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PubMed2005

Toll-like receptor-4: renal cells and bone marrow cells signal for neutrophil recruitment during pyelonephritis.

BACKGROUND: The molecular mechanisms of pathogen recognition that initiate infective pyelonephritis are poorly understood. Toll-like receptor-4 (TLR4) mutant mice infected with uropathogenic Escherichia coli lack renal CXCL2 mRNA expression, subsequent neutrophil recruitment, and renal abscess formation. METHODS: We used a bone marrow transplant approach in order to investigate the contribution of TLR4 in intrinsic renal cells or bone-marrow-derived immune cells to neutrophil recruitment during infective pyelonephritis. RESULTS: Both chimera either expressing mutant tlr4 in intrinsic renal cells and wild-type tlr4 in bone marrow-derived cells or vice versa showed an impaired response to uropathogenic E. coli infection in terms of leukocyturia and renal abscess formation when compared to tlr4 wild-type mice with congenic bone marrow transplants. CONCLUSION: These data suggest that TLR4 is required on both intrinsic renal cells (e.g., tubular epithelial cells) and bone marrow-derived immune cells for the control of ascending uropathogenic E. coli infection by initiating chemokine-driven renal neutrophil recruitment.

Animals↗
PubMed1998

Rapid engraftment after allogeneic transplantation using CD34-enriched marrow cells.

Bone marrow cells expressing the surface antigen CD34 comprise approximately 1% of harvested marrow and are highly enriched for marrow progenitor cells, including the cells believed to be responsible for long-term engraftment following bone marrow transplantation (BMT). Selection of CD34-expressing cells was applied in allogeneic BMT (alloBMT) to decrease the number of T lymphocytes in the infused marrow in an attempt to prevent severe graft-versus-host disease (GVHD). We report 14 patients who underwent HLA-identical sibling-matched alloBMT with marrow-enriched for CD34 cells using the Isolex 300 SA device. Patients received total body irradiation, thiotepa, cyclophosphamide, antithymocyte globulin and methylprednisolone prior to marrow infusion. No post-transplantation immunosuppressive therapy was given except for a 5-week course of steroids. The purity of the infused marrow was 64.9+/-6.0% (mean +/- s.e.m.) CD34-positive cells and patients received a mean of 1.24+/-0.21 x 10(6) CD34 cells/kg. A mean of 9.4+/-1.7 x 10(4) CD3 T cells/kg were present in the CD34-enriched product, representing a 2.7+/-0.1 log depletion. There were no graft rejections and patients achieved a sustained absolute granulocyte count of >500 in a median of 10.5 days and a sustained platelet engraftment of >20000 untransfused in a median of 27 days. Patients were discharged a median of 21.5 days after marrow infusion. There were no instances of grade III or IV graft-versus-host disease (GVHD) and no unexpected adverse events during the transplant hospitalization. With a median follow-up of 12 months, the estimated 100 day survival is 86+/-9%. CD34 selection in alloBMT permits rapid engraftment without unanticipated toxicities.

Adolescent↗
PubMed1985

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells.

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.

Animals↗
PubMed1988

Selective depletion of human myeloma clonogenic stem cells from bone marrow cell preparations by a plasma-cell reactive antibody and complement.

The monoclonal antibody MM4 reacts with human myeloma cells from plasma cell dyscrasia (PCD)-derived cell lines and bone marrow (BM) biopsies from PCD patients, but not with normal BM or peripheral blood mononuclear (PBM) cells. We examined cytotoxicity of MM4 and rabbit complement (MM4:C') on mixtures of normal BM mononuclear cells and myeloma cells from three different PCD-derived cell lines, RPMI 8226, GM 1312, or ARH-77. For cell preparations containing 10% myeloma cells, treatment with MM4 (500 micrograms per 10(5) cells, 4 degrees C, 60 min) and two cycles of complement (1:8, 23 degrees C, 2 x 30 min) consistently eliminated 2 logs or more of clonogenic myeloma stem cells, as determined by colony growth assays and limiting dilution analysis (99.4%, 98.9%, and 99.96% reduction of RPMI 8226, GM 1312, and ARH-77 cells, respectively). The majority of normal marrow progenitors were spared (inhibition of CFU-C: 10-13%; BFU-E: 0%). These observations suggest that MM4 may be useful for selective depletion of human myeloma clonogenic stem cells from bone marrow ex vivo.

Algorithms↗
PubMed1992

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

Alkaline Phosphatase↗
PubMed1981

Stimulation of the proliferation of hemopoietic stem cells in irradiated bone marrow cell culture.

Long-term hemopoiesis was established in bone marrow cell culture in vitro. This culture was shown to support the recovery proliferation of hemopoietic stem cells completely in vitro after irradiation. Hemopoietic stem cells were stimulated into proliferation in culture when normal bone marrow cells were overlayed on top of the irradiated adherent cell colonies. These results indicate that proliferation and differentiation of hemopoietic stem cells in vitro are also supported by stroma-hemopoietic cell interactions.

Animals↗
PubMed1998

Participation of NK1.1+ T cells in the rejection of lpr alphabetaT cells when bone marrow cells of lpr mice are transplanted into B6 mice.

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr-->B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3 IL-2Rbeta+ NK cells and IL-2Rbeta+ CD3int cells (i.e., CD3int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1+ and NK1.1-, among the IL-2Rbeta+ CD3int cells. However, the NK1.1+ subset (i.e., NK1.1- T cells) was much more radioresistant, and the majority of CD3int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM1 antibody to eliminate NK cells alone or both NK cells and NK1.1+ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1+ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4 8 cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.

Animals↗
PubMed2000

Suppressor activity of bone marrow cells and localization of fluorescent-labeled bone marrow cells within ovine endometrial tissue.

Numbers of fluorescein isothiocyanate (FITC)-labeled bone marrow (BM) cells of donor lambs were quantified within endometrial cell suspensions following their administration to ovariectomized (OVX; control-and estradiol-17beta-treated) and intact (estrus, d-14 cyclic and pregnant) ewes. The numbers of fluorescent BM cells were greater (P < .05) for the estrous and d-14 cyclic ewes than for both groups of OVX ewes. Fractionation of the endometrial cells with Percoll revealed that the majority of fluorescent cells were low-density (1.002 to 1.056 g/mL) cells. In coculture experiments, low-density cells from lamb BM not only suppressed the incorporation of thymidine into phytohemagglutinin-treated peripheral blood lymphocytes, but the cells also released suppressor factor into the culture medium. Suppressor activity tended to be reversed (P < .1) by a pan-specific neutralization antibody to transforming growth factor-beta (TGF-beta); however, the activity was unaffected by a neutralization antibody to TGF-beta2. These findings suggest that ovine endometrial suppressor cells may represent a population of low-density BM-derived natural suppressor cells, and their trafficking and localization patterns may depend on an ovarian factor(s). Further, suppressor activity does not seem to be mediated by TGF-beta2.

Animals↗
PubMed2006

Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells.

Distinct signals that guide migration of mesenchymal stem cells (MSCs) to specific in vivo targets remain unknown. We have used rat MSCs to investigate the molecular mechanisms involved in such migration. Rat MSCs were shown to migrate to tumor microenvironment in vivo, and an in vitro migration assay was used under defined conditions to permit further mechanistic investigations. We hypothesized that distinct molecular signals are involved in the homing of MSCs to tumor sites and bone marrow. To test this hypothesis, gene expression profiles of MSCs exposed in vitro to conditioned medium (CM) from either tumor cells or bone marrow were compared. Analysis of the microarray gene expression data revealed that 104 transcripts were upregulated in rat MSCs exposed to CM from C85 human colorectal cancer cells for 24 hours versus control medium. A subset of 12 transcripts were found to be upregulated in rat MSCs that were exposed to tumor cell CM but downregulated when MSCs were exposed to bone marrow CM and included CXCL-12 (stromal cell-derived factor-1 [SDF-1]), CXCL-2, CINC-2, endothelial cell specific molecule-1, fibroblast growth factor-7, nuclear factor-kappaB p105, and thrombomodulin. Exposure to tumor cell CM enhanced migration of MSCs and correlated with increased SDF-1 protein production. Moreover, knockdown of SDF-1 expression in MSCs inhibited migration of these cells to CM from tumor cells, but not bone marrow cells, confirming the importance of SDF-1 expression by MSCs in this differential migration. These results suggest that increased SDF-1 production by MSCs acts in an autocrine manner and is required for migratory responses to tumor cells.

Animals↗
PubMed2001

Use of monoclonal antibodies to refine flow cytometric differential cell counting of canine bone marrow cells.

OBJECTIVES: To evaluate use of monoclonal antibodies to increase accuracy of flow cytometric differential cell counting of canine bone marrow cells. SAMPLE POPULATION: Bone marrow specimens from 15 dogs. PROCEDURES: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Location of fluorescent and nonfluorescent cells within gates of a template developed for canine bone marrow differential cell counting was determined, the template was revised, and 10 specimens were analyzed by use of the old and revised templates and by labeling cells with anti-MHC class-II and anti-CD14. RESULTS: Data confirmed the presumptive location of marrow subpopulations in scatter plots, permitted detection of lymphocytes and monocytemacrophages, and was used to revise the analysis template used for differential cell counting. When differential cells counts determined by the original and revised templates were compared with results of manual differential cell counts, the revised template had higher correlation coefficients and more similar mean values. Labeling cells with anti-MHC class-II and anti-CD14 permitted identification of lymphoid and monocyte-macrophages cells in bone marrow specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the revised flow cytometric analysis template combined with anti-CD14 and anti-MHC class-II antibody labeling provides reliable differential cell counts for clinical bone marrow specimens in dogs. These techniques have potential applications to clinical bone marrow examination and preclinical toxicity studies.

Animals↗
PubMed1990

A simple technique to facilitate the ultrastructural analysis of cells in soft agar culture systems: demonstration of the development in vitro of morphologically mature mast cells and phagocytic macrophages from the bone marrow cells of genetically mast cell-deficient W/Wv or congenic normal mice.

We developed a simple technique that greatly facilitates the ultrastructural examination of cells growing in small, widely dispersed colonies in agar medium, and used the method to examine the development of morphologically mature mast cells and actively phagocytic macrophages in agar cultures of mouse bone marrow cells. The bone marrow cells of genetically mast cell-deficient WBB6F1-W/Wv or congenic normal (+/+) mice were cultured in semisolid agar medium supplemented with supernatants of concanavalin A-stimulated splenocytes. To prepare the colonies of hematopoietic cells for transmission electron microscopy, all the colonies within the agar-containing medium in a 96-well culture plate were removed with a Pasteur pipette and placed in a dilute, mixed aldehyde fixative. After fixation, the agar still enmeshing and separating individual colonies of cells was melted at 94 degrees C, rapidly mixed with molten 2% agar in a microfuge tube, centrifuged for 1 minute, and then the plastic tube was cooled in ice for 30 minutes. The plastic was removed with a razor blade, the agar block was hemisected from top to bottom, and then the blocks were processed for electron microscopy, embedded flat, and sectioned for light and electron microscopy. The culture conditions tested resulted in the development of morphologically mature mast cells and actively phagocytic macrophages, whether cultures were initiated with bone marrow cells from WBB6F1-W/Wv or congenic +/+ mice.

Agar↗
PubMed1993

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: I. Intrathymic repopulating ability of ER-MP12-positive bone marrow cells.

We searched for new cell surface markers that allow a positive identification of thymus-repopulating cells in the bone marrow (BM) of the mouse. Recently we raised two rat monoclonal antibodies (ER-MP12 and ER-MP20) that recognize cell surface antigens expressed by mouse haematopoietic progenitor cells, among which are progenitor cells of the macrophage lineage. Here we show that the ER-MP12 antigen, but not the ER-MP20 antigen, is also expressed by BM cells with thymus-repopulating ability. Using ER-MP12 and ER-MP20 in two-colour immunofluorescence analysis six subpopulations of BM cells can be identified. The thymus-repopulating ability of each BM subpopulation was assessed after fluorescence-activated cell sorting and subsequent intrathymic injection into sublethally irradiated Thy-1 congenic recipient mice. Thymus-repopulating activity appeared to be exclusively confined to two subsets of BM cells expressing either high or intermediate levels of the ER-MP12 antigen, but lacking ER-MP20 antigen expression. These BM subsets comprised 1-2% and 30% of total nucleated BM cells respectively. The frequency of thymus-repopulating cells was maximal in the minor BM subpopulation with the highest level of ER-MP12 antigen expression. We conclude that ER-MP12 detects a hitherto unknown cell surface marker expressed by BM cells with thymus-repopulating ability.

Animals↗
PubMed1984

Cell proliferation in bone marrow cells of severely malnourished animals.

The cell proliferation of bone marrow cells in rats, suffering from severe malnutrition during the lactation period, was investigated by using bromodeoxyuridine (BdUr) mixed with 100 mg of activated charcoal injected i.p. The undernourished rats had significantly more mitosis in the first generation (82.6%) than did well-nourished rats (44.6%) at 18 hours after the BdUr injection, whereas there were more cells in second division metaphase in the well-nourished (54.7%) than in the undernourished group (13.7%). Our results seem to indicate that malnutrition produces a prolongation in the time of proliferation of bone marrow cells during the lactation period. This agrees with other reports that used other cell systems in experimental animals and severely malnourished children.

Animals↗
PubMed1986

Species differences in the expression of caseinolytic proteinases and plasminogen activators by bone marrow cells.

Intact viable bone marrow cells from hamsters (Mesocricetus aruatus), rat (Rattus norvegicus), guinea pig (Cavia porcella) and rabbit (Oryctolagus cuniculus) were analyzed for caseinolytic proteinases and plasminogen activator activity. Species specific quantitative and qualitative differences in caseinolytic activity were detected. Quantitative and qualitative differences in plasminogen activator expression were observed. Cellular fractionation experiments revealed a heterogenous distribution of plasminogen activator activity among subpopulations of cells. The plasminogen activator activity associated with bone marrow cells behaved as ectoenzymes. These results indicated that different species may regulate bone marrow cell proteinase activity in a species-specific manner, compatible with their unique regulatory requirements.

Animals↗
PubMed2000

Role of hepatocyte growth factor in the development of dendritic cells from CD34+ bone marrow cells.

BACKGROUND AND OBJECTIVE: Hepatocyte growth factor (HGF) is known to augment the effects of stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoetin, and granulocyte colony-stimulating factor, all of which are involved in hematopoiesis. HGF is also known to have a role in immune responses. The aim of this study was to investigate whether HGF is involved in the development of dendritic cells (DC) from CD34+ bone marrow cells. DESIGN AND METHODS: CD34+ cells obtained from three healthy donors were incubated in various combinations of HGF, GM-CSF, and tumor necrosis factor (TNF) for 12 days. Developing cell populations were analyzed for surface markers, morphology and functional capacities by flow cytometry, light microscopy and mixed lymphocyte reaction, respectively. RESULTS: Incubation with HGF alone generated greater number of dendritic cells from CD34+ bone marrow cells than incubation with GM-CSF, or a combination of GM-CSF with TNF. HGF was also found to potentiate the effect of GM-CSF on DC and monocyte development. The effects of HGF were inhibited by the concurrent use of TNF. INTERPRETATION AND CONCLUSIONS: HGF appears to be a significant factor in the development of dendritic cells from CD34+ bone marrow cells.

Antigens, CD↗
PubMed2001

Hepatic stem cells: from bone marrow cells to hepatocytes.

Hematopoiesis and the hepatic environment are known to have a close relationship at the time of hepatic development and systemic diseases. Recently, transplanted cells isolated from bone marrow of rodents and humans have been shown to differentiate into oval cells, which are considered to be hepatic stem cells, and hepatocytes in the liver. Then, purified hematopoietic stem cells were shown to have the ability to replace original liver cells in mice with hereditary tyrosinemia. In this review the interactions between hepatic stem cells are summarized and a hypothesis of hepatic differentiation will be proposed.

Animals↗