Cytostatic and cytotoxic effects of tumor necrosis factor alpha on MCF-7 human breast tumor cells are differently inhibited by glucocorticoid hormones.

To investigate the mechanisms of growth inhibition exerted by TNF-alpha on tumor cells in vitro, we analyzed the cytokine effects on growth and cell-cycle parameters of cultured MCF-7 human breast cancer cells. TNF-alpha exerted a dose-dependent inhibition of MCF-7 cell growth, which reached its maximum at 1000 U/ml TNF-alpha concentrations. Flow-cytometric analysis of cell nuclei revealed two main components in TNF-alpha activity: an earlier cytostatic effect (G1/S block), was followed by nuclear shrinkage and cytolysis.

Cellular stress and glucocorticoid hormones protect L929 mouse fibroblasts from tumor necrosis factor alpha cytotoxicity.

Adaptive responses to the environment depend on the induction of the "stress response" in less differentiated organisms and cultured cells and the activation of the hypothalamic-pituitary-adrenal axis in animals and humans. This indicates that adrenal steroids and stress proteins play an important role in regulating cell survival in response to noxious stimuli. In an in vitro model, we analyzed the effects of either dexamethasone (DEX) treatment or environmental changes which can elicit a stress response, on the survival of cultured L-929 mouse fibroblasts exposed to the cytotoxic cytokine tumor necrosis factor alpha (TNF-alpha).

Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis.

Glucocorticoid hormones (GCH) and anti-CD3 monoclonal antibodies (MoAbs) induce in mouse thymocytes and T-cell tumor lines an active process of cell death called apoptosis. Interleukins (IL), including IL-1 and IL-2, have been reported to inhibit such apoptosis. In this study we show that IL-4 also reduced the DNA fragmentation characteristic of dexamethasone (DEX)-induced apoptosis in thymocytes.

PMA inhibits NK cell generation, cytotoxic activity and NK-1.1 expression.

We investigated the role of protein kinase C activator phorbol 12-myristate 13-acetate (PMA) on IL-2-driven NK cell differentiation, by using an in vitro model previously set up by our laboratory. Bone marrow precursor cells, from mice treated with 5-fluorouracil (FUBM), when cultured with IL-2, generated mature NK cells. The biochemical system involved in this process has not yet been defined.

Interleukin-2 induces apoptosis in mouse thymocytes.

Interleukins play a role in the process of T-cell development and, like other cytokines, seem able to modulate apoptosis. Interleukin-2 has been reported to inhibit apoptotic cell death of thymocytes induced in vitro by either activation of CD3/TCR complex or treatment with glucocorticoid hormone. We demonstrate here that IL-2 can provoke DNA fragmentation and cell death of CD4+ CD8+ mouse thymocytes by activating an endogenous apoptotic pathway.

Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival.

The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect.

Long-term cultures of mouse bone marrow cells: a model for studying the generation of natural killer cells.

We investigated the generation of natural killer (NK) cells, using a long-term bone marrow culture (LTBMC) system. Mouse bone marrow cells were cultured for 2 weeks in complete medium without growth factors to obtain an enriched population of NK precursor cells. When these cells were recultured in the presence of interleukin-2 (IL-2) and conditioned medium (CM) from LTBMC, lytic NK cells were generated within 4 days.

Growth and spread of human malignant T lymphoblasts in immunosuppressed nude mice: a model for meningeal leukemia.

Previous work has shown that nude (nu/nu) mice additionally immunosuppressed by splenectomy, sublethal irradiation, and treatment with antiasialo GM1 antiserum (SIA-nu/nu mice) have no detectable natural killer activity and allow the growth of human malignant lymphoblasts. We show here that all SIA-nu/nu mice engrafted intravenously with 5 x 10(6) malignant lymphoblasts originally derived from a child with a T-cell acute lymphoblastic leukemia (PF382) and from a boy with a T-cell lymphoma (ST-4) develop lethal meningeal leukemia and die within 35 days. Histologic examination of moribund SIA-nu/nu mice showed that vertebral and skull bone marrow was always replaced by proliferating human T lymphoblasts.

Heat shock induces apoptosis in mouse thymocytes and protects them from glucocorticoid-induced cell death.

Thymocyte death is a complex phenomenon under the control of different signals and stimuli. We evaluated the effect of elevated temperature (heat shock, HS) on mouse thymocyte apoptosis. Incubation of thymocytes at 43 degrees C for 20 min induced DNA fragmentation and cell death, but it was also able to decrease the apoptosis induced by dexamethasone (DEX), TPA or Ca2+ ionophore.

Glucocorticoid-induced DNA fragmentation: role of protein-kinase-C activity.

Glucocorticoid hormones (GCH) and IL-2 induce apoptotic cell death by a PKC-dependent mechanism. IL-4 counteracts apoptosis by inhibiting PKC activity. GCH and IL-2 show antagonistic effects on apoptosis when administered together.

IL-2-dependent generation of natural killer cells from bone marrow: role of MAC-1-, NK1-1- precursors.

We have previously shown that interleukin-2 (IL-2) is able to induce the generation of natural killer (NK) activity in bone marrow (BM) cell cultures from mice pretreated with 5-fluorouracil (5-FU). Cell fractionation experiments to analyze the nature of BM precursors indicate that MAC-1-, NK1-1- noncytotoxic precursors are induced by IL-2 to proliferate and generate cytolytic NK cells. These data demonstrate that the phenotype and functional characteristics of the IL-2-responsive cells in the FUBM are different from those of mature NK cells in that they are MAC-1+, NK1.

Effect of recombinant murine tumor necrosis factor on the generation of natural killer cells in bone marrow cultures.

We investigated the possible role of tumor necrosis factor-alpha (TNF-alpha) in the interleukin-2 (IL-2)-dependent generation of natural killer (NK) cells from bone marrow precursors. TNF-alpha synergistically augmented both cytotoxic activity against NK-sensitive targets and cell number at the end of the 7-day incubation period. After this time, NK activity was not induced by TNF-alpha in the absence of IL-2.

Interleukins modulate glucocorticoid-induced thymocyte apoptosis.

Glucocorticoid hormones, calcium ionophores and anti-CD3 monoclonal antibodies induce apoptosis in mouse thymocytes. This type of cell death, which is characterized by an extensive DNA fragmentation into oligonucleosomal subunits, occurs in the intrathymic process of negative selection, and is involved in the deletion of autoreactive T-cells during thymic maturation. A number of cytokines are able to modulate apoptosis, and interleukins, including interleukin-1, interleukin-2, and interleukin-4, play a crucial role in thymic maturation and T-cell development.

Immunorestorative properties of ST 789 on experimentally immunosuppressed and infected mice.

ST 789, a newly synthesized chemical characterized by an aminoacidic group joined to the N9 position of the hypoxanthine ring, has recently been shown to be endowed with immunomodulatory properties. In this study we tested ST 789 in vivo for protective effects in Cyclophosphamide-immunosuppressed CD1 mice experimentally infected with several bacterial and fungal pathogens. We found that immunosuppressed mice infected with either fungi or bacteria were significantly protected, as evaluated both by percent mortality and survival time, when treated with doses of ST 789 even as low as 0.

Enhancement of murine NK cell activity generation by ST 789.

We have evaluated the effect of in vivo administration of hypoxanthine derivative ST 789 on the reconstitution of natural killer (NK) cell activity of mice undergoing syngeneic bone marrow (BM) transplantation. Lethally irradiated BD2F1 mice were injected i.v.

Clinical and radiographic evaluation of the Codivilla method of surgical correction of congenital club foot. Medium-term follow-up of 235 cases.

The authors review 30 patients with congenital club foot (CCF) who were treated surgically by a slightly modified Codivilla technique. Clinical morphologic, and functional follow-up was performed an average of 10 years later, and the findings were compared to the radiographic features. The final outcome was rated good in 41% of the cases, fair in 29%, and unsatisfactory in 30%.

Pidotimod stimulates natural killer cell activity and inhibits thymocyte cell death.

Experiments were performed to analyze the possible effect of the immunomodulating agent Pidotimod (3-L-pyroglutamyl-L-thiazolidine-4-carboxylic acid) on mouse Natural Killer (NK) cell activity and glucocorticoid hormone(GCH)-induced thymocyte apoptosis. The results indicate that in vivo treatment with Pidotimod (200 mg/Kg ip for 5 days) causes a significant increase in NK activity and in vitro treatment produces a significant reduction of dexamethasone-induced thymocyte apoptosis. This inhibition appears to be dose-dependent and is also evident against TPA or Ca(++)ionophore-induced apoptosis.

Effect of interleukin-4 on interleukin-2-dependent generation of natural killer cells.

We have previously shown that interleukin-2 (IL-2) is able to induce the generation of natural killer (NK) activity in bone marrow (BM) cells from mice pretreated with 5-fluorouracil. IL-2 alone could dose-dependently induce NK activity in marrow cells and interleukin-4 (IL-4) has dual effect on the NK activity in that, depending on the concentration of IL-2, IL-4 inhibits or stimulates development of NK cells. The inhibitory effect was in part antagonized by interleukin-1 alpha.

A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.

Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts.