In vitro hemopoiesis in human micro long-term bone marrow cultures recharged with either allogeneic, T-cell-depleted allogeneic, or syngeneic bone marrow cells

The production of granulocyte-macrophage colony-forming cells (GM-CFC) and the proliferation period in human long-term bone marrow cultures are inferior to murine cultures. There is also evidence that recharge of the cultures after establishing confluent stromal layers will not greatly improve myelopoiesis. Data in the literature indicate that PHA-responsive T lymphocytes persist for up to 5 weeks in human but not in murine long-term marrow cultures. We therefore analyzed the effects of recharging micro long-term bone marrow cultures with bone marrow cell samples depleted by T lymphocytes. Depletion was performed in a complement-mediated cytotoxicity assay by applying the monoclonal antibody CAMPATH-1. Our data show that regardless of whether T cells were removed only at recharge, at both initiation and recharge, or only at initiation, obvious enhancement could neither be achieved in the GM-CFC production nor in the proliferation period. Furthermore, no advantage was seen when using syngeneic marrow cells. We conclude that in allogeneic long-term marrow cultures hemopoiesis is not limited by immunological incompatibilities.     

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.     

Inhibitory effect of NPY on isoprenaline-induced osteoclastogenesis in mouse bone marrow cells

The effect of neuropeptide Y (NPY), a co-transmitter with noradrenaline in peripheral sympathetic nerve fibers, on the osteoclastogenesis in mouse bone marrow cell cultures treated with isoprenaline, a beta-adrenergic receptor (beta-AR) agonist, was examined. The mouse bone marrow cells constitutively expressed mRNAs for the NPY-Y1 receptor and beta2-AR. NPY inhibited the formation of osteoclast-like cells induced by isoprenaline but not that by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) or soluble receptor activator of nuclear factor-kappaB ligand (RANKL); and it suppressed the production of RANKL and cyclic AMP (cAMP) increased by isoprenaline but not those increased by 1alpha,25(OH)2D3. NPY also inhibited osteoclastogenesis induced by forskolin, an activator of adenylate cyclase; however, it did not inhibit that induced by exogenously supplied dibutyryl cAMP, a cell-permeable cAMP analog that activates cAMP-dependent protein kinase. These results demonstrate that NPY inhibited the isoprenaline-induced osteoclastogenesis by blocking the agonist-elicited increases in the production of cAMP and RANKL in mouse bone marrow cells, suggesting an interaction between NPY and beta-AR agonist in bone resorption.     

The effect of allogeneic spleen cells on the induction of immunologic tolerance

It is shown, in this study, that the transfer of parental immunocompetent spleen cells to F₁ hybrid mice interferes with the induction of tolerance to human gamma globulin (HGG) in the F₁ recipients. The transfer of syngeneic spleen cells did not cause this interference, but the transfer of HGG-tolerant parental spleen cells was as effective as that of their normal counterparts. Hence, the interference with tolerance induction was attributed to the histoincompatibility of donor and host cells and, more specifically, to the graft-vs-host reaction. A mechanism of interference with the expression of tolerance by an antigen-specific T-cell bypass is discussed.     

Acute cytogenetic effect of benzene on rat bone marrow cells in vivo and the effect of inducers or inhibitors of drug-metabolizing enzymes.

Acute cytogenetic effects of benzene in LE rat bone marrow cells in vivo were studied. Chromosome aberrations (CA) induced by benzene consisted mainly of gaps and breaks. Cells with exchanges were rarely observed. The incidence of benzene-induced CA was at its maximum level 12 h after the p.o. or i.p. administration of benzene, dependent on the dose of benzene administered, and higher in male rats than in female rats. However, the sex difference was not observed in the repeated inhalation experiment. Chromosome damage was higher with the p.o. than the i.p. administration. LE rats were more sensitive than Wistar and SD rats to the clastogenic action of benzene. Phenobarbital and Sudan III are well known as inducers of drug-metabolizing enzymes. The peak percentage of benzene-induced CA in the rats pretreated with phenobarbital was observed 6 h after the benzene injection, and it occurred at a higher level than in the rats given only benzene. On the other hand, Sudan III pretreatment suppressed benzene-induced CA at all periods after the benzene injection. SKF-525A (a cytochrome P-450 inhibitor) and cyclohexene oxide (an epoxide hydrase inhibitor) pretreatment also suppressed benzene-induced CA.     

Differences in antibody production after adoptive transfers of syngeneic and allogeneic spleen cells to irradiated mice

Immunologically competent spleen cells from syngeneic or allogeneic donors were transferred to supralethally irradiated mice. At the same time, or 72 h after transfer, sheep red cells were administered intraperitoneally to the hosts. Five days after administration of the sheep red cells, antibody production was determined in host spleen fragments by the method of haemolysis in agarose. It was found that an incompatible allogeneic environment markedly stimulated proliferation of the donor cells and that proliferation of clones of cells reacting specifically to sheep red cells by antibody production was also stimulated.     

Effects of transplanted bone marrow mesenchymal stem cells on the irradiated intestine of mice

We investigated the potency of exogenous bone marrow mesenchymal stem cells (MSCs) to engraft into irradiated intestine, as well as these cells’ effects on radiation-induced enteric injury. MSCs from β-Gal-transgenic mice were transplanted into C57BL/6J recipient mice that received abdominal irradiation (13 Gy). At different time points, recipient intestines were examined for the engraftment of donor-derived cells by immunofluorescence analysis. Additionally, the expression status of chemokines induced by radiation injury was analyzed in the irradiated intestine. Next, MSCs were transduced with an adenoviral vector encoding a certain chemokine receptor gene in order to promote the engraftment rate via chemotaxis. The intestinal permeability and histomorphological alterations were measured to evaluate the therapeutic effect of MSC transplantation. The results demonstrated that infused MSCs possessed the potency to engraft into irradiated enteric mucosa, but the engraftment rate was too low to produce a therapeutic effect. The expression of stromal cell-derived factor-1 (SDF-1) was up-regulated in irradiated intestine. MSCs genetically modified by CXCR4 (the receptor for SDF-1) engrafted into irradiated intestine at a significantly elevated level and ameliorated the intestinal permeability and histopathological damage.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Usefulness of Y-body study on bone marrow smears to demonstrate the origin of fibroblast stromal cells in allogeneic bone marrow transplantation

The value of Y-body study for assessment of stromal cell engraftment was analyzed in 25 patients submitted to allogeneic bone marrow transplantation (BMT) (sex-matched in 12 cases and sex-mismatched in 13). The study was performed weekly on bone marrow smears until day +35, and the results were compared with those obtained in a control group of 20 patients submitted to autologous BMT (12 males and 8 females). Engraftment of haemopoietic cells was documented in all cases. The results of Y-body study on the recipients' fibroblast cells showed a pattern identical to that observed prior to BMT, independent of donor's sex. On the other hand, there were no differences between allogeneic and autologous BMT recipients in regard to percentage of Y-body positive cells. These results indicate that in allogeneic BMT there is no engraftment of the fibroblastic component of bone marrow stroma.     

The inhibitory effect of hydrocortisone on interferon production by rat spleen cells

The effect of hydrocortisone on interferon r(IFN-r) production by rat spleen cells and its mechanism were studied. The results showed that hydrocortisone inhibited IFN-r production at concentrations as low as 5.52 x 10(-10) M, with complete suppression at 5.52 x 10(-8) M, and the total number and survival rate of the cultured spleen cells were not apparently affected by 5.52 x 10(-8) M hydrocortisone. The inhibitory effect was dose-dependent when the concentration was from 5.52 x 10(-10) M to 5.52 x 10(-8) M and could be blocked by RU38486, a competitive antagonist of glucocorticoid. Our results suggested that glucocorticoid may inhibit IFN-r production through a receptor-mediated mechanism.