin at 80 micrograms/ml also decreased the expression of IL-2 receptors measured by immunofluorescence with CD 25 antibodies and a radiolabeled IL-2 binding assay. At the same concentration of ciprofloxacin, there was a very low expression of the transferrin receptor and the cell size increased very little in human lymphocytes after PHA stimulation. The enhanced IL-2 production by 4-quinolones may contribute to side effects reported when these drugs are used for treatment of patients.     

Glucocorticoid resistance in chronic asthma. Peripheral blood T lymphocyte activation and comparison of the T lymphocyte inhibitory effects of glucocorticoids and cyclosporin A

A total of 37 chronic severe asthmatic patients with documented reversible airways obstruction were classified as glucocorticoid sensitive or resistant according to changes in the FEV1 following a course of oral prednisolone. The phenotype and expression of activation molecules on peripheral blood T lymphocytes from these patients just before the course of prednisolone were studied using flow cytometry. The resistant patients had significantly elevated percentages of T lymphocytes expressing the activation molecules IL-2R and HLA-DR compared to the sensitive patients. There were no differences between the patient groups in the percentages of peripheral blood T lymphocytes expressing the phenotypic markers CD4 and CD8. Peripheral blood mononuclear cells (PBMC) from 29 patients were cultured in vitro with the T lymphocyte mitogen PHA in the presence or absence of dexamethasone or cyclosporin A. Dexamethasone (10(-7) mol/L) significantly inhibited the proliferation of T lymphocytes from the sensitive but not the resistant asthmatic subjects. In contrast, cyclosporin A (500 ng/ml) inhibited proliferation of T lymphocytes from both the sensitive and the resistant asthmatic subjects, although the effect was less marked in the latter group. Inhibition of elaboration of interleukin-2 and interferon-gamma by mitogen-stimulated T lymphocytes from sensitive and resistant asthmatic patients was also studied. Dexamethasone (10(-7) mol/L) significantly inhibited the production of interleukin-2 and interferon-gamma by proliferating T lymphocytes isolated from the glucocorticoid-sensitive but not the resistant chronic asthmatic patients. Cyclosporin A (500 ng/ml) inhibited the elaboration of both lymphokines by T lymphocytes derived from both patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the phytohemagglutinin-induced proliferating lymphocyte subpopulations in chronic lymphocytic leukemia patients using a clonogenic agar technique and monoclonal antibodies

Peripheral blood lymphocytes from normal donors and patients with chronic lymphocytic leukemia, B-cell type, were purified into T, helper T, and suppressor T lymphocytes by fluorescence-activated cell sorting using OKT3, OKT4, and OKT8 monoclonal antibodies. The maximum response of the purified subpopulations to stimulation by phytohemagglutinin (PHA) was determined by measuring the production of colonies when the stimulated cells were grown on agar. The helper T cells in normal and CLL patients were the most responsive to PHA stimulation, although the responsiveness of helper T cells to PHA was decreased in CLL. Purified CLL B cells responded minimally to PHA stimulation, but normal B lymphocytes did not. The abnormal response of CLL lymphocytes to PHA appears to be due abnormal helper T cells, and, to a smaller extent, to the ability of CLL B lymphocytes to respond.     

T cells in B-cell chronic lymphocytic leukemia: quantitative assessment of cytotoxic and interleukin-2-producing lymphocyte precursors by limiting dilution analysis

Controversy exists as to the functional capacity of T lymphocytes in patients with B-cell chronic lymphocytic leukemia (CLL). We have used a limiting dilution (LD) culture approach to quantitatively assess frequencies of proliferating lymphocyte precursors (PLP), cytotoxic lymphocyte precursors (CLP), and interleukin-2 (IL-2)-producing helper lymphocyte precursors (HLP). Unseparated mononuclear cells (MNC) or purified T cells (E+) and leukemic B cells (E-) were cocultured under LD conditions with irradiated OKT3 hybridoma cells in the absence (determination of HLP) or presence of recombinant IL-2 (determination of PLP and CLP). Under these conditions, low frequencies of PLP, HLP, and CLP (f = 1/65 to 1/4600) were measured in unseparated MNC of CLL patients. In contrast, purified T cells (50% to 92% CD3+) contained precursors of proliferating, IL-2-producing and cytotoxic T cells in similar frequency as did T cells from healthy control donors (f = 1/4 to 1/24). Leukemic B cells rigorously depleted of T cells did not give rise to measurable frequencies of PLP, HLP, or CLP (f less than 1/50.000) except in one CLL patient where a significant frequency (f = 1/1700) of HLP was consistently present in E- cells, despite the absence of growth-inducible PLP and CLP. Taken together, these results indicate that comparable numbers of IL-2-producing helper T cells and cytotoxic T cells are present in B-CLL patients and healthy controls, respectively. The data are discussed with respect to reported T cell abnormalities in B-CLL.     

Lamina propria T cell activation: role of the costimulatory molecule CD2 and its cytoplasmic tail for the regulation of proliferation and apoptosis

Background/aims Accumulation of T lymphocytes in the gut is a hallmark of inflammatory bowel disease probably caused by insufficient T cell apoptosis. Activated peripheral T cells, or “resting” lamina propria T lymphocytes (LPLs), are highly susceptible to apoptosis induction, e.g., using the mitogenic anti-CD2 monoclonal antibody (mAb) pair T11₂₊₃. It is, however, unknown how CD2-mediated LPL apoptosis is related to proliferation and whether the whole CD2 molecule is required for apoptosis induction.Materials and methods Mapping of anti-CD2 mAb was performed using erythrocyte rosetting assays and cross-blocking enzyme-linked immunosorbent assay (ELISA). Lamina propria mononuclear cells (LPMNCs) or phytohemagglutinin (PHA) blasts were stimulated with a panel of 18 anti-CD2 mAbs followed by apoptosis analysis [Annexin V expression on propidium iodide (PI)-negative cells, 4c6-diamidino-2-phenylindole·2HCl (DAPI) staining]. Proliferation was measured by [³H]-thymidine incorporation. For structural analysis, EL4 cells were used which were transfected with human CD2 (wild type (WT), cytoplasmic-deficient, cytoplasmic CD28). Sorting was performed employing standard techniquesResults All three mitogenic anti-CD2 mAb pairs induced apoptosis of LPMNC and PHA blasts. Two out of four submitogenic anti-CD2 mAb, AICD2.M3, and ICRFCD2.3 lead to LPMNC proliferation but no apoptosis. Importantly, apoptosis was also detected in cytoplasmic-deficient CD2 tg or CD2/CD2/CD28 tg EL4 cells. Sorted CD45^high huCD2 WT EL4 had higher apoptosis rates compared to WT huCD2tg EL4 cellsConclusion LPMNC apoptosis induction via CD2 is always associated with proliferation, although proliferation is not necessarily associated with apoptosis. The cytoplasmic tail of CD2 is not required, and CD45 appears to transmit apoptotic signals entering the T cell via CD2.     

Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haemopoietic cell development

Basic fibroblast growth factor (FGF-2) and acidic fibroblast growth factor (FGF-1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF-1 or 2 on normal human marrow cell proliferation in vitro , only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34⁺ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF-R) expression on CD34⁺ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF-1 or 2 on survival of early progenitor cells in serum-free culture. To explain these findings, we examined progenitor cells for expression of the FGF-R at the mRNA and protein level using RT-PCR and flow cytometry. Primitive CD34⁺/KIT⁺ MNC had no detectable FGF-R (FGF-R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF-R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF-R1 expression was detected on subpopulations of MNC and on cells derived from day-6 CFU-GM and BFU-E colonies. Accordingly, FGF-R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF-Rs are expressed on more mature cells.     

Effect of a short-term fast on hepatic microsomal glutathione-insulin transhydrogenase

The effect of nutritional state on the hepatic insulin degrading enzyme glutathione-insulin transhydrogenase (GIT) was assessed by comparing the distribution of GIT activity between its nonlatent and latent forms in fractionated liver microsomes from ad lib fed (n = 11) and overnight fasted (n = 11) rats. In fed state microsomes, treatment with the membrane disrupting agent phospholipase-A2 (PLA2) over a range of PLA2 concentrations (less than or equal to 2.0 micrograms/ml) caused biphasic release of GIT with a peak activity of 651 +/- 58 U/mg microsomal protein (n = 11) occurring at PLA2 = 1.0 microgram/ml. In total liver microsomes from fasted animals, GIT release in response to PLA2 was sigmoidal over the entire range of PLA2 concentrations, with a plateau of activities (450 U/mg microsomal protein) occurring at PLA2 greater than or equal to 0.75 microgram/ml. Peak activities (478 +/- 88 U/mg prot., n = 11, PLA2 = 1.0 microgram/ml) were 30% lower as compared to the fed state (p less than .05). In untreated (intact) microsomes from fed rat liver nonlatent activity was 126 +/- 8 U/mg protein, representing 19.9 +/- 1.2% of the total GIT activity. In contrast, nonlatent activity measurable in suspensions of intact microsomes from fasted rat liver (110 +/- 6 U/mg) expressed as a % of total activity was significantly increased (p less than .05) being 23.3 +/- 1.1%. Similar fasting-induced changes were also apparent in isolated smooth microsomes but not in rough membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hepatocyte growth factor on early human haemopoietic cell development

Hepatocyte growth factor (HGF) stimulates cell proliferation, differentiation and migration by binding to its receptor, MET R. Whether the HGF/MET R axis plays an important regulatory role in human haemopoietic cell growth is an unresolved issue. To investigate this situation, we employed several complementary strategies including RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). We found that very primitive, FACS sorted, CD34⁺ Kit⁺ marrow mononuclear cells (MNC) failed to express RT-PCR detectable MET R mRNA. In contrast, MET R expression was easily detectable by RT-PCR in marrow stroma fibroblasts, in cells isolated from BFU-E and CFU-GM colonies, and in unselected normal MNC. Subsequent FACS analysis revealed that MET R protein was detectable on ∼5% of the latter cells. HGF, at concentrations of 1–50 ng/ml, had no demonstrable effect on survival or cloning efficiency of normal CD34⁺ MNC in serum-free cultures. Antisense ODN mediated perturbation of MET R mRNA expression in normal CD34⁺ MNC, with FACS documented decline in protein expression, had no effect on the ability of these cells to give rise to haemopoietic colonies of any lineage. We also examined the biology of HGF/MET R expression in malignant haemopoietic cells. Using the strategies described above, we found that MET R mRNA was expressed in many human haemopoietic cell lines, and that the protein was expressed at high levels on HTLV transformed T lymphocytes. Wild-type CML and AML blast cells also expressed MET mRNA, and HGF was able to co-stimulate CFU-GM colony formation in ∼20% of cases studied. Therefore, although the HGF/MET R axis appears to be dispensable for normal haemopoietic cell growth, it may play a role in the growth of malignant haemopoietic progenitor cells.