Mice Receiving Bone Marrow Cells Research Articles

Aromatase deficiency in transplanted bone marrow cells improves vertebral trabecular bone quantity, connectivity, and mineralization and decreases cortical porosity in murine bone marrow transplant recipients.

Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17β-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

Influence of donor mouse age on the establishment of murine acute graft versus host disease model after allogenic hematopoietic stem cell transplantation

This study was purposed to elucidate the influence of donor mouse age on the establishment of murine acute graft versus host disease (aGVHD) model after allogenic hematopoietic stem cell transplantation. The male mice with 2-week-old, 10-week-old and 18-week-old mice (BALB/cH-2Kb) were taken as donors. The 8-week-old mice (BALB/c, H-2Kd) were selected as recipients. Each group animals were irradiated with 7.5 Gy (60)Co for total body, the recipient mice were injected intravenously with 1 × 10⁷ bone marrow cells and 1 × 10⁷ spleenoctyes from various donors in 4-5 hours after irradiation. Mouse transplant characteristics and survival were observed every day. The white blood cell number in peripheral blood of each group were counted at day 5, 10, 15, 20, 25 and 30 after transplantation. Furthermore, the pathological damage in the liver, spleen, lung and intestines were evaluated by sectioning and in situ hematoxylin-eosin (HE) staining. The results showed that compared with the 2-week-old and 10-week-old donor groups, mice received bone marrow (BM) cells and splenocytes from 18-week-old mice showed higher incidence of aGVHD, lower clinical GVHD scores and suffered from diarrhea, ruffled hair, a hunched posture, and diminished body weight. In contrast, mice received BM cells and splenocytes from 2-week-old donor mice indicated attenuated GVHD symptoms and survived longer. The histo-pathological analysis in 18-week-old donor group demonstrated the most serious pathological damage in the liver, spleen, lung and intestines. It is concluded that the donor age has been confired to have an obvious influence on the establishment of murine aGVHD model. This study lay an important foundation for establishing animal models and may be helpful for further study.

Hermansky-Pudlak Syndrome Interstitial Pneumonia

Hermansky-Pudlak Syndrome Interstitial Pneumonia

Aging-Induced Collateral Dysfunction: Impaired Responsiveness of Collaterals and Susceptibility to Apoptosis via Dysfunctional eNOS signaling

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (p < 0.001 and p = 0.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.

Steroid treatment alters adhesion molecule and chemokine expression in experimental acute graft-vs.-host disease of the intestinal tract

Acute graft-vs.-host disease (aGVHD) is a major complication after allogeneic bone marrow transplantation (allo-BMT) that is characterized by high morbidity and mortality. Systemic treatment with steroids has been the mainstay of first-line therapy of aGVHD, although controlled experimental data in this context are limited. Using a haploidentical murine BMT model, steroid effects on hepatic and intestinal inflammation during aGVHD have been investigated. Lethally irradiated B6D2F1 mice received bone marrow cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. Intraperitoneal administration of prednisolone (2 mg/kg body weight every day) early after onset of GVHD from day +10 until day +42 resulted in reduced clinical GVHD severity and improved survival of allogeneic recipients. Although the liver was barely affected by prednisolone treatment, aGVHD-related histopathologic injury of the gastrointestinal tract was strongly reduced in association with diminished expression of interferon-γ, tumor necrosis factor, CXCL 9-11, CCL2-3, mucosal addressin cell adhesion molecule-1, and intercellular adhesion molecule-1. Prednisolone-induced reduction of adhesion molecule expression in the gut manifested earlier than seen for cytokines or chemokines. Interestingly, when starting steroid treatment on day +28, the course of GVHD was unchanged and no major differences in cyto- or chemokine expression in gastrointestinal tract or liver on day +42 were seen. When started early after GVHD onset, prednisolone-related beneficial effects can affect aGVHD target organs differently, involving divergent regulation of inflammation and leukocyte migration. Specifically, a change in adhesion properties between leukocytes and endothelial cells in the gastrointestinal tract may be one of the initial steps in a cascade of steroid-related aGVHD-attenuating events.

Bone marrow cells are a source of undifferentiated cells to prevent Sjögren's syndrome and to preserve salivary glands function in the non-obese diabetic mice

Non-obese diabetic (NOD) mice develop Sjögren's-like syndrome (Ss) and a gradual loss of saliva secretory function. Our previous study showed that injections of matched normal spleen cells with Complete Freund's Adjuvant (CFA) reversed salivary gland dysfunction in 14-week-old NOD mice, which had established Ss. The spleen and bone marrow are closely related organs, and both are among the first sites of hematopoiesis during gestation. Noticing a rapidly increasing number of clinical trials using bone marrow (BM) cells treatments for autoimmune diseases, we tested if BM cells can prevent Ss and restore salivary glands’ function. We injected CFA and MHC class I-matched normal BM cells in 7-week-old NOD mice, which had not yet developed Ss. We found at week 52 post-treatment that all NOD mice receiving BM cells and CFA had a recovery of salivary flow and were protected from Ss and diabetes. BM cells-treated mice had their salivary function restored quantitatively and qualitatively. Saliva flow was higher ( p < 0.05) in BM cells-transplanted mice when compared to control mice, which continued to deteriorate over time. Total proteins, epidermal growth factor, amylase, and electrolytes concentrations in saliva of BM cells-treated mice were not significantly changed at week 44 and 52 post-therapy when compared to pre-therapy (when the mice did not have Ss). Restoration of salivary flow could have resulted from a combination of rescue and paracrine effects from BM cells. This study suggests that a combined immuno- and cell-based therapy can permanently prevent Ss and restored salivary function in NOD mice.

Cooperation between Multiple Microbial Pattern Recognition Systems Is Important for Host Protection against the Intracellular PathogenLegionella pneumophila

Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.

Abstract 5557: Lack of Interleukin-1 Receptor Antagonist in Inflammatory Cells Causes Spontaneous Femoral Artery Aneurysms in Mice

Background: Arterial aneurysms represent a complex degenerative disorder involving chronic arterial wall inflammation and destructive remodeling of structural connective tissue. The relative balance of pro- and anti-inflammatory cytokines likely controls inflammation in the aneurysm wall. The interleukin (IL)-1 receptor antagonist (IL-1Ra) negatively regulates IL-1 signaling. However, the role of IL-1Ra in aneurysm is poorly understood. Methods and Results: IL-1Ra-deficient (IL-1Ra−/−) mice (backcrossed 8 generations to the B6 background) and wild-type (WT) mice did not differ with regard to systolic arterial pressure and plasma lipid levels. However, IL-1Ra−/− mice had spontaneously developed femoral artery aneurysms after 8-month-old, and both maximal vessel area and the intimal + medial area of femoral arteries in IL-1Ra−/− mice were significantly larger compared with WT mice at 9 months of age. Real-time PCR of 9-month-old IL-1Ra−/− mice revealed significantly increased mRNA levels of IL-1 β (6.6-fold, P&lt;0.05), tumor necrosis factor (TNF)- α (12.4-fold, P&lt;0.05) and matrix metaroproteinase-9 (6.0-fold, P&lt;0.01) compared with WT mice. Histological analysis revealed numerous inflammatory cells around the femoral artery aneurysm in IL-1Ra−/− mice, and elastin staining showed destruction of the internal and external elastic lamina in IL-1Ra−/− mice. Immunostaining revealed many macrophages and some CD4-positive T cells in the adventitia. Then we studied the function of macrophage and T cell. After LPS (1 μ g/ml) stimulation, IL-1Ra deficient macrophages produced much higher levels of TNF- α than those from WT mice. Furthermore TNF- α production of T cells from IL-1Ra−/− mice stimulated with anti-CD3 antibody was also higher than those from WT mice. Finally, we performed bone marrow (BM) cell transplantation. We detected femoral artery aneurysms with many inflammatory cells in the adventitia in several WT mice receiving BM cells from IL-1Ra−/− (44%) but not WT mice (0%). Conclusions: This study demonstrates that IL-1Ra-deficiency in inflammatory cells disrupts immune system homeostasis, resulting in the development of autoimmunity and inflammation. Then the inflammation causes spontaneous femoral artery aneurysms in B6 mice.

Porphobilinogen deaminase over-expression in hepatocytes, but not in erythrocytes, prevents accumulation of toxic porphyrin precursors in a mouse model of acute intermittent porphyria

Acute intermittent porphyria (AIP) is characterized by hepatic porphobilinogen deaminase (PBGD) deficiency resulting in a marked overproduction of presumably toxic porphyrin precursors. Our study aimed to assess the protective effects of bone marrow transplantation or PBGD gene transfer into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. Lethally irradiated AIP mice were intravenously injected with 5x10(6) nucleated bone marrow cells from wild type or AIP donor mice. To achieve liver gene transfer, AIP mice received via hydrodynamic injection plasmids expressing human PBGD or luciferase, driven by a liver-specific promoter. Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Hepatic PBGD activity increased 2.2-fold after hydrodynamic injection of therapeutic plasmid. Mice injected with the luciferase control plasmid showed a high excretion of porphyrin precursors after phenobarbital administration whereas just a small increase was observed in AIP mice injected with the PBGD plasmid. Furthermore, motor disturbance was almost completely abolished in AIP mice treated with the therapeutic plasmid. PBGD deficiency in erythroid tissue is not associated with phenotypic manifestations of acute porphyria. In contrast, PBGD over-expression in hepatocytes, albeit in a low proportion, reduced precursor accumulation, which is the hallmark of acute porphyric attacks. Liver-directed gene therapy might offer an alternative to liver transplantation applicable in patients with severe and recurrent manifestations.

DNMT3b's Role in Hematopoietic Stem Cell Self-Renewal.

DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b's function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

Blocking of IL-21 Signal Attenuates Graft-Versus-Host Disease but Not Graft-Versus-Leukemia Effect in a Mouse Model

Immunological effects of interleukin-21 (IL-21) on T-, B-, and NK-cells have been reported, but the role of IL-21 in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) remains obscure. Here, we demonstrate that splenocytes from IL-21R−/− mice show attenuated GVHD symptoms as compared to splenocytes from wild type mice. In addition, the survival of mice receiving IL-21R−/− splenocytes is significantly longer than of those receiving wild type splenocytes. Both types of splenocytes appear to efficiently eliminate leukemic cells, suggesting the retention of GVL effects. Our results suggest that CD4 T-cells augment GVHD by autocrine stimulation via IL-21, and that production of IL-17 is downregulated in IL-21R−/− splenocyte recipients. To develop a treatment model in mice, we generated a decoy receptor for IL-21, in which most of the intracellular domain is deleted. Mice receiving bone marrow cells transduced with this decoy receptor survived longer than those receiving cells transduced with an empty retrovirus.These results suggest that IL-21 promotes GVHD and that the blockade of IL- 21 signaling might be a useful treatment for GVHD, without causing loss of the GVL effect.

Apoptotic Cell Death and Regeneration in the Newborn Retina After Irradiation Prior to Bone Marrow Transplantation

Purpose: We studied the contribution made by circulating bone marrow (BM)-derived cells to the newborn and mature retinas of BM-transplanted mice. Methods: Newborn and adult C57BL/6J mice were administered a lethal dose of total-body irradiation, after which pathologic changes to the retinas were periodically assessed. In addition, mice received BM cells from 8-week-old green fluorescent protein (GFP) transgenic mice, and the subsequent differentiation of GFP+ cells was studied. Results: Within 5 hr after irradiation of newborn mice, retinal cells began to die due to apoptosis. By contrast, irradiation of adult mice elicited no histologic changes in the retina. BM cells generally did not differentiate in adult mice, but numerous GFP+ BM cells were integrated into the retinal tissue of newborn mice, where they expressed various cell type–specific markers. Finally, examination of whole retina mounts showed that GFP+ cells also contributed to retinal vascularization. Conclusions: Our findings underscore the importance of careful evaluation of the biological effects of irradiation in models making use of BM transplantation.

Transfer of Endothelial Progenitor and Bone Marrow Cells Influences Atherosclerotic Plaque Size and Composition in Apolipoprotein E Knockout Mice

Recent clinical trials use cell therapy with bone marrow (BM) cells or endothelial progenitor cells (EPCs) for ischemic syndromes. We explored the effect of BM cell- or spleen cell-derived EPC transfer on plaque size and stability markers in the apolipoprotein E knockout (apoE KO) mouse model. ApoE KO mice aged 10 weeks served as recipients. Labeled BM cells and spleen cell-derived EPCs from age-matched apoE KO mice were injected intravenously to 2 groups of recipient mice each. Additional mice served as controls receiving saline. Both protocols were repeated 3 times at 2 weekly intervals. On killing, plaque size and character were studied, lipid profile analyzed, and serum and aortic cytokines assayed. Spleen cell-derived cells contained a significantly larger number of endothelial cell precursors. Labeled EPCs and BM cells were found abundantly in the spleens, yet also in the lesions of the recipient mice. Aortic sinus lesion size was significantly increased in mice receiving BM cells (n=10) in the EPC-treated group (n=10) compared with controls (n=10; a 54% and a 34% increase in aortic sinus plaque area, respectively). Mice receiving EPCs exhibited plaques with larger lipid cores and thinner fibrous caps and a higher number of infiltrating CD3 cells. RT-PCR analysis of aortas revealed reduced expression of mRNA for interleukin-10 (IL-10) in both cell transfer groups. Higher serum concentrations of IL-6 and monocyte chemoattractant protein-1 were found in sera from BM recipients, whereas lower IL-10 levels were found in mice transfused with spleen-derived EPCs. Transfer of BM cells and EPCs may result in an increase in atherosclerotic lesion size, whereas EPC transfer could also potentially influence plaque stability.

Restoration of C1q levels by bone marrow transplantation attenuates autoimmune disease associated with C1q deficiency in mice

C1q deficiency in both humans and mice is strongly associated with autoimmunity. We have previously shown that bone marrow transplantation (BMT) restored C1q levels in C1q-deficient (C1qa(-/-)) mice. Here, we studied the effect of BMT on autoimmunity in C1qa(-/-) mice. Following irradiation, young C1qa(-/-) or wild-type MRL/Mp mice received bone marrow cells (BMC) from strain-matched wild-type or C1qa(-/-) animals. C1q levels increased rapidly when C1qa(-/-) mice received BMC from wild-type mice. Conversely, they decreased slowly in wild-type mice transplanted with C1qa(-/-) BMC. C1qa(-/-) animals transplanted with C1qa(-/-) BMC demonstrated accelerated disease when compared with wild-type mice given wild-type BMC. In contrast, a significant delay in the development of autoantibodies and glomerulonephritis was observed in C1qa(-/-) mice reconstituted with wild-type BMC, and the impaired clearance of apoptotic cells, previously described in C1qa(-/-) mice, was rectified. Moreover, the autoimmune disease was accelerated in wild-type mice given C1qa(-/-) BMC compared to animals transplanted with wild-type cells. These results provide supporting evidence that BMT may be a therapeutic option in the treatment of autoimmunity associated with human C1q deficiency.

Oncogenic interaction between BCR-ABL andNUP98-HOXA9 demonstrated by the use of an in vitro purging culture system

Chronic myelogenous leukemia (CML) is a clonal stem cell disease caused by the BCR-ABL oncoprotein and is characterized, in its early phase, by excessive accumulation of mature myeloid cells, which eventually leads to acute leukemia. The genetic events involved in CML's progression to acute leukemia remain largely unknown. Recent studies have detected the presence of the NUP98-HOXA9 fusion oncogene in acute leukemia derived from CML patients, which suggests that these 2 oncoproteins may interact and influence CML disease progression. Using in vitro purging of BCR-ABL-transduced mouse bone marrow cells, we can now report that recipients of bone marrow cells engineered to coexpress BCR-ABL with NUP98-HOXA9 develop acute leukemia within 7 to 10 days after transplantation. However, no disease is detected for more than 2 months in mice receiving bone marrow cells expressing either BCR-ABL or NUP98-HOXA9. We also provide evidence of high levels of HOXA9 expressed in leukemic blasts from acute-phase CML patients and that it interacts significantly on a genetic level with BCR-ABL in our in vivo CML model. Together, these studies support a causative, as opposed to a consequential, role for NUP98-HOXA9 (and possibly HOXA9) in CML disease progression.

Bone Marrow-Derived Hepatocytes: Rare but Promising

Bone Marrow-Derived Hepatocytes: Rare but Promising

Muscle regeneration by reconstitution with bone marrow or fetal liver cells from green fluorescent protein-gene transgenic mice.

The myogenic potential of bone marrow and fetal liver cells was examined using donor cells from green fluorescent protein (GFP)-gene transgenic mice transferred into chimeric mice. Lethally irradiated X-chromosome-linked muscular dystrophy (mdx) mice receiving bone marrow cells from the transgenic mice exhibited significant numbers of fluorescence(+) and dystrophin(+) muscle fibres. In order to compare the generating capacity of fetal liver cells with bone marrow cells in neonatal chimeras, these two cell types from the transgenic mice were injected into busulfantreated normal or mdx neonatal mice, and muscular generation in the chimeras was examined. Cardiotoxin-induced (or -uninduced, for mdx recipients) muscle regeneration in chimeras also produced fluorescence(+) muscle fibres. The muscle reconstitution efficiency of the bone marrow cells was almost equal to that of fetal liver cells. However, the myogenic cell frequency was higher in fetal livers than in bone marrow. Among the neonatal chimeras of normal recipients, several fibres expressed the fluorescence in the cardiotoxin-untreated muscle. Moreover, fluorescence(+) mononuclear cells were observed beneath the basal lamina of the cardiotoxin-untreated muscle of chimeras, a position where satellite cells are localizing. It was also found that mononuclear fluorescence(+) and desmin(+) cells were observed in the explantation cultures of untreated muscles of neonatal chimeras. The fluorescence(+) muscle fibres were generated in the second recipient mice receiving muscle single cells from the cardiotoxin-untreated neonatal chimeras. The results suggest that both bone marrow and fetal liver cells may have the potential to differentiate into muscle satellite cells and participate in muscle regeneration after muscle damage as well as in physiological muscle generation.

Intravenous injection of apoptotic leukocytes enhances bone marrow engraftment across major histocompatibility barriers

AbstractCross-tolerization of T lymphocytes after apoptotic cell uptake by dendritic cells may be involved in self-tolerance maintenance. Furthermore, immunosuppressive properties are attributed to apoptotic cells. This study evaluated the consequences of apoptotic leukocyte administration in a restrictive engraftment model of murine bone marrow (BM) transplantation. Sublethally irradiated recipients received a limited number of allogeneic BM, with or without irradiated apoptotic leukocytes of different origins. No graft-versus-host disease was observed. Whereas only a low proportion of mice receiving BM cells alone engrafted, addition of apoptotic irradiated leukocytes, independently of the origin (donor, recipient, third-party mice, as well as xenogeneic peripheral blood mononuclear cells), significantly enhanced engraftment. Similar results were obtained after infusion of leukocytes rendered apoptotic by UVB irradiation or by anti-Fas monoclonal antibody stimulation, thus confirming the role of apoptotic cells in engraftment facilitation. Overall, these results suggest that apoptotic leukocytes can nonspecifically facilitate allogeneic BM engraftment. Such a simple approach could be of interest in BM transplantation settings involving an important HLA donor/recipient disparity, a T-cell–depleted graft, or reduced conditioning regimen intensity.

Reversion of hepatobiliary alterations By bone marrow transplantation in a murine model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is characterized clinically by cutaneous photosensitivity and biochemically by the accumulation of excessive amounts of protoporphyrin in erythrocytes, plasma, feces, and other tissues, such as the liver. The condition is inherited as an autosomal dominant or recessive trait, with a deficiency of ferrochelatase activity. A major concern in EPP patients is the development of cholestasis with accumulation of protoporphyrin in hepatobiliary structures and progressive cellular damage, which can rapidly lead to fatal hepatic failure. The availability of a mouse model for the disease, the Fech(m1Pas)/Fech(m1Pas) mutant mouse, allowed us to test a cellular therapy protocol to correct the porphyric phenotype. When Fech/Fech mice received bone marrow cells from normal animals, the accumulation of protoporphyrin in red blood cells and plasma was reduced 10-fold but still remained 2.5 times above normal levels. Interestingly, in very young animals, bone marrow transplantation can prevent hepatobiliary complications as well as hepatocyte alterations and partially reverse protoporphyrin accumulation in the liver. Bone marrow transplantation may be an option for EPP patients who are at risk of developing hepatic complications.

The fate of donor T-cell receptor transgenic T cells with known host antigen specificity in a graft-versus-host disease model.

The expansion of transgenic donor CD8+ T-cells with known allospecificity against a host MHC class I alloantigen was examined in a murine graft-versus-host disease (GVHD) model. Lethally irradiated, Ld+ BALB/c mice received bone marrow cells and spleen cells from anti-Ld 2C T-cell receptor (TCR)-transgenic B6 mice, alone or with normal B6 spleen cells. Transgenic TCR-bearing T-cell expansion, apoptosis, and function were monitored at various time points and were correlated with clinical outcome. Fifteen-fold clonal expansion of 2C CD8 cells occurred by day 4 after bone marrow transplantation. Between days 4 and 7, increasing proportions of 2C CD8 cells underwent apoptotic cell death, coincident with a 7-15-fold decline in their numbers. CD8 and TCR expression were down-regulated on 2C CD8 cells by day +4 after bone marrow transplantation, and they were anergic to TCR-mediated stimulation. Clinically, the BALB/c recipients of 2C spleen cells exhibited only minimal chronic GVHD. In contrast, lethally irradiated BALB/c mice receiving similar numbers of non-transgenic B6 bone marrow cells and spleen cells exhibited severe GVHD (median survival time: 28 days). The addition of a small number of 2C spleen cells to the inoculum accelerated GVHD mortality, and 2C CD8 cells showed a similar time course of expansion and decline to that observed in recipients of larger numbers of 2C cells alone. Initial clonal expansion, down-regulation of CD8 and TCR, anergy, and later deletion of graft-versus-host-reactive CD8 cells via apoptosis occurs in lethally irradiated recipients. Expansion of a single CD8 clone produces much less severe GVHD than that induced by a polyclonal, mixed CD4 plus CD8 response. These results have implications for GVHD pathogenesis and its sometimes self-limited nature.