Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.     

Adenovector-mediated expression of human thrombopoietin cDNA in immune-compromised mice: insights into the pathophysiology of osteomyelofibrosis

Thrombopoietin (TPO) cDNA can be effectively delivered in vivo by adenovectors. Immune normal mice (BALB/c) and syngeneic mice with variable degrees of immune dysfunction nu, SCID, and NOD-SCID) were treated with an adenovirus vector expressing the human TPO cDNA (AdTPO). Platelet peaks were significantly higher in SCID and NOD-SCID mice compared with BALB/c and nu mice. Human plasma TPO concentration correlated with the platelet counts. SCID and NOD-SCID mice exhibited also granulocytosis and increased numbers of hemopoietic progenitors in bone marrow. Following platelet peak, BALB/c mice developed autoantibodies against murine TPO leading to thrombocytopenia and depletion of megakaryocytes and hemopoietic progenitors in bone marrow. AdTPO-treated SCID mice developed osteomyelofibrosis and extramedullary/extrasplenal hemopoiesis. In contrast, NOD-SCID mice with a similar magnitude of TPO overexpression did not show fibrotic changes in bone marrow. We conclude, first, that a chronic high level of TPO overexpression stimulates megakaryocytopoiesis and myelopoiesis leading to thrombocytosis and granulocytosis. Second, increased megakaryocytopoiesis is not sufficient for development of secondary osteomyelofibrosis. The functionally deficient monocytes and macrophages of NOD-SCID mice probably prevented fibrotic marrow changes. Third, immune deficiency enhances expression of adenovirally mediated transgenes, and fourth, xenogeneic transgene delivered by adenovector to a host with normal immune functions may induce loss of immune tolerance and autoimmune phenomenon.     

EblacZ tumor dormancy in bone marrow and lymph nodes: active control of proliferating tumor cells by CD8+ immune T cells

A well-defined lacZ gene tagged DBA/2 lymphoma (EblacZ) was used to examine the role of host immune responses in controlling tumor dissemination and persistence, as well as metastasis. In s.c. and intra-ear pinna-inoculated mice, low numbers of EblacZ cells homed to the bone marrow and lymph nodes. The frequency of bone marrow-residing tumor cells did not change with the growth of primary tumor or with multiple inoculations of tumor cells. The bone marrow-residing tumor cells expressed the proliferation-associated Ki67 antigen and expanded upon CD8+ depletion. In contrast, inoculation of nu/nu or severe combined immunodeficiency mice or of immune-suppressed DBA/2 mice led to the rapid outgrowth of EblacZ cells in the bone marrow and their metastasis to other organs. Transfer of bone marrow from EblacZ immunized MHC congenic or syngeneic DBA/2 donors, but not from naive donors, protected s.c.-inoculated DBA/2 mice. Protection was abrogated by in vitro depletion of CD8+ T cells prior to transfer of bone marrow. These experiments show that bone marrow and lymph nodes are privileged sites where potentially lethal tumor cells are controlled in a dormant state by the immune system. Metastasis may be a consequence of the breakdown of this immune control.     

Clinical and MRI findings of the temporomandibular joint in relation to occlusion in young adults

Although considerable evidence suggests that carcinogenic polycyclic aromatic hydrocarbons are immunosuppressive compounds, limited information is available regarding precise effects of these agents on immune cells. In the present report, the effect of subchronic exposure to benzo[a]pyrene (B[a]P) on immune cells in bone marrow, thymus and spleen of B6C3F1 mice was investigated. B[a]P treatment was found to reduce thymic cellularity and also to significantly alter normal thymocyte differentiation, as indicated by expression of CD4 and CD8 cell-surface antigens. Exposure to B[a]P also reduced cellularity of the bone marrow, including decreased percentage and absolute number of CD45R+ B-lineage lymphocytes. Further, B[a]P treatment dramatically increased the percentage of CD44hi cells in bone marrow, while proportionately reducing CD44lo cells. These results may indicate CD44lo bone marrow cells, including prolymphocytic cells, represent sensitive targets of B[a]P. The spleens of treated mice were found deficient in both Thy 1.2+ T lymphocytes and CD45R+ B lymphocytes, effects that correlate well with the chemical-induced alterations observed in thymus and bone marrow, respectively.     

Enhanced acceptance of regenerating bone marrow of random bred newborn mice by random bred adult mice

Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i.p. injections of hydroxyurea (HU) (2 x 100 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2x10(6) nucleated cells per femur as compared with 08.-2x10(5)). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 x 10(4) i.v., 10(5) in vitro and 5 x 10(4) per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S (5-10 x 10(4) cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better" than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content of activated CFU-S and/or pre-CFU-S in the regenerating bone marrow     

Multiple minor histocompatibility antigen disparities between a recipient and four HLA-identical potential sibling donors for bone marrow transplantation

A patient with acute leukemia and her family including four HLA-identical siblings were analyzed to select a donor who was not only HLA- but also minor histocompatibility (mH) antigen compatible for allogeneic bone marrow transplantation (BMT). The HLA-A2 restricted mH antigen-specific HA-1, -2, -4, and -5 cytotoxic T-lymphocyte (CTL) clones were used to type the family members for expression of these mH antigens. The patient and one HLA-identical sibling were compatible for these mH antigens. This sibling was selected as the bone marrow donor. The patient engrafted promptly but developed acute and chronic graft-versus-host disease. To study the presence of other mH antigen disparities between recipient and donor, host-versus-graft CTL lines and clones were generated by stimulation of recipient peripheral blood lymphocytes (PBLs) with donor bone marrow cells, and graft-versus-host CTL lines were generated after BMT by stimulation of PBLs of donor origin with recipient bone marrow cells. These CTL lines were cytotoxic to cells from the bone marrow donor and from the recipient, respectively, and to cells from several other family members. T-cell lines, generated from the patient after BMT by stimulation of recipient-derived PBLs with donor bone marrow cells, exhibited no specific cytotoxicity to donor or recipient cells. Chimerism studies after BMT revealed that the PBLs and T-cell lines generated after BMT were of donor origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy and toxicity of 9-beta-D-arabinofuranosylguanine (araG) as an agent to purge malignant T cells from murine bone marrow: application to an in vivo T-leukemia model

9-beta-D-Arabinofuranosylguanine (araG), an analog of deoxyguanosine which is not degraded by purine nucleoside phosphorylase, has been previously shown in in vitro studies by our laboratory to be effective in purging malignant T cells from human bone marrow (1). We now describe studies in a murine model of T-cell acute lymphoblastic leukemia (ALL) in which we tested whether bone marrow, contaminated with malignant T cells and purged ex vivo with araG, could reconstitute both the lymphoid and myeloerythroid lineages in the absence of leukemic relapse. The model utilized 6C3HED tumor cells, derived from a Thy 1.2+ malignant murine T-cell line, which were shown to cause lethal leukemia in C3H/HeN mice. Intravenous injection of 10(6) 6C3HED cells resulted in 100% mortality within 18 days, with autopsy revealing tumor infiltration of multiple organs. 100% of non-leukemia bearing lethally irradiated C3H/HeN mice transplanted with syngeneic bone marrow, treated ex vivo with 100 microM of araG for 18 hours, survived > 365 days post-transplant with full lymphohematopoietic reconstitution. Evidence of araG's ability to purge bone marrow of malignant tumor cells without causing significant toxicity to normal marrow derived hematopoietic progenitor cells was documented in experiments in which 75% of lethally irradiated mice transplanted with syngeneic bone marrow, contaminated with 6C3HED tumor cells and treated ex vivo with 100 microM araG for 18 hours, survived for > 250 to > 400 days. Death in 25% of mice was secondary to infection which developed before marrow reconstitution occurred. Reconstitution of the lymphoid, myeloid, and erythroid lineages with donor cells in surviving mice was documented. The data presented indicate that araG may effectively purge bone marrow of malignant T cells without irreversible toxicity to hematopoietic stem cells. This purging regimen is recommended for consideration for clinical trials in patients with T-cell malignancies undergoing autologous bone marrow transplantation and may also be a viable option for T-cell depletion as a strategy to prevent graft-versus-host disease.     

A nonlethal conditioning approach to achieve engraftment of xenogeneic rat bone marrow in mice and to induce donor-specific tolerance

BACKGROUND: The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance. METHODS: C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting. RESULTS: One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80 x 10(6) Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks. CONCLUSION: The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.     

Misrepresentation of publications by applicants for radiology fellowships: is it a problem?

In light of the recently described experimental technique of in vivo bone reconstitution with biotechnologic methods (from bone marrow stromal cells) and the prefabrication flap procedures, the possibility to obtain autologous bone growth in a myocutaneous flap, thus creating a composite osteomyocutaneous preformed flap, is postulated. Human bone marrow stromal cells were delivered into the latissimus dorsi of athymic mice by a porous hydroxyapatite ceramic model. Eight weeks after the implantation, histologic examination revealed the presence of spongious bone tissue. A simple myocutaneous flap was thus transformed into a composite osteomyocutaneous flap. This flap is called the biotechnologic prefabricated flap, because it was the result of ex vivo expanded osteogenic precursor cells and in vivo bone tissue neoformation. The shape of the bone flap was exactly the same as the shape of the ceramic model used. A possible clinical application may be the correction of skeletal defects. The advantages of this procedure are simple surgical execution, the possibility of preshaping the graft to the exact characteristics of the defect, and the availability of autogenous donor tissue without donor site morbidity.     

Correction of neutropenia and hypogammaglobulinemia in X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation

X-linked hyper-IgM (X-HIM) syndrome is a primary immunodeficiency disease characterized by defects in both cellular and humoral immunity. X-HIM is caused by mutations in the gene for CD40 ligand (CD40L), a T cell membrane protein that mediates T cell-dependent immune functions. We report the case of a 6-year-old male with X-HIM due to an intronic mutation resulting in aberrant CD40L RNA splicing and absence of detectable CD40L protein. The patient had a history of multiple infectious complications and chronic neutropenia requiring treatment with recombinant granulocyte colony-stimulating factor, and underwent allogeneic bone marrow transplantation from an HLA-matched sibling donor. Following successful engraftment, T cell CD40L expression and immunoglobulin isotype switching were reconstituted and neutropenia resolved. Allogeneic bone marrow transplantation can correct neutropenia and reconstitute immune function in X-HIM.     

Leukocyte low density lipoprotein receptor (LDL-R) does not contribute to LDL clearance in vivo: bone marrow transplantation studies in the mouse

The targeted disruption of the low density lipoprotein (LDL) receptor gene in mice results in accumulation of plasma LDL cholesterol and in predisposition to diet-induced aortic atherosclerosis. Although the liver is the central organ for receptor mediated clearance of LDL, the in vivo role of other organs and tissues in LDL catabolism has not been directly studied. Since bone marrow-derived cells such as blood leukocytes and tissue macrophages express LDL receptors and contribute a large mass to the body, we designed bone marrow transplantation (BMT) experiments to reconstitute LDL receptor null mice [LDL-R(-/-)] with marrow obtained from LDL-R wild-type mice [LDL-R(+/+)] and evaluate the effects on parameters of plasma lipid metabolism. Although reconstitution of the transplanted mice with donor bone marrow cells was complete, no differences in plasma lipid levels and lipoprotein distribution were found between groups, irrespective of the diet used, and turnover studies using 125I-labeled LDL showed that LDL receptor expression by leukocytes and macrophages does not significantly contribute to plasma LDL clearance. The complementary experiment of transplanting LDL-R(-/-) marrow into C57BL/6 recipients [LDL-R(-/-)-->LDL(+/+)], performed to evaluate the role of leukocyte LDL-R in normocholesterolemic condition, also produced no effects on plasma lipid parameters. LDL binding studies using macrophages isolated from transplanted mice showed a lack of LDL-R expression. Thus, despite their large number and wide distribution, bone marrow-derived cells do not significantly influence receptor-mediated clearance of plasma LDL.     

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.     

Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. Complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor

MRL/MP-Ipr/Ipr (MRL/Ipr) mice possess radioresistant (9.5 Gy) abnormal stem cells and show a recurrence of autoimmune diseases within 5 mo of conventional allogeneic bone marrow transplantation. We recently have found that the MHC preference exists between hemopoietic stem cells and stromal cells; when bones are engrafted, donor-derived stromal cells present in the engrafted bones can migrate into the recipient bone marrows, which are replaced with both donor-derived stromal cells and hematopoietic cells. Based on these findings, we attempted to prevent the recurrence of autoimmune diseases in MRL/Ipr mice by the transplantation of both bone marrow cells and bone (as a source of stromal cells). MRL/Ipr mice were irradiated (8.5 Gy) and then reconstituted with C57BL/6 bone marrow cells plus bone grafts. The mice survived more than 48 wk after this treatment. Immunohistologic studies revealed that the mice were completely free from both lymphadenopathy and autoimmune diseases such as lupus nephritis and rheumatoid arthritis. Sera from these mice showed normal levels of circulating immune complexes and rheumatoid factors. Normal functions of both T cells and B cells were noted. Abnormal T cells such as Thy-1+B220+ cells present in nontreated MRL/Ipr mice could not be seen in the thus-treated mice. In addition, to our surprise, spleen cells from treated mice showed completely normal in vitro primary anti-SRBC responses. These results indicate that stromal cells in allogeneic bone marrow transplantation play a crucial role not only in the prevention of graft failure but also in the successful cooperation among APCs, T cells, and B cells. Although MRL/Ipr mice are radiosensitive and usually die of interstitial pneumonia or fatty liver due to the side effects of radiation, it should be noted that this strategy allows a reduction in the radiation dose (9.5 Gy-->8.5 Gy), and that these mice can survive more than 48 wk without showing any symptoms of autoimmune diseases.     

Gender-specific effects of prenatal chlordane exposure on myeloid cell development

Work previously reported by this laboratory indicated that prenatal chlordane exposure affected macrophage function in young adult mice. Because these macrophage effects were due to exposure during the development of the immune system, the possibility of a persistent effect on the development of myeloid stem and progenitor cells was considered. Female mice were treated with either 0 or 8 mg of chlordane per kilogram body weight daily for 18 days during pregnancy. Myeloid hemopoietic activity of bone marrow cells from 6-week-old offspring was evaluated for in vitro colony-forming units-in-culture in response to exogeneously added recombinant forms of the cytokines granulocyte/macrophage-colony stimulating factor, macrophage-CSF, and interleukin 3 (IL-3). There was a significant depression of the numbers of bone marrow colony forming units-granulocyte/macrophage (CFU-GM), CFU-IL-3, and CFU-macrophage (CFU-M) in only the female offspring. Male offspring consistently demonstrated no difference in the CFU-GM, CFU-IL-3, or CFU-M. Prenatal treatment with chlordane did not significantly affect the number of recoverable viable bone marrow cells in either male or female mice.     

Chronic myelogenous leukemia

Chronic myelogenous leukemia is a clonal hematopoietic malignancy characterized by a balanced translocation between chromosomes 9 and 22 that results in the generation of an abnormal bcr/abl fusion protein with increased tyrosine kinase activity. This abnormal fusion protein has transforming activity for hematopoietic cells in vitro and causes chronic myelogenous leukemia-like myelopoiesis in mice. Chronic myelogenous leukemia progenitor cells display abnormalities in their interactions with bone marrow stroma, perhaps due to defective adhesion molecule function. Conventional therapies for chronic myelogenous leukemia include hydroxyurea, busulfan, or interferon. Treatment with interferon may prolong overall survival, especially in patients who achieve a cytogenetic response. Related donor marrow transplantation can result in long-term survival in more than 65% of patients treated early in the course of disease. For patients without an available matched sibling donor, unrelated donor marrow transplantation or autologous marrow transplantation are alternative therapeutic options.     

A stable RAG1-RAG2-DNA complex that is active in V(D)J cleavage

A well-known characteristic of gamma delta T cells is that they are produced in waves during ontogeny, with cells expressing T-cell receptor V gamma 5 appearing early in fetal thymic ontogeny, followed by V gamma 6, then by other gamma delta T-cell types. In addition, evidence exists to suggest that the potential of haemopoietic precursors to generate different types of gamma delta T cells changes in ontogeny. We have used these observations as the basis for an extensive study of the potential for haemopoietic precursors isolated from fetal liver, neonatal spleen and adult bone marrow to reconstitute severe combined immunodeficient (SCID) mice. Mice that were reconstituted as newborns with fetal liver cells most closely resembled normal C.B-17 mice with respect to both lymphocyte numbers and subsets, while mice reconstituted with adult bone marrow had fewer cells than normal mice. This deficit spanned both T and B cells in all organs examined. Among the gamma delta T-cell subsets examined, the ability to reconstitute V gamma 4+ cells was particularly dependent on the ontogenic age of the reconstituting presursors, with fetal liver cells having the greatest capacity to generate V gamma 4+ cells, and adult bone marrow cells the least. The vast majority of the T cells produced in the reconstituted mice were of donor origin, and the level of reconstitution was found to be dependent upon some factor other than the presursor frequency.     

Ultrastructural characteristics of Fc epsilon R-positive basophils in the spleen and bone marrow of mice immunized with goat anti-mouse IgD antibody

BACKGROUND: Previous work showed that injection of mice with goat anti-mouse IgD antibodies results in increased numbers of Fc epsilon R-positive, non-B, non-T cells in the spleen and Fc epsilon R-positive cells in the bone marrow, and that some of these cells had ultrastructural features of basophils. Fc epsilon R-positive, non-B, non-T cells express virtually all of the capacity of mouse splenic "non-B, non-T cells" to produce interleukin-4 in response to stimulation by cross-linking of Fc epsilon R or Fc gamma R, or by the calcium ionophore, ionomycin. EXPERIMENTAL DESIGN: The present study is a detailed ultrastructural analysis of Fc epsilon R-positive bone marrow cells or Fc epsilon R-positive splenic non-B, non-T cells sorted from mice injected with goat anti-mouse IgD antibody and of Fc epsilon R-positive bone marrow cells or spleen cells pooled from normal mice not injected with goat anti-IgD. RESULTS: Basophils represented the majority (90%) of the granulated cells present in the Fc epsilon R-positive splenic non-B, non-T cells or Fc epsilon R-positive bone marrow cells of goat anti-IgD-injected mice. In contrast, the cytoplasmic granule-containing Fc epsilon R-negative cells sorted from spleen or bone marrow of goat anti-IgD-injected animals contained predominantly a mixture of neutrophils, eosinophils, monocytes and their precursors. Both the Fc epsilon R-positive and -negative preparations contained rare (< 5%) cells with ultrastructural features of very immature mast cells. Basophils were also identified in Fc epsilon R-positive cells sorted from total bone marrow cells or spleen cells of normal mice not injected with goat anti-IgD. CONCLUSIONS: Taken together with data concerning the numbers of Fc epsilon R-positive, non-B, non-T cells in the spleen, and Fc epsilon R-positive B220-negative cells in the bone marrow, these ultrastructural findings indicate that injection of mice with goat anti-IgD results in increased numbers of basophils, particularly in the spleen, that exhibit an 8-fold increase in basophils as a result of injection of goat anti-IgD.     

Local synthesis of C3 within the splenic lymphoid compartment can reconstitute the impaired immune response in C3-deficient mice

Mice bearing a disrupted C3 locus (C3-/-) have an impaired Ab response to T-dependent Ags (bacteriophage phiX 174 and nuclear protein-keyhole limpet hemocyanin) characterized by a reduction in number and size of germinal centers and impaired retention of Ag by follicular dendritic cells. To test the importance of C3 synthesized locally within the lymphoid compartment during an immune response to T-dependent Ag, we reconstituted C3-/- mice with wild-type bone marrow of MHC-identical littermates. Engraftment not only restored local C3 synthesis in the spleen, but also rescued the impaired humoral response. The major source of C3 mRNA was MOMA-2+ macrophages localized within the white pulp areas of the spleen. Interestingly, C3 expression is apparently regulated as C3 mRNA was not detected in splenic sections of nonimmune mice. Furthermore, local C3 synthesis by donor macrophages reversed the impaired Ag trapping by splenic follicular dendritic cells in C3-deficient mice.     

Specific tolerance induction and transplantation: a single-day protocol

Bone marrow transfusion is a well-established method for induction of mixed hematopoietic chimerism and donor-specific tolerance in animal models. This procedure, however, is inapplicable in clinical transplantation using cadaveric donors due to the interval (1 week to 7 months) between tolerance induction and organ transplantation. For clinical use, it is essential that allografts be placed at the time of bone marrow transfusion. In the present study, we performed skin transplantation within 1 hour after a nonlethal conditioning regimen. Recipient mice were treated with anti-CD3, anti-CD4, low-dose total body irradiation (3 to 6 Gy TBI) and fully mismatched or haploidentical donor bone marrow cells. Stable multilineage chimerism and specific T-cell nonresponsiveness developed. Donor skin grafts were permanently accepted. These results suggest that this single day protocol has clear potential for application in both cadaveric and living-related organ transplantation.     

Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood

Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult "competitor" marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.