Migration of erythroblastic islands toward the sinusoid as erythroid maturation proceeds in rat bone marrow

It has been hitherto considered that mature erythroblasts migrate toward the sinusoid along the cytoplasmic processes of macrophages of erythroblastic islands in bone marrow. Our previous report, however, has demonstrated the morphological features of a mature erythroblastic island passing through the sinusoidal endothelium. In this study, the possibility of migration of erythroblastic islands toward the sinusoid was examined in rat bone marrow by light microscopical histoplanimetry. As a result, the more mature erythroblasts were not regularly arranged in the peripheral direction of the erythroblastic islands. Immature erythroblasts were populated more in the erythroblastic islands away from the sinusoid than in those islands neighboring the sinusoid, whereas mature erythroblasts were more in erythroblastic islands neighboring the sinusoid. These findings suggest that the formation of erythroblastic islands occurs in a region away from the sinusoid, and that erythroblastic islands migrate towards the sinusoids as erythroid maturation proceeds.     

Immunohistological demonstration of erythroid cells in canine bone marrow

In an immunohistological/cytological study of canine bone marrow, the aim was to demonstrate canine erythroid cells with the help of various commercially available antibodies against human antigens (monoclonal antibody against glycophorin A, polyclonal antibodies against haemoglobin and spectrin). In order to preserve possible cross-reacting epitopes various fixation methods (cross-linking, precipitating and dehydrating fixing agents, partly in combination with unmasking measures), decalcification techniques [acid or ethylenediaminetetraacetic acid (EDTA) decalcification] and tissue-embedding methods (paraffin embedding, cryostat sectioning technique) were used. Alternative methods, such as the preparation of cell smears and immunoblotting, were also employed. The only result that was of use for routine diagnostic procedures (paraffin sections) was that obtained by using polyclonal antibodies against haemoglobin. Best results were achieved when tissue was fixed in a formaldehyde-glutaraldehyde mixture, decalcified in EDTA and treated with microwave irradiation. The primary antibody was used in a dilution of 1:500 and incubated for 16 h. With the exception of mature red blood cells and proerythroblasts, different stages of erythrocytopoietic cells in canine bone marrow were shown to be arranged in erythrons. The polyclonal antibody against spectrin also showed clear cross-reactivity, but was only employable in other systems (immunoblotting). The monoclonal antibody against glycophorin A reacted only when used on human tissue or cells.     

The in vitro growth of erythroid colonies from dog bone marrow.

Enriched methyl cellulose media together with either human urinary erythropoietin or serum collected from phlebotomized dogs exposed to hypoxia was used in the study of the erythroid colony forming (CFU-E) capacity of dog marrow. The dog serum erythropoietin was found to be more efficient in stimulating CFU-E than comparable concentrations of human urinary erythropoietin. Numbers of CFU-E were directly related to the culture concentration of the stimulating serum and to the number of cells per plate. Sheep plasma erythropoietin was also found to be effective in stimulating CFU-E growth. The system described is chemically better defined and produced more consistent results than has been reported for the plasma clot method.     

Type of smear may influence thrombopoietic cell counts in the bone marrow of clinically healthy dogs

BACKGROUND: The expected number of thrombopoietic cells in normal canine bone marrow is poorly defined and there is no consensus on the most appropriate way to prepare cytologic smears to evaluate these cells nor on the optimum method for their quantification. OBJECTIVE: The purpose of this study was to determine total and differential counts of thrombopoietic cells in the bone marrow of clinically healthy Beagle dogs by comparing 4 different smear types and bone marrow core biopsies. METHODS: Twenty-two clinically healthy, male Beagle dogs, 10 to 12 months old, were used in the study. Following bone marrow aspiration and core biopsy from the iliac crest, Giemsa-stained smears were prepared by 4 techniques: drop-squash, particle-squash, buffy coat, and fat-layer smears. Thrombopoietic cells were counted in up to 100 low-power fields (LPF, X10 objective) in the aspiration smears and in all possible high-power fields (HPF, X40 objective) in H&E-stained biopsy sections. RESULTS: Mean total thrombopoietic cell counts were 2.76 cells/LPF (drop-squash), 1.55 cells/LPF (particle-squash), 8.05 cells/LPF (buffy coat), and 3.08 cells/LPF (fat-layer). Core biopsies yielded 5.31 cells/HPF but frequently failed to provide interpretable specimens. There was a significant difference in cell counts among the 4 smear types (P <.001). Based on evaluation of buffy coat smears, thrombopoietic cells included 1.23% megakaryoblasts, 8.77% promegakaryocytes, and 90% megakaryocytes, with a mean maturation index of 0.11. CONCLUSION: Thrombopoietic cell counts in canine bone marrow are influenced by the smear technique. Buffy coat and fat-layer smears may be useful to obtain cellular smears in hemodiluted or small aspirate samples.     

Caspase activation in equine influenza virus induced apoptotic cell death.

Equine influenza virus (EIV) is the leading cause of acute respiratory infection in horses worldwide. In recent years, the precise mechanism by which influenza infection kills host cells is being re-evaluated. In this report, we examined whether caspases, a group of intracellular proteases, are activated following EIV infection and contribute to EIV-mediated cell death. Western blotting analysis indicated that a nuclear target of caspase-3, poly(ADP-ribose) polymerase (PARP) was proteolytically cleaved in EIV-infected MDCK cells, but not in mock-infected cells. In comparison with caspase-3 specific inhibitor Ac-DEVD-CHO, a general caspase inhibitor Boc-D-FMK provided much stronger inhibition of EIV-induced cytopathic effect and apoptosis. Our results suggest that EIV may activate more than one caspase. Caspase activation and cleavage of its cellular targets may play a critical role in EIV-mediated cytotoxicity.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

Characterization of fibroblast colony-forming units in bone marrow from healthy cats

The adherent cell layer of bone marrow from healthy cats was characterized in vitro, and the mean fibroblast colony-forming unit (CFU-F) was determined. The majority (82%) of the cells in the adherent cell layer were spindle-shaped fibroblastic cells. These cells were weakly positive for acid phosphatase activity and negative for alpha-naphthyl butyrate esterase and alkaline phosphatase activities. They did not phagocytose latex beads. The remaining cells (18%) in the adherent cell layer resembled macrophages. They were strongly positive for acid phosphatase and alpha-naphthyl butyrate esterase activities, and they phagocytosed latex beads. The mean CFU-F per 10(6) mononuclear cells in bone marrow from healthy kittens and adult cats was 62 and 65, respectively. The CFU-F assay was linear over a range of 0.25 to 1.25 x 10(6) bone marrow mononuclear cells cultured. Variation in the feline CFU-F assay was similar to that reported for the human CFU-F assay. Bone marrow collections repeated at 1-month intervals (from the same bone) did not affect CFU-F concentration. A difference was not observed between CFU-F cultured from the feline humerus or femur. Bone marrow adherent cells in cats resembled those described for other species. Results of the feline CFU-F assay were consistent and repeatable and were similar to those reported for other species.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

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.     

Effect of incorporation of serum from dogs with renal impairment on canine erythroid bone marrow cultures

Serum from dogs with surgically induced renal impairment was incorporated into the medium for erythroid bone marrow cultures. A significant correlation was found between serum activities of erythropoietin and numbers of erythroid colony-forming units grown in culture. Serum creatinine concentrations had no correlation, and serum parathyroid hormone activities had a negative correlation with numbers of erythroid colony-forming units that was below the level of significance. Purified 1-84 parathyroid hormone added to bone marrow cultures was found to be stimulatory to erythroid colony-forming unit growth in higher concentrations, but decreased the number of burst-forming units. Unmeasured substances in the canine serum appeared to have a greater effect on the canine erythroid bone marrow cultures than did creatinine or parathyroid hormone values.     

Evaluation of bone marrow cytology and stainable iron content in healthy adult llamas

Complete blood counts and sternal bone marrow aspirates were obtained from healthy adult llamas ranging in age from 2.5 to 8.4 years. Megakaryocyte numbers and erythroid and myeloid series maturation and morphology appeared similar to other mammalian species. The particles contained 50 to 75% marrow cells with the remainder composed of lipocytes and stromal cells. In samples with numerous particles and adequate cellularity, M/E ratios ranged from 0.9 to 2.9. Samples with higher white blood cell counts and fewer particles had higher M/E ratios. Eosinophils comprised 14.3% of the myeloid series which is higher than other domestic mammals. Hemosiderin granules were numerous in marrow particles.     

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Characterization of neuron-like cells derived from canine bone marrow stromal cells

Regenerative therapy using bone marrow stromal cells (BMSCs) has begun to be clinically applied in humans and dogs for neurological disorders such as spinal cord injury. Under appropriate conditions in vitro, BMSCs differentiate into neuronal cells, which may improve the effects of regenerative therapy. In this study, we evaluated canine neuron-like cells (NLCs) derived from BMSCs. We speculated on their suitability for neuro-transplantation from the point of view of their morphological features, long-term viability, abundant availability, and ability to be subcultured. Canine NLCs were differentiated as follows: third-passage BMSCs were maintained in pre-induction medium containing 2-mercaptoethanol and dimethylsulfoxide for 5 h, and then cells were transferred to neuronal induction medium containing fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, dibutyryl cyclic AMP, and isobutylmethylxanthine for 7 or 14 days. Canine NLCs fulfilled the transplantation criteria and expressed markers of both immature neurons (nestin, 84.7 %) and mature neuronal cells (microtubule-associated protein-2, 95.7 %; βIII-tubulin protein, 12.9 %; glial fibrillary acidic protein, 9.2 %). These results suggest that canine BMSCs can be induced to differentiate into neuronal cells and may be suitable for neuro-transplantation. This study may provide information for improving cellular therapy for neurological diseases.     

Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity

Fucoidan is a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and has a variety of biological effects including mobilization of hematopoietic progenitor cells. Recently, we demonstrated that fucoidan stimulates the antigen-presenting functions of dendritic cells. In this study, we investigated the radioprotective effects of fucoidan on bone marrow cells (BMCs), which are the main cellular reservoir for the hematopoietic and immune system. To evaluate the effects of fucoidan, we assayed cell viability and immune responses. In a viability assay, fucoidan significantly increased the viability of BMCs. Based on the results of flow cytometric analysis, the increased viability of fucoidan-treated BMCs was attributed to the inhibition of radiation-induced apoptosis. Furthermore, fucoidan altered the production of immune-related cytokines from BMCs and increased the capability of BMCs to induce proliferation of allogeneic splenocytes. Taken together, our study demonstrated that fucoidan has radioprotective effects on BMCs with respect to cell viability and immunoreactivity. These results may provide valuable information, useful in the field of radiotherapy.     

硝基苯致小鼠骨髓细胞的突变作用

为探讨硝基苯对哺乳动物的致突变性，将12周龄雄性昆明小鼠随机分为染毒组（26、52、104mg／kg）、空白对照组（生理盐水）、溶剂对照组（花生油）和阳性对照组（环磷酰胺），连续灌胃染毒7d，检测骨髓细胞微核率、骨髓细胞染色体畸变率以及外周血网织红细胞数。结果显示，随着染毒剂量的增加，骨髓细胞微核率、染色体畸变率均有增加，外周血网织红细胞数减少，各组与对照组差异均极显著（P〈0．01），并呈剂量-效应关系。表明，硝基苯可导致小鼠骨髓细胞发生突变。     

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.     

Detection and distribution of apoptotic cell death in normal and diseased canine cranial cruciate ligaments

One of the possible initiating factors in canine cranial cruciate ligament (CCL) rupture could be an abnormal pattern of ligament cell death. This study compared apoptotic cell death in sections of ruptured CCLs and normal controls, and examined nitric oxide (NO) production in joint tissues and correlated this to apoptosis. CCLs and cartilage from the lateral femoral condyle were harvested from 10 healthy dogs and 15 dogs with CCL rupture and ligaments were further processed to detect cleaved caspase-3 and to determine supernatant NO production in explant cultures. Apoptotic activity was greater in ruptured ligaments compared to controls. NO in ligaments showed a moderate but significant positive correlation with caspase-positive cells. The results suggest that increased apoptosis has a role in CCL rupture and that apoptosis may be influenced by local NO production.