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Bone marrow capacity for bone cells and trabecular bone turnover in immobilized tibia after sciatic neurectomy in mice.

Trabecular bone turnover and bone marrow capacity for the development of bone cells in the tibia were assessed after sciatic neurectomy (NX) in mice. The right hindlimbs of 6-week-old DDY mice were neurectomized and left hindlimbs were sham-operated and served as NX controls. Histomorphometrical analyses of the trabecular bone of the proximal tibia demonstrated the initial decrease in bone formation rate for the first 14 days and the subsequent increase in osteoclast surface for the next 14 days. The number of adherent stromal cells per tibia obtained for the NX limbs was reduced on days 7 and 10 postsurgically, and then recovered on day 12. However, the alkaline phosphatase activity of the cells was persistently depressed. The formation of osteoclast-like multinucleated cells in the marrow cultures obtained from NX limbs at days 10, 12, and 14 showed a significant increase in the medium containing parathyroid hormone (PTH). The number of colonies cultured for colony forming units-fibroblastic (CFU-f) that developed from the marrow cells did not differ in the NX and the contralateral limbs at any time during the period. On the other hand, the number of colonies cultured of colony forming units for granulocytes and macrophages (CFU-GM) was markedly increased for both the NX and the contralateral tibiae at days 12 and 14. This study clearly demonstrates that there are two stages in the development of osteopenia after NX. During the first 14 days, trabecular bone formation and number of marrow stromal cells are reduced. In the second 14 day period, the trabecular osteoclast number is increased and osteoclast formation from the bone marrow cells is enhanced in the presence of PTH. However, neither the CFU-f nor the CFU-GM assay could identify the changes in osteogenic or osteoclastogenic potential of the bone marrow. These in vitro assays provide limited information on the shifts in bone marrow cell lineages and the local environment producing osteopenia in the immobilized limb in vivo.

Alkaline Phosphatase↗

Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow.

Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue-nonspecific alkaline phosphatase (TNAPase)-immunopositive osteoblastic cells and osteopontin immunopositive-extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF-induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin-immunoreactivity was observed at cement lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase-positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF-induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts.

Acid Phosphatase↗

Absence of detectable P450 2E1 in bone marrow of B6C3F1 mice: relevance to butadiene-induced bone marrow toxicity.

The observation that the concentration of butadiene monoepoxide (BMO) is greater in bone marrow than in the blood following inhalation of 1,3-butadiene (BD) by B6C3F1 mice prompted the present investigation into whether cytochrome P450 2E1, the isozyme believed to be involved in the epoxidation of BD, is present in the bone marrow of B6C3F1 mice. The bone marrow of male and female B6C3F1 mice was analyzed by Western blot analysis and immunohistochemistry to determine whether cytochrome P450 2E1 is present, and, if present, in which cell types. Both Western blot and immunohistochemical analyses revealed the apparent absence of P450 2E1 in B6C3F1 mouse bone marrow. This observation suggests that BD is converted to BMO outside of bone marrow and is subsequently concentrated in the bone marrow, or that the conversion of BD to BMO occurs by an alternate enzymatic pathway within the bone marrow.

Animals↗

[Cellular aspects of bone regeneration: role of bone marrow periostium].

Bone regeneration is only possible if stem cells give rise to progenitors of osteoblasts, chondroblasts or chondroidocytes. Stem cells and osteogenic progenitors were evidenced in bone marrow while only progenitors can be found in periosteum. Bone marrow stem cells did show an amazing plasticity and some cells of the bone surrounding tissues such as perivascular cells, adipocytes, muscle cells or even circulating cells are able to transdifferentiate in osteoblasts when submitted to an osteogenic environment. We have shown that the destruction of both bone marrow and periost impairs the bone healing. It indicates that the periost and bone marrow destruction removes the predetermined osteogenic cells and the informative factors able to induce the transdifferenciation of the cells contained in the peri-osseous tissues.

Bone Marrow↗

Multiple myeloma: an immunologic profile. II. Bone marrow studies.

Bone marrow mononuclear cell populations were studied in 35 patients without myeloma, 39 patients with multiple myeloma, and 15 patients with benign monoclonal gammopathy. Bone marrow mononuclear cell receptors, responses to mitogens or allogeneic stimuli, and suppressive effects on in vitro peripheral blood lymphocyte (PBL) function were studied. In bone marrow cell populations from patients with untreated multiple myeloma, the percent of complement receptor-bearing cells and the pokeweed mitogen- and concanavalin A-stimulated responses were significantly greater than were those in bone marrow cell populations from patients without myeloma. Sheep red blood cell receptor-bearing cells were significantly greater in marrow populations from treated multiple myeloma patients compared to those from untreated multiple myeloma patients. Sheep red blood cell receptor-bearing cells from the bone marrow of multiple myeloma patients suppressed responses of the multiple myeloma patients' PBL's to autologous mitomycin C-treated bone marrow plasma cells and to allogeneic stimuli in one-way mixed leukocyte culture. Complement receptor-bearing cells suppressed the response to pokeweed mitogen. The presence of lymphocytes in the marrow compartment that are capable of suppressing the response of myeloma patients' PBL's to plasma cell antigens may be significant in the pathogenesis of multiple myeloma.

Bone Marrow↗

Bone marrow necrosis.

Bone marrow necrosis has been regarded as a rare entity in specimens obtained from living patients and has been associated with poor prognosis. In contrast, we believe that it is a commonplace finding in bone marrow specimens which is frequently overlooked and which occurs in patients with multiple acute and chronic disorders. It is postulated that bone marrow necrosis eventuates from vascular occlusion of small blood vessels as a result of a number of causes. When bone marrow necrosis is prolonged, it may be associated with the development of bone marrow fibrosis and serve as a predisposing lesion for idiopathic myelofibrosis. Additional investigation of this phenomenon is required to determine its usefulness in the diagnosis of disease states and its role in the pathophysiology of a number of disorders.

Animals↗

Use of photosensitive, antibody directed liposomes to destroy target populations of cells in bone marrow: a potential purging method for autologous bone marrow transplantation.

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry.

Bone Marrow↗

Characterization of a B cell progenitor present in neonatal bone marrow and spleen but not in adult bone marrow and spleen.

The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin- CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin- CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin- CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin- CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.

Aging↗

Early B cell precursors in long-term bone marrow culture: selective development in the bone marrow of irradiated recipients.

The primary site for the growth and differentiation of B cell precursors in irradiated recipient mice was investigated. Bone marrow (BM) cells from lipopolysaccharide (LPS)-responder C57BL/6 mice were transferred into irradiated LPS-nonresponder C57BL/10ScCr mice, and the generation of donor-derived B cells in the recipient was monitored by determining the immunoglobulin-producing cells developed in the LPS-stimulated cultures of recipient's spleen cells as well as BM cells. As previously stated, the transfer of fresh BM cells resulted in the development of LPS-reactive cells both in spleen and BM simultaneously. On the other hand, when long-term cultured BM cells which were shown to be devoid of B cells and pre-B cells were used as the donor cells, the development of LPS-reactive cells was first observed only in BM, and subsequently in both BM and spleen. The failure to detect LPS-reactive cells in the spleen in the early phase, or day 11 after irradiation and reconstitution, was not attributed to the culture condition or the existence of suppressive activity in day 11 spleen cells. These results indicate that B cell precursors lodge only in the BM of irradiated recipients, grow and differentiate in the same place, and then the differentiated progeny migrate to peripheral lymphoid organs.

Animals↗

Thymus-dependent lymphocytes in human bone marrow.

Human bone marrow is known to contain significant numbers of bursa-dependent lymphocytes. The presence of thymus-dependent (T) cells is controversial. Bone marrow cells obtained from healthy volunteers was fractionated by density centrifugation. A lymphocte-enriched subpopulation was shown to be reactive to alloantigens in mixed lymphocyte culture and to contain substantial numbers of T lymphocytes. The T lymphocytes were detected by cell surface markers (rosette formation with sheep RBC) and by response to the mitogens phytohemagglutinin and concanavalin A. Bone marrow T cells exhibited functional characteristics quantitatively different from peripheral blood T cells, suggesting that they may represent a particular subpopulation of T cells. The lymphocyte-enriched fraction additionally contained committed granulopoietic stem cells capable of colony formation in semisolid gel. The presence of T cells in human bone marrow is consistent with findings in other mammals and may explain the high incidence of graft versus host disease in bone marrow transplant recipients.

Adult↗

Prevention of cyclosporine-induced syngeneic graft-versus-host disease in bone marrow transplantation by UV-B irradiated bone marrow cells.

UV-B irradiation of allogeneic rat bone marrow cells (BMC) transplanted into lethally gamma-irradiated recipients prevents GVHD and induces stable complete hematopoietic chimerism. Cyclosporine (CsA), an effective immunosuppressive agent, causes an autoimmune syndrome termed syngeneic GVHD in syngeneic radiation chimeras following discontinuation of CsA. To understand the in vivo interactions of CsA with UV-B modulated syngeneic bone marrow transplant (BMT), as this is essential before clinical use, we studied the effects of CsA therapy in recipients of UV-B irradiated donor BMT in the rat model. Lethally irradiated (10.5 Gy) Lewis recipients of naive or UV-B irradiated syngeneic BMT (admixture of 10(8) BMC and 5 x 10(6) spleen cells) were treated with CsA (i.m. 12.5 mg/kg/day) for 30 consecutive days after BMT. The results show that all irradiated Lewis recipients of syngeneic BMT modulated with 700 J/m2 UV-B were hematologically fully reconstituted in 25-35 days and survived their normal life span. In contrast, all lethally irradiated recipients of untreated BMT that received CsA for 30 days developed lethal acute syngeneic GVHD 7-12 days after CsA withdrawal. Of interest is the finding that while higher doses of UV-B (500-700 J/m2) irradiation of BMC prior to transplantation into CsA-treated animals prevented hemopoietic reconstitution, lower doses (100-300 J/m2) allowed for hemopoietic recovery and, in addition, prevented the development of syngeneic GVHD following the discontinuation of CsA. The development of syngeneic GVHD was dependent on the presence of the thymus and did not occur in thymectomized recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Basic and clinical studies on patients who have undergone autologous or allogeneic bone marrow transplantation].

Bone marrow transplantation (BMT) is now increasingly applied not only to hematologic disorders such as leukemias and aplastic anemia but also to some of solid tumors. We experienced ten cases of bone marrow harvest and six cases of autologous or allogeneic BMT during the period from June, 1986 to August, 1987. For autologous BMT cases, bone marrow cells were harvested and cryopreserved at -196 degrees C in liquid nitrogen tank and colony forming assays were examined to ascertain the colony forming ability of harvested bone marrow stem cells. Recovery of colony forming ability after cryopreservation in colony forming unit in culture and burst forming unit in erythrocyte is about 63 to 73% and well preserved. Among 6 cases undergone BMT, five cases are patients with advanced solid tumors including breast cancer, seminoma, lung cancer, malignant lymphoma and brain tumor and these 5 cases have undergone autologous BMT. Conditioning regimen for each cancer in autologous BMT differs based upon conventional chemotherapy regimen and two to four fold dose of conventionally used chemotherapy regimen were administered and after two or three days of high dose chemotherapy, cryopreserved bone marrow cells were thawed and infused. Peripheral white blood cells and platelets were recovered to more than 1000/microliters and 5 x 10(4)/microliters, respectively, within 21 days after bone marrow cell infusion. Disappearance or decrease in tumor size was observed in all cases except brain tumor even though those cases were advanced ones. After autologous BMT, recurrence occurred within three to five months in three cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Antigens, CD34↗

Bone marrow monocytes in histiocytosis X acquire some phenotypic features of Langerhans cells in long term bone marrow cultures.

Bone marrow cells of a patient with Letterer-Siwe disease were cultured for three weeks in long-term bone marrow culture (LTBMC) conditions and examined at one-week intervals with a large panel of monoclonal antibodies by immunohistochemistry and by the immunogold transmission electron microscopy (immunoTEM) technique. Although at diagnosis the bone marrow showed a slight increase of monocytes with a normal phenotype, a rapid expansion of cells expressing CD1a and CD1c was observed already after 1 week of culture. A progressive increase in CD4, CD11b and CD11c expression was also observed. ImmunoTEM of cultured cells demonstrated that CD1a+ cells had macrophage-like morphology, and did not contain Birbeck granules. These findings indicate that bone marrow monocytes acquire some phenotypical features of Langerhans cells in LTBMC and support the hypothesis that these cells may derive directly from a bone marrow monocytic precursor.

Aged↗