Interleukin 2 regulation following semi-allogeneic bone marrow transplantation in mice

Success of allogeneic and autologous bone marrow transplantation (BMT) is hampered by susceptibility to infection during the first two post-treatment years. Further, in treating malignant diseases, impaired anti-host reactivity for donor cells may contribute to a high rate of relapse. Both complications are a consequence of immune deficiency involving B and T lymphocytes. The present study evaluates several key parameters of the immunologic reconstitution mechanism in mice subjected to myeloablative total body irradiation following semi-allogeneic (parental) BMT. This resulted in a gradual reduction of splenic CD3, CD4 and CD8 cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15–20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT.     

Splenocytes and bone marrow cells from T-cell deficient Nu/Nu mice secrete interleukin 3 activity after stimulation in vitro

We show here that the combination of Concanavalin A (Con A), phorbol myristate acetate (PMA), and Ionomycin (Iono) reproducibly stimulated splenocytes from Nu/Nu mice and bone marrow cells from both normal and Nu/Nu mice to secrete interleukin 3 (IL-3) in vitro. IL-3 was measured by its property of supporting the growth of four different clones known to grow only in IL-3. None of the agents indicated above nor several other types of stimuli tested could induce the cells to secrete IL-3 activity. IL-3 activity from induced cells of either tissue was detected after 24 hr of culture, peaked at 48 hr and either declined by 72-96 hr of culture (bone marrow cells) or remained relatively constant through the 4-day culture period (splenocytes). The cells participating in the production of IL-3 activity in Nu/Nu spleen were THY1+, L3T4-, LyT2-, B-220-, J11d-, Ia-, and those in the marrow from either normal or Nu/Nu mice were THY1+, J11d+, L3T4-, LyT2-, B-220-, Ia-. Finally, we present evidence that Ia-positive cells negatively regulate the production of IL-3 activity by both splenocytes and marrow cells. We conclude that Nu/Nu splenocytes and bone marrow cells from both normal and Nu/Nu mice can secrete IL-3 activity after proper stimulation in vitro and that such property is negatively regulated by Ia-positive cells.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

Linomide administration following bone marrow transplantation in mice

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.     

Immunoreconstitution after human bone marrow transplantation

Immunologic reconstitution was studied in 24 patients who underwent bone marrow transplantation, 17 allogenic and 7 autologous. The GVHD prophylaxis consisted of methotrexate and prednisone. The complete immune evaluation was to be carried out prior to transplantation at 1, 2, 3, 6, 9, 12 months after BMT and subsequently every 6 months up to 4 years. The investigated immunological parameters included total lymphocyte count, B-lymphocytes, T3-, T4-, T8-lymphocytes, T4/T8 ratio, natural killer cell activity, ADCC, lymphocyte blastogenic response and serum-IgG, -IgA, -IgM. Absolute lymphocyte count, B-lymphocytes, T3-lymphocytes recovered to normal levels after 6 months. T4-lymphocytes decreased significantly during the first 180 days posttransplant. T8-lymphocytes increased after 6 months to values higher than normal and the T4/T8 ratio decreased significantly and continued below 0.8 for 48 months. Patients without and with GVHD had low lymphocyte response to PHA and Con A for the first 6 months.     

Production of and responsiveness to interleukin 2 in autoimmune BXSB mice

BXSB male mice serve as one of several murine models of human systemic lupus erythematosus. T-cell abnormalities in these mice involve decreased production of and responsiveness interleukin 2 (IL-2) and are age-related. The studies presented here investigated the mechanism of these T-cell defects. The results suggest that excessive suppressor-T-cell activity as well as soluble inhibitors of IL-2 production and activity, including PGE, are not responsible for the low levels of IL-2 observed in culture supernatants of Con A-stimulated lymphocytes from "old" (3-6 months) BXSB male mice. Supplementation of Con A-stimulated lymphocyte cultures from BXSB male mice with human IL-1 or normal murine accessory cells did not augment IL-2 production. Reduced proliferative responses were observed in bulk cultures of Con A- or alloantigen-stimulated "old" BXSB male lymphocytes, which were not enhanced by exogenous IL-2. Limiting dilution analysis revealed reduced frequencies of Con A- and alloantigen-inducible IL-2-reactive T cells in these mice. These results suggest intrinsic defects in the ability of T cells from "old" BXSB male mice to be activated to produce and respond to IL-2.     

Retroviral marking of human bone marrow fibroblasts: in vitro expansion and localization in calvarial sites after subcutaneous transplantation in vivo

Amplification of multipotential stem cells, with or without ex vivo gene transfer, offers the potential for their use for beneficial repopulation of a host in which there is specific cellular deficiency or functional impairment. The aims of the current study were to immunoselect, genetically mark, and determine the fate of fibroblastic progenitor cells in vivo. A monoclonal antibody, HOP-26, which has high reactivity with a cell surface antigen present on human osteoprogenitors in bone marrow fibroblast populations, was used to select these cells by immunopanning. Following culture in 10% FCS in alphaMEM containing ascorbate-2-phosphate and dexamethasone the amplified cells expressed the osteoblast phenotype as determined by expression of osteocalcin protein determined immunohistochemically, and Type I collagen and osteocalcin mRNA expressions determined by RT-PCR analysis. The selected cells were genetically labeled using a murine leukemia virus (MuLV) encoding a reporter gene (lacZ) with a selective marker gene (neo(r)) using a triple transient transfection protocol. Transfected cells were implanted in CB17 scid/scid mice by local subcutaneous injection over the calvariae. Localization of the genetically marked cells within the calvarial tissues was detected by beta-galactosidase histochemistry and immunocytochemistry. Genetically marked cells were observed within the periosteal layer in close association with the osteoblast layer, covering mineralized bone surfaces and within bone osteoid at 5 and 7 days after injection. This study demonstrates the successful selection, expansion, and retroviral-marking of human osteoprogenitors and their migration and localization within calvariae of SCID mice following in vivo implantation. These basic studies indicate the migration of these cells to skeletal sites and support possibilities for future uses of human osteoprogenitors in therapy of bone deficiency diseases and the potential for development of gene therapy procedures in these conditions.     

Graves-Basedow disease after allogeneic bone marrow transplantation

One severe aplastic anaemia case who presented autoimmune thyroid disease after allogeneic bone marrow transplantation (alloBMT) is described. A 19 year old Polish girl developed Graves' hyperthyroidisms 19 months after allogeneic BMT for severe aplastic anaemia (SAA) donated from her brother. Her serum was positive for thyroid stimulating antibody (TSAb) and anti-thyroid peroxidase autoantibodies (aTPO) while her brother remained euthyroid, seronegative for TSAb, and showed no clinical signs of thyroid pathology. The genetic studies of lymphocytes FISH (fluorescence in situ hybridization) and analysis of STR (short tandem repeated) fragments suggested, that lymphocytes responsible for hyperthyroidisms were of donor origin.     

The onset of hemoglobin synthesis in spleens of irradiated mice after bone marrow transplantation.

Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.     

Lymphokines and the acute-phase response in clinical bone marrow transplantation.

This paper reviews the role of the acute phase response and of cytokines in clinical bone marrow transplantation. Data are discussed from the literature and from the authors experience which show that measurement of C-reactive protein is a rather non-specific marker of tissue injury, but that it is elevated in graft-versus-host disease, and especially in infection. Cytokines are clearly implicated in several aspects of transplantation, and tumour necrosis factor in particular may be important. Although there are some data which associate high TNF levels with severe graft-versus-host disease, this association may not hold true in individual patients.     

Growth and growth hormone in children after bone marrow transplantation

Growth and growth hormone (GH) were investigated every year in 24 children after allogeneic bone marrow transplantation (BMT) for severe aplastic anemia (SAA) or leukemia. Conditioning included total body irradiation (TBI) in all cases of leukemia. The young leukemic children grew poorly. At 4 years after BMT, the mean standard deviation score for attained height had decreased from 0 to -1.73. GH deficiency was diagnosed with provocation tests. Three years after BMT, 10/18 children had a subnormal response. Ten children were further investigated with 24-hour GH profiles. Children with SAA had normal growth and GH levels. TBI seemed to be the major factor responsible for impaired growth.