Cloning of stem cells in the bone marrow of irradiated mice

Various numbers of cells of intact or irradiated bone marrow from syngeneic donors were injected into mice irradiated with a lethal dose. A linear relationship was found between the number of 8–9 day colonies growing in the femoral marrow and the number of injected cells, and an exponential relationship was found with the dose of irradiation. The pattern of cloning of the stem cells in the bone marrow was similar to that in the spleen. Differences were found in the radiosensitivity of colony-forming units (CFU) depending on the organ (spleen, bone marrow) in which they formed the colonies. CFUs settling in the bone marrow were more resistant (D₀ 160–200 R) than CFUs settling in the spleen (D₀ 80–100 R). Differences in the radiosensitivity of the CFUs are due, it is postulated, to the presence of a heterogeneous population of stem cells and also to specific features of the organ (spleen, bone marrow) in which the colonies are formed.     

Graft-versus-host reactions in mice. IV. Thymus cell suppression of antibody formation

The ability of transplanted marrow-thymus cell mixtures to generate antibody-forming cells in irradiated syngeneic or F₁ hybrid mice when immunized with sheep erythrocytes 18 hours later was determined. Much fewer anti-sheep plaque-forming cells (PFC) were detected in spleens of F₁ hybrid mice. Adrenalectomy, use of infant recipient mice, or preimmunization of donors or hosts did not prevent the suppression; the grafting of irradiated donor-type spleen cells (source of “accessory” cells) produced only an additive effect. Parental marrow and thymus cells were able to generate new precursors of PFC and specific inducer cells, respectively, in spleens of F₁ hybrid mice, as detected by two-step experiments utilizing parent-strain secondary recipient mice. The suppression depended upon transferring parental strain thymus cells into F₁ hybrid mice and was seen irrespective of the marrow donor strain. When irradiated mice were immunized twice (on the day of transplantation and 4 days later), there was only marginal suppression of antibody production when marrow cells only or marrow plus thymus cells were transplanted. Thus, it appears that an excess of thymus-derived “suppressor” cells is generated upon exposure to alloantigens and inhibit terminal differentiation of antibody-forming cells in a noncytotoxic manner. Mature PFC themselves were not the targets of suppression. The method of immunization probably determines the relative functional capacity of thymus-derived “helper” and suppressor cells.     

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1α,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1α,25(OH)₂D₃ were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1α,25(OH)₂D₃, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that (1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1α,25(OH)₂D₃ exhibit non-functional basic features of osteoclast morphology, and (2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P=0.002). Ten of twelve (83%) patients with metastatic disease (stage M₁) and six of eight (75%) patients without any overt metastases (M₀) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Blastogenic response of lymphocytes separated from bone marrow to allogeneic lymphoid cells in vitro

Fractions of cells were separated from the bone marrow and spleen of Lewis rats by brief centrifugation in linear sucrose-serum density gradients and were cultured in vitro either alone or mixed with F₁ (Lewis × Brown Norway) hybrid rat lymphoid cells. After 4 days the incorporation of [³H]thymidine into DNA was measured by scintillation counting, and the morphology and incidence of [³H]thymidine-labelled cells were determined in radioautographs.

Lymphocyte-rich, slowly-sedimenting fractions of Lewis rat bone marrow cells showed increased incorporation of [³H]thymidine and the development of many large proliferating blast-like cells when cultured with F₁ hybrid cells. This blastogenic response was greater than could be ascribed to contaminating intravascular blood lymphocytes. Slowly-sedimenting fractions of spleen cells, consisting mainly of small lymphocytes, showed a greater blastogenic responsiveness to F₁ hybrid cells than that of whole spleen, while rapidly-sedimenting spleen cell fractions, containing many large cells, showed a reduced responsiveness. Paradoxically, rapidly-sedimenting marrow cell fractions containing few small lymphocytes, showed an increment in [³H]thymidine incorporation when cultured with F₁ cells but this was due to active macrophage proliferation as well as to blastogenesis.

The results demonstrate that some parenchymal bone marrow lymphocytes undergo blastogenic transformation in response to allogeneic lymphocytes in vitro.

     

Cytology and histology of haemopoietic cell transplantation under the influence of ALS in mice. II. The effect of marrow from donors pretreated with antilymphocyte serum (ALS) on the recipient

Cytology and histology of recipients of allogeneic bone marrow were studied 13. 20, 24, 30 and 34 days after transplantation. The developing chronic secondary disease was characterized by increased numbers of myeloid cells, by lymphopenia and by erythroblastopenia in the bone marrow and the spleen. Erythroblastopenia together with lymphopenia and augmented myeloid cells also occurred in irradiated F₁-hybrids suffering from a chronic homologous disease. The latter model eliminated the following as a cause of erythroblastopenia in secondary disease: (1) host-versus-graft reaction against donor-type erythroblasts for immunogenetical reasons, and (2) graft-versus-host reaction against recipient-type erythroblasts since they had already been destroyed by irradiation. Treatment of the donor with ALS resulted in a suppression of homologous disease in F₁-hybrids with a cytology resembling that of recipients of syngeneic spleen cells. The same treatment only delayed the onset of chronic secondary disease in recipients of allogeneic bone marrow. These allogeneic recipients, in contrast to the F₁-hybrid recipients, died with the typical morphology of a chronic secondary disease.

In recipients of syngeneic bone marrow from donors treated with ALS, repopulation with lymphocytes was somewhat delayed and transient erythroblastosis in the spleen occurred 20 days after transplantation.

     

Superoxide anion (O2-) production and bactericidal capacity of polymorphonuclear neutrophils obtained from patients subjected to glucagon test

Superoxide anion (O₂^-) production and bactericidal capacity of morphologically mature bone marrow polymorphonuclear neutrophils (PMN) were evaluated in 30 haematologically normal individuals. These same parameters of peripheral PMNs were estimated in 15 healthy volunteers before and after glucagon-induced marrow granulocyte reserve mobilization. Bone marrow PMN in comparison with cells obtained from peripheral blood manifested impaired superoxide anion production and diminished bactericidal capacity. The admixture of bone marrow PMN released into the circulation by the use of glucagon administration significantly lowered both estimated PMN functions.     

Studies on the organization and regeneration of bone marrow: Origin,growth, and differentiation of endocloned hematopoietic colonies

Hematopoietic colonies were studied by light microscopy in the marrow of alternate fraction x-irradiated mice (C576J/B1) to investigate the microenvironmental organization of marrow and identify early hematopoietic cellstromal cell interactions. Undifferentiated colonies (UC) were detected at 3 days postirradiation, showed a marked predilection for bone surfaces, and disappeared as differentiated colonies developed. Some UC occurred along marrow arteries. Neutrophilic granulocyte colonies (GC) occurred in all areas at 3 days but grew rapidly only subosteally. Few eosinophilic colonies (GC_e) occurred. Erythrocytic colonies (EC) appeared at 4 days as dispersed populations of motile cells within a localized area of marrow; these tended to proliferate initially in intermediate and central marrow zones. Macrophage colonies (M?C) of two “subtypes” were detected, peaking in relative frequency at 4 days. These appeared active in stromal repair and monocytopoiesis. Megakaryocyte colonies (MC) originated along bone and differentiated away from bone. From 3–5 days, the frequency of GC > UC > M?C ? MC ? GC_e. All colony types except UC, M?C, and central GC increased in size and became mixed in differentiation by 12–14 days. For several weeks, however, erythropoiesis concentrated toward central areas, whereas granulopoiesis and thrombopoiesis concentrated along bone. Some mixed colonies showed an abrupt transition from erythrocytic, centrally, to granulocytic, subosteally. These results were interpreted as evidence that in x-irradiated marrow: (1) hematopoietic microenvironments (HMs) for stem-cell proliferation and commitment to differentiation, with the possible exception of HMs determining erythroid differentiation, occur in endosteal and periarterial regions; (2) a proliferative and/or chemotactic stimulus to erythroid progenitors exists in intermediate and central marrow regions; and (3) some subosteal regions may exclude erythropoiesis, or preferentially support nonerythroid differentiation. Elaborate associations occurred between macrophages and early UC, GC, and EC, but not MC hematopoietic cells. UC and GC often associated with osteoclasts. Reticular and other fibroblastic cells associated with the cells of all colony types.     

Induction of Auto-reactive Regulatory T Cells by Stimulation with Immature Autologous Dendritic Cells

We have shown in ex vivo studies in donor bone marrow-infused kidney transplant recipients, that chimeric cells of either donor or recipient origin taken from the recipient's bone marrow down-regulated the recipient's cellular immune responses. In the present study, we have now induced regulatory T cells from peripheral blood mononuclear cells (PBMC) of renal transplant recipients or laboratory volunteers by multi-stimulation with autologous immature dendritic cell (iDC) enriched populations derived from either bone marrow cells (BMC) of the (immunosuppressed) kidney transplant recipients or PBMC of the laboratory volunteers (i.e., ibDC and ipDC, respectively). These regulatory T cells, induced by ibDC and ipDC, were autoreactive and designated as T_Ab and T_Ap with similar phenotypes and functional profiles. They were largely CD4 + CD25^high, CD45RA low and CD45RO high, and uniformly expressed intracellular CTLA-4, and message of IL-4, IL-10, Foxp3, and differentially expressed TGFβ. Their proliferative responses to autologous mature dendritic stimulating cells (mDC) were ～two-fold stronger than to allogeneic mDC, and to allogeneic mDC were significantly lower than those of (control) autologous T_PBL, suggesting an anergic state. T_Ab and T_Ap were not cytotoxic to autologous cells expressing Epstein-Barr virus (EBV) antigens, but were able to inhibit (regulate) the effector phase of this T_PBL response to both autologous and allogeneic EBV lymphoblasts. This regulation appeared to require cell-to-cell contact.     

17β-雌二醇对骨髓基质细胞骨形态发生蛋白受体ⅠA、B mRNA表达的影响

目的:研究骨髓基质细胞在向成骨细胞分化的介质中,17β-雌二醇(E₂)对其骨形态发生蛋白受体(BMPR)ⅠA,ⅠBmRNA表达的影响,探讨雌二醇对成骨细胞生成的作用。方法:用1,25(OH)₂D₃和地塞米松(DEX)诱导大鼠骨髓基质细胞向成骨细胞分化,应用半定量RT-PCR技术,观察不同浓度E₂对骨髓基质细胞分化过程中BMPR-ⅠA,ⅠBmRNA表达的影响,以α-磷酸奈酚为底物测定细胞碱性磷酸酶的活性,VanGieson染色法显示Ⅰ型胶原的含量。结果:E₂能明显抑制骨髓基质细胞分化过程中BMPR-ⅠAmRNA的表达,且呈剂量依赖性,随E₂浓度增加,BMPR-ⅠAmRNA从(23.7±1.7)%降至(16.3±1.5)%(P<0.05)和(8.3±1.2)%(P<0.01);BMPR-ⅠBmRNA随E₂浓度增加而增加,从(1.3±0.6)%增至(5.7±2.0)%(P<0.05)和(15.3±3.0)%(P<0.01)。ALP活性随E₂浓度增加而降低,从(42.6±2.5)U·L^-1·g^-1protein降至(10.8±1.5)U·L^-1·g^-1protein和(3.6±1.7)U·L^-1·g^-1protein(P<0.01);细胞Ⅰ型胶原的含量随E₂浓度增加而减少。结论:E₂能明显抑制体外培养的骨髓基质细胞分化过程中BMPR-ⅠAmRNA的表达,降低成骨细胞生成。     

Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts.

Two types of host reactivities not requiring immunization in the mouse and not mediated by T lymphocytes were compared: resistance of irradiated and nonirradiated F₁ hybrids to accept parental grafts of normal or malignant hemopoietic cells (Hh system), and the natural killer cell activity against mouse lymphomas (NK system). The effects of six independent variables known to influence resistance to marrow grafts were investigated in the NK system using YAC-1 lymphoma cells as targets. The following properties were shared: (a) maturation during the fourth week of life; (b) low sensitivity to acute total body irradiation; (c) dependence on the integrity of bone marrow as demonstrated by reduced reactivity in ⁸⁹Sr-treated mice; (d) suppression by a single injection of rabbit anti-mouse bone marrow serum; (e) suppression by a single injection of the anti-macrophage agents silica and i-carrageenan; and (f) suppression by multiple injections of parental spleen cells into F₁ mice. These positive correlations are particularly significant because most of the variables have either opposing or no effect on conventional immunity. F₁ mice rendered specifically unresponsive to parental marrow grafts, could retain NK cell activity, and genetically susceptible mice could be rendered hyporeactive in terms of NK cells, indicating that the specificities of YAC-1 and Hh-1 incompatible targets were different. It is extremely unlikely that this remarkable parallelism is fortuitous. These results indicate that either a very similar, or more likely a common mechanism is operative in the two cell-mediated natural reactivities: effector cells in the NK and Hh systems do not bear B or T lymphocyte markers but are nevertheless endowed with “specificity”. They are dependent for generation in vivo (presumably by maturation or by recruitment) on the interaction with nonlymphoid accessory cells not endowed with specificity, capable of also interacting in vitro with Thy-1-positive F₁ hybrid prekiller cells specific for parental targets. Because of thymus independence in vivo and apparent restriction to target cells of the hemopoietic system, these reactivities should be effective in the regulation of hemopoiesis and surveillance over leukemogenesis.     

Attempts to immunize F1 hybrid mice against their parents

Attempts were made to immunize F₁ hybrid mice to their inbred parental strains by active immunization with either living or dead parental cells and by adoptive transfer of isogenic (F₁) lymph node cells to irradiated hybrids that subsequently received parental bone marrow. Cumulative mortality studies and erythrocyte serotyping revealed that many mice survived and retained their parental grafts for long periods of time. In most experiments reported here, ⁵⁹Fe uptake by erythrocytes and spleens of chimeras 1 week after marrow transplantation was used as a measure of erythropoiesis and thereby of success or failure of the marrow graft. Pre-treatment (`pre-immunization'') of hybrids with parental spleen cells produced no evidence of specific immunization. However, pre-treatment with spleen cells from parent 1 was detrimental to subsequently transplanted marrow from parent 2 and vice-versa. Adoptive transfer of viable F₁ lymph node cells failed to decrease growth of parental cells in irradiated F₁ hybrids. These findings indicate that the poor-growth phenomenon of particular parent—F₁ combinations cannot be explained in terms of classical immune rejection of parental cells by F₁ hybrids. Instead, growth of parental cells in the F₁ environment is inhibited or delayed.     

Development of cell therapy using autologous bone marrow cells for liver cirrhosis

The plasticity of bone marrow has been confirmed by the autopsy of a female recipient of bone marrow cell transplantation from a male donor. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model named the green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model. Using the GFP/CCl₄ model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and differentiated into albumin-producing hepatocytes under persistent liver damage induced by carbon tetrachloride. Moreover, bone marrow cell transplantation into mice with liver cirrhosis improved liver function and liver fibrosis with the strong expression of matrix metalloproteinases (MMPs), especially MMP-9 activity, resulting in an improved survival rate. Results from the GFP/CCl₄ model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. A summary of findings from the GFP/CCl₄ model is described.     

Studies on the production of complement

The production of complement was investigated using a modification of Jerne's haemolytic plaque technique. Guinea-pig spleen and bone marrow cells were suspended in layers of agarose containing, as indicator cells, sensitized sheep erythrocytes (EA) or intermediate products consisting of EA and some of the components of complement. R reagents or serum, in which certain components of the C′₃ complex had been inactivated, were added after incubation. No plaque formation due to release of complement components was observed. Bone marrow and spleen cells produced plaques when incubated with sensitized or non-sensitized erythrocytes and a reagent containing cobra venom.

A method was developed for the preparation of thin films of agarose in which tissue cells and erythrocytes were suspended in a single cell layer. Single cells and clumps of cells present in the peritoneal exudate of normal guinea-pigs produced plaques when incubated with EA or EAC′₄ and R₁. Occasionally, similar clumps of cells also produced plaques with EA or EAC′₁ and R₄. The plaque-forming cells appeared to be macrophages. Plaques were not detected with R₂ or R₃.

No plaques were produced by spleen cells under similar experimental conditions. Bone marrow cells produced plaques with both sensitized and non-sensitized erythrocytes after prolonged incubation.

Optimal plaque formation occurred when R₁ or R₄ was added before incubation. Plaques were not produced if the tissue cells were pre-incubated in suspension cultures for 2 hours or longer before plating. Treatment of the peritoneal exudate cells with KCN or with dinitrophenol prevented plaque formation.

     

A murine model for bone marrow metastasis established by an i.v. injection of C-1300 neuroblastoma in A/J mice

A reproducible tumor model for bone marrow metastasis has been developed by an injection of murine C-1300 neuroblastoma (C-1300 NB) cells into the tail vein of syngeneic A/J mice. The animals died with liver metastases at 18–21 days after an injection of 10⁵ tumor cells and often had bone marrow metastasis in the femur. N-methylformamide (NMF), a maturational agent, was administered to inhibit liver metastases and to extend survival in mice with advancing bone metastasis. Histological examination of bone marrow metastasis, demonstrated lesions varying from a few small colonies of C-1300 NB cells either in metaphysis or diaphysis to large foci replacing normal hematopoietic bone marrow, simultaneously invading epiphysis or cortex of bone as bone metastasis. This assay demonstrated the ability to detect neuroblastoma cells in the bone marrow histologically and could determine bone marrow TD₅₀ by extraction of bone marrow cells after treatment with various doses of drug. Fifty per cent of mice injected with cyclophosphamide (CY) developed bone marrow metastasis without liver metastasis. Treatment with tamoxifen, an anti-calmodulin drug, suppressed tumor takes in the recipient mice with tamoxifen-dose-dependent fashion. This experimental system allows for investigations into the therapeutic response and biology of neuroblastoma metastases in the bone marrow.     

补肾活血中药对免疫介导再障小鼠骨髓CD45+细胞的影响

目的:观察补肾活血中药对免疫介导再障小鼠CD₄₅⁺细胞的影响。方法:建立免疫介导再障小鼠模型,随机分组胃饲生理盐水、高剂量中药、低剂量中药、环孢素A(CsA),并设正常对照,第10d分别称体重、计算外周血细胞和骨髓有核细胞数、采用流式细胞技术测定骨髓CD₄₅⁺细胞。结果:中药组小鼠体重、外周血细胞、骨髓有核细胞数、骨髓CD₄₅⁺细胞显著高于空白模型组和CsA组,而接近正常对照组。结论:补肾活血中药对免疫介导再障小鼠的骨髓造血细胞有保护作用。     

Effects of ‘Bordetella pertussis’ on hemic colony-forming cells and the immune response

Single injections of ‘Bordetella pertussis’ were administered to BDF₁ mice, and levels of colony forming cells (CFC) in bone marrow and spleen were determined at selected time intervals after injection. Our studies demonstrated that such injections have multiphasic effects on the levels of CFC present in bone marrow and spleen. To test whether the immune response was altered by ‘B. pertussis’, cultured spleen cells from treated mice were measured for their ability to produce plaque forming cells (PFC), in vitro, and to participate in mixed cell interactions, in vitro. Augmentation or suppression of the immune response in vitro depended on the inoculum size of cultured cells as well as the time of assay after injection.     

Differential β3 and β1 Integrin Expression in Bone Marrow and Cortical Bone of Estrogen Deficient Rats

Integrin‐based (β₃) attachments to the extracellular matrix (ECM) on osteocyte cell processes have recently been proposed to play an important role in facilitating osteocyte mechanosensation. However, it is not yet known whether integrin expression is altered in the mechanoregulatory osteocytes during osteoporosis. The objective of this study was to test the hypothesis that the expression of integrin‐based mechanosensory complexes (β₁ and β₃ integrins) is altered as a direct response to estrogen deficiency, in an estrogen deficient animal model of osteoporosis. Four weeks post‐operatively, immunohistochemistry was used to detect for β₁ and β₃ integrin subunits in bone tissue and marrow of ovariectomized (OVX; N = 4) and SHAM (N = 4) operated animals. A tartrate resistant acid phosphatase (TRAP) control stain was performed to quantify the presence of osteoclasts in the bone marrow and bone surfaces. Image analysis was performed to quantify expression patterns in different biological compartments, that is, bone marrow, endosteum, and cortical bone. Our results showed that β₁ integrins were ubiquitously expressed throughout the bone and marrow, for both OVX and SHAM groups. β₃ integrin subunit expression was lower in bone cells from osteoporotic animals compared to controls, whereas β₃ expression in marrow cells did not differ significantly between groups. At the endosteum no difference was observed in β₃ integrin subunit expression. As expected, the number of osteoclasts was higher in the OVX group validating an imbalance in bone remodeling. We propose that a reduction in β₃ integrin expression in osteocytes might impair mechanosensation by bone cells during estrogen deficiency. Anat Rec, 298:1548–1559, 2015. © 2015 Wiley Periodicals, Inc.     

Macrophages in normal human bone marrow and in chronic myeloproliferative disorders: An immunohistochemical and morphometric study by a new monoclonal antibody (PG-M1) on trephine biopsies

Summary An immunohistochemical and morphometric study was performed on routinely processed trephine biopsies of the bone marrow in 30 normal individuals and in 90 patients with various subtypes of chronic myeloproliferative disorder. Using a new monoclonal antibody (PG-M₁) directed against a formalin-resistant epitope on macrophages and by employment of the Prussian blue reaction, quantitation of this cell population was feasible. Morphometric analysis revealed that the number of iron-laden macrophages represented only a fraction of the total number of histiocytic reticular cells. As could be expected, in polycythaemia rubra vera, no haemosiderin deposits were detectable, but the content of macrophages slightly exceeded that of the normal bone marrow. In chronic myeloid leukaemia 9 of 30 patients showed a significant increase in PG-M₁-positive reticular cell elements. These were consistent with pseudo-Gaucher cells, sea-blue histiocytes and intermediate cell types. Primary (idiopathic) myelofibrosis-osteomyelosclerosis was characterized by a significant increase in macrophages (25 of 30 patients). Involvement of macrophages in the complex mechanisms generating bone marrow fibrosis and angiogenesis and in bone remodelling (osteosclerosis) may be responsible for this finding.