Yumiko Ohta

Thymosin α1 exerts protective effect against the 5-FU induced bone marrow toxicity

Abstract Thymosin α 1 was shown to prevent the 5-fluorouracil(5-FU)-induced bone marrow toxicity in BDF 1 mice, as determined by the cellularity, haemopoietic stem cells (CFU-s) and granulocyte-macrophage colony forming unit (GM-CFU). Furthermore, thymosin α 1 increased the levels of colony stimulating factor (CSF) in sera or in culture media of spleen cells derived from 5-FU-treated mice. The treatment of spleen cells with anti-Thy 1,2 antibody plus complement abolished completely the CSF production. The in vivo treatment of donor mice with anti-Thy 1,2 antibody following 5-FU abolished completely the capability of their bone marrow cells to save lethally irradiated recipients. Thymosin α 1 treatment prevented the damage by such combined treatment. The present study indicates that thymosin α 1 exerts its protective effect against the 5-FU-induced bone marrow toxicity, at least partially, through its effect on the maturation of immature T cells to functional T cells which produce various kinds of lymphokines including CSF.

Immunomodulating activity of thymosin fraction 5 and thymosin α1 in immunosuppressed mice

SummaryWe found that both thymosin from calf thymus and its constituent peptide α1 prepared by chemical synthesis restore cell-mediated immunity following its suppression in mice by injection of 5-FU. Conditions suitable for assessing the thymosin activity by means of footpad reaction were established in such immunosuppressed mice. In this new animal model, thymosin α1-peptide showed activity at a low dose of 5–50 μg/kg, which was 100–1,000 times less than that required for thymosin F-5 preparations.Further studies utilizing the adoptive transfer technique showed that α1-peptide corrects the 5-FU-induced suppression of mature T cells, transferring the DTH response as well as that of macrophage function responsible for the expression of footpad reaction. Furthermore, regeneration of lymph node and bone marrow cells as well as CFU-c (progenitor cells of macrophages and granulocytes) was enhanced by thymosin α1 in the 5-FU-treated mice. All these results indicate that thymosin α1 accelerates the replenishment and maturation of haematopoietic cells, including not only T cells but also macrophages, when they have been severely damaged by the 5-FU treatment.

Thymosin α1 enhances haemopoietic colony formation by stimulating the production of interleukin 3 in NU/NU mice

Abstract We have studied the action mechanism of thymosin α 1 in modulation of haemopoietic system. The present study demonstrated that thymosin α 1 enhanced the colony formation as determined by CFU-s, GM-CFU and T-CFU, as well as the production of IL-3, when administered twice a week for a total of six or twelve times into nu/nu mice. The incubation of bone marrow cells with thymosin α 1 in vitro did not cause an increase of CFU-s, in contrast to IL-3 which caused a marked increase of CFU-s during the incubation of 72 h. These experiments indicate that thymosin α 1 exerts its effect on colony formation of haemopoietic stem cells (CFU-s) indirectly through the enhancement of the IL-3 production. The present finding may support clinical applications of thymosin α 1 in a wide range, since IL-3 is known to be the growth factor for many kinds of haemopoietic precursor cells including not only CFU-s but also GM-CFU, BFU-E, Eos-CFU, Meg-CFU, Mast cells, Pre B-cells and Pre T-cells.

Voltage-gated K+ channels in the mouse interleukin 3-dependent cell line, FDC-P2

SummaryThe electrical properties of a mouse interleukin (IL)-3-dependent cell line, FDC-P2, were examined using the tightseal whole-cell clamp technique. Under current clamp conditions with 140mM K+ in the pipette, the cells had a resting potential of ∼−30 mV. Under voltage-clamp conditions, a transient outward current was elicited upon depolarization from a holding potential of −80 mV. The current was activated at potentials more positive than −10 mV and had a delayed-rectifying property. It showed rapid activation and slow inactivation during command steps. The current was abolished by Cs+ in the pipette, indicating that K+ is the charge carrier. The K+ current was suppressed by tetraethylammonium withKi of <0.1mM and was not affected by scorpion toxin. Recovery from inactivation was steeply voltage dependent: As the holding potential was more hyperpolarized, the recovery became faster. Thus, with a holding potential of −80 mV, the current showed slight use-dependent inactivation, while the current decreased prominently by repetitive depolarization at a holding potential of −40 mV. These properties of the K+ current are similar to those of thel-type K+ channel current in mature T lymphocytes. The K+ current in FDC-P2 cells was dramatically reduced after culture in the IL-3-free medium for 1–2 days. When IL-3 was re-added to the medium, the current was re-expressed. These observations suggest that expression of the K+ current depends on extracellular IL-3, and that the current may play some roles in proliferation of these cells.

BINDING SITES OF INTERLEUKIN-3-MIMETIC MONOCLONAL AUTOANTIBODIES DERIVED FROM A MRL/lpr MOUSE

MRL/lpr mouse-derived interleukin-3 (IL-3)-mimetic monoclonal antibodies were examined for their binding sites. One of these five antibodies (B10, F8, F9, F12, H11), F9 interacted with the IL-3 receptor, as if it were an anti-idiotypic antibody; the IL-3-mimetic activity of F9 was blocked by a neutralizing rat monoclonal anti-IL-3 antibody. IL-3 mRNA was not detected in hybridoma F9, as analyzed by the S1 protection assay, Thus, the activity neutralized by the rat antibody is of the F9 antibody itself but not the IL-3 type. Such blocking was not observed with the IL-3-mimetic activity of the other MRL/lpr-derived monoclonal antibodies. On the other hand, the binding of all these monoclonal antibodies to IL-3-dependent cells was inhibited by each other and vice versa, as analyzed by two-color flow cytometry. This indicates that the binding sites of the five monoclonal antibodies are located so close to each other that the binding of one would interfere with the binding of any one of the others (since the binding experiment was done on ice, it is unlikely that the inhibition is due to down-modulation of the receptors). Taken together the results obtained by the enzyme digestion study, we discussed that all five IL-3-mimetic monoclonal antibodies are directed to the IL-3 receptor, but only F9 binds to the portion directly responsible for the binding of IL-3 and the other antibodies (B10, F8, F12, H11) bind to different portions, respectively, which are adjacent or overlapping to the binding site of F9.

Efficacy of Thymosin α1 in Animal Models

The thymus gland produces many factors which are known to play a major role in the differentiation and maturation of T cells (Trainin et al., 1983). Considering the importance of T cells in immunoregulatory systems, thymic factors are expected to be useful as pharmaceutical agents to counteract immunodeficiency states. Thymic factors have been given in many clinical instances and shown to improve immunodeficiency diseases, particularly those with congenital T-cell defects (Wara et al., 1975). However, their efficacy in patients with the secondary immunodeficiencies has not been fully investigated.