Effects of levamisole on T lymphocyte colony formation by cells from bone marrow aspirates.

Levamisole has been added to cultures of bone marrow and peripheral blood mononuclear cells stimulated by phytohaemagglutinin (PHA). T lymphocytes colony-forming cells (TL-CFC) in the peripheral blood produce a characteristic biphasic dose--response curve in cultures containing levamisole but the characteristics of the dose--response curve seen when levamisole is added to mononuclear cells obtained from bone marrow aspirates are strikingly different. The results suggest that the mixtures of bone marrow and blood obtained by aspiration contain different proportions of the cells which influence TL-CFC than either blood, or by implication, bone marrow alone.     

脐带血的T淋巴细胞集落培养

本文应用细胞培养和免疫间接荧光法测定脐血，正常人体外周血和成人骨髓的Ｔ淋巴细胞集落形成情况及培养前后淋巴细胞亚群的变化，结果表明：脐血的ＣＦｕ－ＴＬ显著低于正常人外周血（Ｐ〈０．０１），略低于成人骨髓（Ｐ〉０．０５）培养脐血的ＣＤ３，ＣＤ４细胞含量均显著低于正常人外周血（Ｐ〈０．０１），与成人骨髓相近似（Ｐ〉０．０５）脐血的ＣＤ８细胞含量与正常人外周血和成人骨髓相似（Ｐ〉０．０５）；培养后的ＣＦｕ     

Erythroid colony growth from peripheral blood and bone marrow in polycythaemia.

Erythroid colony growth in the presence and absence of erythropoietin was compared in 23 patients with primary proliferative polycythaemia (PPP), nine with idiopathic erythrocytosis, 10 with secondary polycythaemia, 15 with pseudopolycythaemia and in 76 normal subjects. Erythroid colonies growing without erythropoietin stimulation (endogenous erythroid colonies) from peripheral blood (BFU-E) were found in 20 of 22 patients with PPP and in two of seven with idiopathic erythrocytosis. None was found in secondary polycythaemia, pseudopolycythaemia, or in normal subjects. Small numbers of endogenous colony forming units-erythroid (CFU-E) (though not BFU-E) were cultured from the bone marrow of three of 24 normal subjects, suggesting that peripheral blood cultures provide a more specific indicator of clonal erythropoiesis. Peripheral blood endogenous erythroid colony growth is an effective and convenient means of distinguishing patients with clonal erythrocytosis and may be of particular value when iron deficiency obscures the diagnosis of PPP on conventional criteria.     

Inhibitory effects of peripheral blood cells on in vitro colony formation by autologous bone marrow in aplastic anaemia: relation with response to immunosuppressive therapy.

The inhibitory activity of peripheral blood lymphocytes on autologous bone marrow was studied in 27 patients with aplastic anaemia after treatment with androgen. Inhibitory activity was hard to assess in 10 patients studied during the first year of treatment. The colony count was too low to be certain of differences between the samples incubated with or without lymphocytes. Among the 17 patients who had more than 10 colonies per 2 x 10(5) mononuclear bone marrow cells, nine showed inhibitory activity by peripheral blood lymphocytes. After 12 months of androgen therapy each of these patients showing inhibitory activity of bone marrow colony forming cells by peripheral lymphocytes responded to antithymocyte globulin. None of nine patients with few colony forming cells or no inhibitory activity of lymphocytes responded to immunosuppression.     

The effect of cyclosporin A on peripheral blood T cell subpopulations in renal allografts.

Treatment with cyclosporin A (CyA) produces a reversal of the normal ratio of OKT4+ (inducer type) to OKT84 (suppressor-cytotoxic type) cells so that renal allograft recipients on CyA alone develop a four-fold increase in the absolute number of circulating OKT8 positive cells. Conventional immunosuppression with azathioprine and prednisolone reduces both populations of T cells without altering the ratio of OKT4+ to OKT8+ cells. This effect of CyA may help to explain its action as an immunosuppressive agent.     

Inhibition of human T lymphocyte colony formation by methylprednisolone

Methylprednisolone, at very low concentrations, inhibits colony formation by PHA-stimulated human blood lymphocytes in vitro. The inhibition is exerted at an early stage in transformation. It results not from direct effects on colony-forming cells but from inhibition of production of a soluble factor produced by accessory cells.     

Genetic influence on peripheral blood T lymphocyte levels

T lymphocytes are a major component of the adaptive immune system. CD4 positive T cell subpopulations regulate B cell and macrophage effector function while CD8 positive T cells are largely responsible for anti-viral cytotoxic activity. The degree of natural variation in the levels and ratios of the various T cell subpopulations is a possible risk factor for the development of autoimmune disease, infectious disease and cancer. There is some evidence from studies of inbred strains of mice and humans which suggests that variation in T cell subpopulations is genetically influenced. However, family studies alone cannot distinguish between common environmental and shared genetic influences and provide less robust estimates of the heritability than twin studies. To comprehensively examine genetic influences on a selection of important T cell phenotypes, we investigated variation in levels of total lymphocytes, CD3+, CD4+, CD8+, CD3+CD4+, CD3+CD8+ lymphocytes and in CD4:CD8 ratio as a proportion of lymphocytes and of T cells using the classical twin model approach. Healthy female twin pairs were sampled from the St. Thomas' UK Adult Twin Registry. A maximum of 103 monozygotic (MZ) and 186 dizygotic (DZ) twins aged 18-80 years participated in the study. Whole blood samples were analysed for T cell subsets by flow cytometry. The relative genetic contribution to these phenotypes was estimated using a variance components model-fitting approach. Heritability estimates were calculated of 65% for CD4:CD8 T cell and lymphocyte ratios, around 50% for absolute lymphocyte, CD3+ and CD4+ counts, and 56% for CD8+ numbers. Unique (rather than shared) familial environment explains the remainder of the variance. Genetic factors have a major influence on the variation in peripheral T cell subset numbers. Polymorphism dictating such variation should be taken into account when assessing risk factors for T cell immune-mediated disease with a genetic background.     

Suppressor effect of prostaglandins on T colony formation

This study evaluated the action of prostaglandins on T colony formation. A single step culture process was used which involved direct seeding of freshly isolated peripheral blood mononuclear cells (MC) in semi-solid agar culture medium containing phytohaemagglutinin (PHA). Suppression of endogenous production of prostaglandins with 10(-6) M indomethacin increased T colony formation by up to 100%. Similarly, addition of synthetic prostaglandin E (PGE) to the culture system demonstrated a dose-dependent reduction of T colony formation by PHA-stimulated non-adherent cells. The 50% inhibitory dose (ID 50) was 10(-7) M for PGE2 and 1.3 x 10(-7) M for PGE1. Prostaglandin F had no effect on T colony formation. The synthesis of PGE by adherent cells can be increased two- to three-fold in the presence of T colony promoting activity released by PHA-stimulated lymphocytes. We conclude that monocyte produced PGE is responsible for the suppressor effect exerted by these cells on T colony formation. The PGE inhibitory role is interpreted as a feedback mechanism, modulated by lymphokines released by PHA-activated lymphocytes.     

Inhibitory effect of human blood granulocyte glycosaminoglycans on colony-forming activity of bone marrow fibroblasts

Inhibitory effect of glycosaminoglycans isolated from peripheral blood granulocytes of patients with chronic myeloleukemia and normal subjects on increment in colony-forming units of fibroblasts in monolayer cultures of rat bone marrow is determined by the degree of sulfation of their main component chondroitin-4-sulfate and by increased content of heparan sulfate. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 3, pp. 319–322, March, 1998     

Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoproterenol

The present study was designed to elucidate the mode of action of isoproterenol (Isp; adrenergic beta-agonist) and to characterize the effect of the calcitonin gene-related peptide (CGRP; sensory neuropeptide) on osteoclast formation induced by Isp in a mouse bone marrow culture system. Treatment of mouse bone marrow cells with Isp generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) capable of excavating resorptive pits on dentine slices, and caused an increase in receptor activator of NF-kappaB ligand (RANKL) and a decrease in osteoprotegerin (OPG) production by the marrow cells. The osteoclast formation was significantly inhibited by OPG, suggesting the involvement of the RANKL-RANK system. CGRP inhibited the osteoclast formation caused by Isp or soluble RANKL (s-RANKL) but had no influence on RANKL or OPG production by the bone marrow cells treated with Isp, suggesting that CGRP inhibited the osteoclast formation by interfering with the action of RANKL produced by the Isp-treated bone marrow cells without affecting RANKL or OPG production. This in vitro data suggest the physiological interaction of sympathetic and sensory nerves in osteoclastogenesis in vivo.     

人外周血T淋巴细胞自噬现象的观察

目的：观察分离培养的人外周血T淋巴细胞的自噬现象。方法：密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞，分空白组及地塞米松（DXM）组，培养72小时后观察细胞光镜及电镜形态学、MDC荧光染色，并用流式细胞仪检测自噬细胞比例变化。结果：①通过自然培养后人外周血T淋巴细胞可出现典型自噬细胞形态学改变。②空白组及DXM组72小时自噬细胞发生率与0小时比较有显著性差异。③DXM组与空白组72小时自噬细胞发生率有显著性差异。结论：人外周血T淋巴细胞存在自噬现象，DXM可诱导人外周血T淋巴细胞自噬。     

蚕蚀性角膜溃疡患者外周血淋巴细胞免疫表型和T细胞活化的研究

目的：观察蚕蚀性角膜溃疡患者外周血淋巴细胞免疫表到和T细胞活化的变化,进一步探讨本病患者的免疫功能。方法：用流式细胞术检测8例活动性蚕蚀性角膜溃病患者外周血淋巴细胞对项免疫表型变化,用植物凝集素和沸被醉酯作为刺激剂,以HLA－DR,CD69、CD71、CD25的表达为T细胞活化的指标,用流式细胞术分析。结果：蚕蚀性角膜溃疡患者外周血CD4、CD5、CD8、CD10、CD11b、CD15、CD16、CD19、CD23、CD25、CD41、CD69、HLA-DP、HLADR抗原表达阳性淋巴细胞以及HLA－DR、CD69、CD71、CD25在CD3和CD8细胞的表达均明显高于正常对照组。结论：蚕蚀性角膜溃疡患者具有增强的细胞免疫和体液免疫,外周血T细胞及其亚群出现异常活化,反映患者体内存在自身免疫反应而支持该病发病之自身免疫学说。     

Humoral nature of the colony-stimulating action of bone marrow cells on stromal colony formation in bone marrow culture

N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR D. S. Sarkisov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 11, pp. 509–510, November, 1990.     

Benzene-induced micronuclei formation in mouse fetal liver blood, peripheral blood, and maternal bone marrow cells

The transplacental cytogenetic effects of benzene were studied by using the micronucleus test of polychromatic erythrocytes (PCE) found in both fetal liver and fetal peripheral blood, and were compared with PCE from maternal bone marrow. Timed-pregnant mice received single intraperitoneal doses of benzene (0, 109, 219, 437, or 874 mg/kg bw) on the 14th day of gestation and were sacrificed 21 hr after injection. Benzene elicited a significant increase (P less than 0.01) in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in fetal liver blood cells (0.55 to 1.36%, control 0.18%) at doses of 219 to 874 mg/kg, and in fetal peripheral blood cells (0.49 to 0.58%, control 0.25%) and maternal bone marrow cells (0.53 to 0.70%, control 0.10%) at doses of 437 and 874 mg/kg. The data demonstrate that benzene is a moderate transplacental clastogenic agent, and that the mouse transplacental micronucleus test using fetal liver blood cells is a potentially more sensitive indicator of the genotoxicity of benzene than either fetal peripheral blood or maternal bone marrow cells.     

Comparison of phorbol myristate acetate and phytohaemagglutinin as stimulators of in vitro T lymphocyte colony formation of human peripheral blood lymphocytes. I. Surface markers of colony cells.

Human T lymphocyte colonies were grown in methylcellulose semi-solid cultures in the presence of phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA). Surface marker analysis showed lower percentages of OKT3- and OKT4-positive cells in PMA-induced colonies than those in PHA-induced colonies. The percentage of OKIa1-positive cells in PMA-induced colonies was approximately twice that in PHA-induced colonies. The percentage of OKT9-positive cells in PMA-induced colonies was significantly lower than that in PHA-induced colonies. These data suggest that the subsets of PMA-induced colony cells express a more immature phenotype than that of PHA-induced colony cells and that, among PMA-induced colony cells, there are fewer T cells in the proliferative status at the time tested. When 3 X 10(5)/ml monocyte-depleted T cells, at which concentration of seeded cells neither PHA nor PMA could induce colony growth, were cultured in the presence of both PHA and PMA, T cell colony growth was observed. In T cell colonies induced by a combination of PHA and PMA, the percentages of OKT3-, OKT4- and OKT8-positive cells were different from those in colonies induced by either PHA or PMA alone. These results suggest that PMA acts not only as a substitute for monocytes and/or interleukin-1, but may directly affect lymphocyte proliferation induced by a combination of PHA and PMA.     

T lymphocyte reconstitution following autologous bone marrow transplantation.

Peripheral blood T lymphocyte subpopulations were analysed using OKT3, OKT4 and OKT8 monoclonal antibodies in eleven patients undergoing autologous bone marrow transplantation following high dose chemotherapy +/- total body irradiation for bronchial carcinoma or as intensification therapy for acute myeloblastic leukaemia. Marked inversion of the OKT4:OKT8 ratio was noted in all patients following the procedure. This is due to both a fall in the number of OKT4 positive cells and a rise in the number of OKT8 positive cells in all but one patient. Sequential analysis of six patients shows a return to the normal ratio in two with time and a trend towards normalization in the others. We suggest that the subpopulation inversion noted following bone marrow transplantation is an effect of marrow regeneration and is not causally related to graft versus host disease.     

T lymphocyte regeneration after transplantation of T cell depleted allogeneic bone marrow

The regeneration of T cell subsets was studied with double immunofluorescence marker methods in 37 patients who received HLA matched T lymphocyte depleted bone marrow transplants (BMT) as part of the treatment for their haematological disease. A cocktail of anti-pan-T (CD6: MBG6) and anti-suppressor/cytotoxic-T cell (CD8: RFT8) monoclonal antibodies was used with rabbit serum as a source of cytolytic complement to achieve selective T cell lysis. The T8+ cells reached low normal values around 60 days post-transplant and remained within the normal range throughout the study (greater than 150 days). This observation is in contrast to our previously published results in patients who, after receiving BMT without efficient T cell depletion, had increased numbers of circulation T8+ cells from 60 days post-transplant. In the present study Leu-7+, RFT8- cells reached normal values rapidly but the reconstitution of T4+ lymphocytes was slow: low normal levels were reached only around day 150 following BMT. The degree of graft-versus-host disease (GVHD) seemed to be related to the number of residual T cells infused: two of the three patients who received the highest numbers of T cells developed Grade II III; otherwise GVHD was minimal. Among the clinical parameters studied cytomegalovirus (CMV) immune status moderately influenced reconstitution: at 55-90 days post-transplant T8+ cells were present at the upper normal levels in seven out of 15 patients receiving BMT from CMV seronegative donors, but in none of the 16 individuals receiving BMT from seropositive donors. CMV related complications were relatively uncommon. Thus the most significant factor in preventing 'T8+ cell overshoot' and T cell imbalance during regeneration appears to be the depletion of T (including T8+) lymphocytes from marrow. The differences of T8+ cell reconstitution in this and previous studies may reflect a different regeneration pattern from T cell precursors as opposed to inoculated mature T cells.