Transitional B cell subsets in human bone marrow

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as ‘transitional B cells’. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight‐colour flow cytometry. T1 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD^lo) and T2 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD⁺) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24^hiCD38^hi, the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.     

Phenotypes of Human Large Granular Lymphocytes as Defined by Monoclonal Antibodies

Four monoclonal antibodies VEP8, VEP9, VIM-D5, VIB-C5 against antigens expressed on human mature myeloid cells (polymorphonuclear leukocytes [PMNL] and/or monocytes) as well as on immature cells in the bone marrow were tested for reactivity with cell preparations highly enriched for large granular lymphocytes (LGL). These cells are known to be the main effector cells responsible for natural killer (NK) cell activity in human peripheral blood. Using indirect membrane immunofluorescence (IMF), none of these antibodies showed any reactivity at all. In addition, LGL-enriched cell preparations were tested with the anti-lymphocyte monoclonal antibodies OKT6, anti-Leu1, anti-Leu2a, anti-Leu3a, and anti-human Lyt3, and also with OKM1 antibody. Significant reactivity was found with anti-Leu2a (59 ± 8 %), anti-Lyt3 (55 ± 4 %) and OKM1 (81 ± 11 %) antibodies, whereas T6, Leu1, and Leu3a antigens were less pronounced or missing on LGL. As a further approach, another monoclonal antibody, VEP13, which reacts with LGL, granulocytes but not monocytes and is therefore different in its specificity from OKM1 and OKT10, was used for identification of LGL. The coexpression of antigens as defined by the above-mentioned antibodies and OKT10 on VEP13⁺ cells was studied. Again, phenotypes similar to those observed on LGL enriched by Percoll^® gradient centrifugation were found: of VEP13⁺ cells 84 ± 6 % reacted with OKM1, 82 ± 5 % with OKT10, 52 ± 17 % with anti-human Lyt3, and 48 ± 14 % with anti-Leu2a, whereas VEP8, VEP9, VIM-D5, VIB-C5, T6, Leu1, Leu3a antigens were not expressed on VEP13⁺ cells. Taken together as an overall evaluation of phenotypic characteristics, our data indicate that LGL cannot be integrated into one of the known lymphocytic or myelomonocytic lineages. LGL show an intermediate phenotype depending possibly on varying differentiation or activation stages of haemopoietic cells. However, the possibility also exists that LGL belong to a separate, yet undefined cell lineage.     

Effect of AC II,an Herbal Formulation in Cyclophosphamide-Induced Immunosuppression in BALB/c Mice—Implication in HIV Treatment

Effect of AC II, herbal drug formulation in reducing immunosuppression caused by administration of cyclophosphamide was studied. Mice were injected cyclophosphamide (CTX) 50 mg/kg b.wt. for 14 days with or without the drug and total WBC, bone marrow cellularity and α-esterase positive cells were determined. On day 15, total WBC count in cyclophosphamide treated mice was 1500 ± 420 cells/mm³, while in AC II-treated mice it was 7658 ± 376 cells/mm³. On day 16, administration of cyclophosphamide reduced bone marrow cellularity to 3.42 ± 0.38 × 10⁶cells/femur from the normal value of 13.83 ± 0.96 × 10⁶ cells/femur. In AC II treated group bone marrow cellularity was increased to 8.05 ± 0.7 × 10⁶ cells/femur. The number of α-esterase positive cells was found to be reduced to 177 ± 25 cells per 4000 cells in CTX treated groups. But in AC II-treated group the number of α-esterase positive cells were raised to 843 ± 86 cells per 4000 cells, which was closer to that of normal (710 ± 49 cells per 4000 cells). Results indicate the usefulness of AC II to combat immunosuppression induced by chemical and biological agents.     

Bone Marrow Harvest in Pediatric Sibling Donors: Role of Granulocyte Colony-Stimulating Factor Priming and CD34+ Cell Dose

To ensure optimal clinical outcomes for patients while retaining adequate protection for donors, the National Marrow Donor Program developed guidelines specifying that up to 20?mL/kg of bone marrow can be harvested from donors. These guidelines, originally developed for unrelated adult donors, are followed in children as well. We studied the impact of granulocyte colony-stimulating factor (G-CSF) priming on the cellular composition of harvested bone marrow, sought to develop an algorithm to optimize bone marrow harvest volume from pediatric matched sibling donors, and studied the impact of CD34⁺ cell dose on clinical outcomes. We analyzed data from 92 bone marrow harvests and clinical outcomes for 69 sibling recipient-donor duos, The mean age of recipients was 9.85?±?5.90 years, and that of donors was 11.85?±?6.36 years. G-CSF priming was not associated with higher yield of CD34⁺ cells/µL. The median CD34⁺ cell count obtained from donors was 700 cells/µL (range, 400-1700 cells/µL) in donors age <6 years, 360 cells/µL (range, 100-1100 cells/µL) in donors age 6 to 12 years, and 300 cells/µL (range, 80-800 cells/µL) in donors age >12 years (P?<?.001). The number of CD34⁺ cells infused had no impact on traditional clinical outcomes; however, it was significantly related to graft-versus-host disease/relapse/rejection-free survival. Our investigation revealed that ultimately, a CD34⁺ cell count of approximately 3 to 5 × 10⁶/kg was a threshold beyond which increasing CD34⁺ cell dose did not impact outcome. In this study, we addressed the broad question of whether harvesting up to 20?mL/kg of bone marrow from a child donor is truly necessary for optimal outcomes in every pediatric case.     

Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens

We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1?×?10⁵ cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1?×?10⁵ cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.     

Development of cytotoxic T lymphocyte precursors in the absence of the thymus

Reconstitution of adult thymectomized and lethally irradiated C3H or (C 3 H × DBA/2) F₁ mice with syngeneic adult bone marrow cells or 18 to 20-day-old fetal liver cells resulted almost regularly in the generation of large numbers of cytotoxic T lymphocyte precursors (CTLP) against trinitrophenylated C3H cells and three types of allogeneic stimulator and target cells. The responses against 2,4,6- trinitrophenylated cells were restricted and in so far typical for cytotoxic T lymphocytes. The failure of bone marrow cells from athymic C 3 H mice to reconstitute significant cytotoxic activity indicated that the CTLP had developed from “cytotoxic T lymphocyte preprogenitors” (CTLPP) which were derived from the donor and which had already been under thymic influence. The stem cell preparations in themselves contained frequencies of mature CTLP far too low to account for the reactivity and CTLP frequencies in the reconstituted mice. This fact implied that the CTLPP proliferated extensively and/or matured in the recipient in the absence of the thymus. Individual mice failed to respond to one or more of these stimulator cells. The failure of individual mice to respond was randomly expressed for the four specificities even when the mice were reconstituted with the same stem cell preparation suggesting that CTLPP with defined specificity and low frequency (about 1/10⁸ in fetal liver) were contained in the stem cell preparation and transferred randomly into the irradiated recipients. This would imply that one CTLPP gives rise to 5 × 10³–20 × 10³ CTLP within 5 weeks. The data suggest that the peripheral pools, at least of certain T cell subclasses, may be maintained largely by post-thymic proliferation of CTLPP.     

Phenotypic Analysis and Characterization of CD34+ Cells from Normal Human Bone Marrow,Cord Blood,Peripheral Blood,and Mobilized Peripheral Blood from Patients Undergoing Autologous Stem Cell Transplantation

Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34⁺ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34⁺ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 × 10⁴ to 0.65 × 10⁴/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamidemobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34⁺ CD19⁺ (Leu 12) CD10⁺ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 ± 16.9%, mean ± 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34⁺ cells defined a CD45RA^-CD71⁺ population containing 89 ± 6.3% (n = 4) BFU-E and a CD45RA⁺CD71⁺ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13⁺ CD45RA⁺ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34⁺ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34⁺ cells.     

Refinement and verification of test conditions for colony-forming unit assay in dog bone marrow cells with preservable colony specimens

The colony-forming unit assay reported by Kubota is a potentially useful method for toxicity evaluation since the method enables us to preserve the specimens. However, no study has yet reported on the utility of Kubota’s method in conducting the CFU assay in toxicity evaluation. Therefore, we examined the optimum test conditions for CFU assays by Kubota’s method using bone marrow cells from dogs, a widely used species in toxicity studies, to establish a useful hematopoietic toxicity evaluation system in vitro. We established test conditions for colony-forming unit granulocytes–macrophage assay and colony-forming unit erythroid assay. Optimum conditions for colony-forming unit granulocytes–macrophage assay were 20 % of colony-stimulating dog serum, 1?×?10⁵ cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for colony-forming unit erythroid assay were 2 IU/ml recombinant human erythropoietin, 0.265–2.5?×?10⁴ cells/dish, and a culture period of 2–4 days. We also examined the response to bone marrow toxicants doxorubicin and azidothymidine in these systems. Both compounds reduced the numbers of colonies in a concentration-dependent manner in both assays. Taken together with these results, we confirmed that our method efficiently functioned as an evaluation system for bone marrow toxicity in vitro.     

Human Fetal/Neonatal Suppressor Activity: Relation Between OKT Phenotypes and Sensitivity to Prostaglandin E2 in Maternal and Neonatal Lymphocytes

ABSTRACT: T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4⁺ subpopulation exerted only a marginal suppressor activity (12 ± 7 as compared to 73 ± 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8⁺ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E₂ (PGE₂) at doses varying between 1.4 × 10^?5 and 1.4 × 10^?9 M. Both maternal OKT4^? and OKT8^? T-cell subsets were highly sensitive to suppression by PGE₂. In contrast, cord OKT8^? T cells were essentially nonsensitive at all doses of PGE₂ used, whereas cord OKT4^? T cells were significantly suppressed at four out of five concentrations tested (1.4 × 10^?6 through 1.4 × 10^?9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE₂.     

人骨髓间质干细胞体外扩增和向内皮细胞定向诱导分化的研究

目的：体外分离、扩增成人骨髓间质干细胞（MSCs）和向内皮细胞（ECs）定向诱导分化，开辟心血管组织工程种子细胞的新来源。 方法： 采用Percoll(1 073 g/L)从正常成人骨髓中分离出MSCs，纯化和扩增后流式细胞仪鉴定其纯度；用血管内皮生长因子(VEGF)诱导MSCs向ECs分化，Ⅷ因子(vWF)免疫组化和透射电镜(TEM)鉴定细胞性质。 结果： 5.0×105个MSCs在体外扩增15代后，获得8.0×1012个MSCs，扩增了约1.6×107倍；MSCs在加入VEGF诱导培养大约14-21 d，80%-90%的诱导细胞对Ⅷ因子相关抗原呈阳性反应；TEM可观察到胞浆内有Weible-palade小体，证实为ECs。 结论： 成人骨髓MSCs在体外具有定向诱导分化为ECs的潜能，这为心脏组织工程瓣体外构建, 尤其是在小儿先天性心脏病组织工程研究中种子细胞的来源提供了可能性。     

骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫抑制

背景：骨髓间充质干细胞对再生障碍性贫血患者T细胞增殖的影响国内报道较少,而骨髓间充质干细胞是否通过抑制T细胞增殖实现对再生障碍性贫血患者的免疫调节目前尚无定论。 目的：观察人骨髓间充质干细胞对再生障碍性贫血患者T细胞的免疫调节作用。 方法：体外分离培养、扩增人骨髓间充质干细胞并通过形态学特征以及流式细胞术进行表面标志鉴定,将骨髓间充质干细胞分别与正常人和再生障碍性贫血患者外周血提取的T淋巴细胞共培养7 d。应用ELISA法检测各培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素、白细胞介素4及白细胞介素10水平。 结果与结论：再生障碍性贫血组患者培养上清液中T淋巴细胞分泌的白细胞介素2、γ-干扰素水平明显高于正常人(P < 0.05),白细胞介素4、白细胞介素10低于正常人(P < 0.05)。骨髓间充质干细胞可下调白细胞介素2、γ-干扰素表达,同时上调白细胞介素4、白细胞介素10表达,从而调节再生障碍性贫血患者的免疫紊乱。     

Haematology of Persian Fallow Deer ( Dama mesopotamica )

The haematology of Persian fallow deer was studied and the means of various parameters were determined for sex and age groups. For total samples, the mean ± standard deviation of haematological parameters were: red blood cells (RBC), 7.42 ± 1.27 × 10¹²/l; haematocrit (PCV), 38.83 ± 7.38%; haemoglobin (Hb), 148.0 ± 17.3 g/l; mean cell volume (MCV), 50.84 ± 7.26 fl; mean corpuscular haemoglobin (MCH), 20.17 ± 1.98 pg; mean corpuscular haemoglobin concentration (MCHC), 38.71 ± 3.88%; white blood cells (WBC) 3.200 ± 1.697 × 10⁹/l; neutrophils, 1.410 ± 0.446 × 10⁹/l, lymphocytes, 1.382 ± 1.116 × 10⁹/l, monocytes, 0.048 ± 0.100 × 10⁹/l, eosinophils, 0.341 ± 0.339 × 10⁹/l, basophils, 0.009 ± 0.005 × 10⁹/l, neutrophil:lymphocyte ratio (N:L), 2.11 ± 2; platelets, 354.8 ± 116.62 × 10⁹/l and fibrinogen, 2.855 ± 1.126 g/l. Three types of haemoglobin were separated by electrophoresis: HbC with higher migration, HbB and HbA with lowest movement. Significant differences were seen for RBC, MCV, MCH, HbC, and HbB concentrations between sexes.     

Transient Extremity Ischemia Augments CD34+ Progenitor Cell Availability

Peripheral blood is an easily accessed source for stem cell production; however, the number of cells produced is relatively low. We hypothesized that ischemic preconditioning may serve as a safe method to increase the number of CD34+ cells that can be harvested and cultured in a short period. This study was conducted to test this hypothesis by examining the safety and efficacy of brief, transient ischemia of the lower limbs to augment the number of cells that can be produced from blood of healthy volunteers. Following induction of ischemia, blood samples were withdrawn at baseline, 30 min, 12 h and 24 h. The number of progenitor cells was determined by flow cytometry after the harvested cells were cultured for 5 days. We also analyzed the blood samples to determine IL-8 and VEGF concentrations. No serious adverse events were observed. The total number of cells increased from 0.46 ± 0.1 × 10⁶ cells/ml in the pretreatment blood samples to 0.7 ± 0.1 × 10⁶ cells/ml in blood taken 12 h after the conclusion of transient ischemia, p = 0.0029. The number of CD34+ cells increased from 4.23 ± 0.8 × 10⁴ cells/ml in the pretreatment samples to 7.17 ± 1.34 × 10⁴ cells/ml in blood taken 12 h after ischemia, p = 0.0001. The harvested stem cells maintained their ability to construct tubular structures. The augmentation in the number of CD34+ cells was positively correlated with the increase of IL-8, but not with VEGF concentrations. Ischemic preconditioning is a safe and effective technique to increase the availability of stem cells for therapeutic purposes.     

DR.lyp/lyp bone marrow maintains lymphopenia and promotes diabetes in lyp/lyp but not in +/+ recipient DR.lyp BB rats

Lymphopenia is due to a frameshift mutation in Gimap5 on rat chromosome 4 and is linked to type 1 diabetes in the diabetes prone (DP) BB rat. The hypothesis that bone marrow derived cells confer the lymphopenia phenotype was tested by reciprocal bone marrow transplantation in 40-day-old lethally irradiated diabetes resistant (DR) congenic DR.^lyp/lyp (lymphopenia and diabetes) and DR.^+/+ (no lymphopenia and no diabetes) rats.In two independent series of transplants, all DR.^lyp/lyp rats (n = 5 and 4) receiving DR.^lyp/lyp bone marrow retained lymphopenia and developed insulitis (5/5 and 4/4) as well as diabetes in some (2/5 and 3/4). Both DR.^+/+ and DR.^lyp/lyp rats receiving DR.^+/+ bone marrow cells as well as DR.^+/+ rats receiving DR.^lyp/lyp bone marrow cells showed no lymphopenia or diabetes. In accordance with earlier studies in non-congenic BB rats, the DR.^+/+ rats receiving DR.^lyp/lyp bone marrow cells recapitulated an intermediary phenotype rather than the +/+ or lyp/lyp phenotypes.Our data demonstrate that BBDP rat lymphopenia and diabetes are transferred by bone marrow transplantation to syngeneic DR.^lyp/lyp but not DR.^+/+ recipients. The intermediary recapitulation of DR.^lyp/lyp T cells in recipient DR.^±/± rats suggests that radiation resistant ±/± T cells, the Gimap5 mutation in bone marrow cells, or both may not support the development of lymphopenia.     

CD4+ T cells migrate from airway to bone marrow after antigen inhalation in rats

BACKGROUND: IL-5-producing T lymphocytes increase in rat bone marrow after inhalational challenge with allergen. OBJECTIVE: To test the hypothesis that T cells migrate from the airways to the marrow, we examined the trafficking of T cells in Brown Norway rats after sensitization and challenge with ovalbumin. METHODS: Purified CD4+ T cells, harvested from cervical lymph nodes of naive and ovalbumin-sensitized donors, were labeled with carboxy fluorescein diacetate succinimidyl ester; 20 x 10(6) cells were placed in the trachea of naive or sensitized recipients under anesthesia, and 18 hours later, animals were challenged with inhaled ovalbumin. Cells were harvested 24 hours later from the bone marrow, bronchoalveolar lavage fluid, lungs, the lung blood pool of cells, lung draining lymph nodes, peripheral blood, and spleen. RESULTS: The number of carboxy fluorescein diacetate succinimidyl ester-positive cells, measured by fluorescence-activated cell sorter, in the bone marrow of ovalbumin sensitized, primed T-cell recipients was higher than either the sham-sensitized, primed T-cell recipients or sham-sensitized, naive T-cell recipients (P < .05). The number of eosinophils in both bone marrow and bronchoalveolar lavage fluid was increased in ovalbumin-sensitized, primed T-cell recipients. The expression of the T-cell chemoattractants eotaxin and IL-16, evaluated by immunohistochemistry, was higher in the bone marrow of ovalbumin-sensitized, primed T-cell recipients. CONCLUSIONS: CD4+ T cells travel from airway to bone marrow after antigen inhalation. The homing of the CD4+ T cells might be facilitated by eotaxin and IL-16 expression in the bone marrow and might contribute to the stimulation of eosinophilopoiesis after airway allergen exposure.     

Correlations of cortical bone microstructural and mechanical properties with water proton fractions obtained from ultrashort echo time (UTE) MRI tricomponent T2* model

Mechanical and microstructural evaluations of cortical bone using ultrashort echo time magnetic resonance imaging (UTE‐MRI) have been performed increasingly in recent years. UTE‐MRI acquires considerable signal from cortical bone and enables quantitative bone evaluations. Fitting bone apparent transverse magnetization (T2*) decay using a bicomponent model has been regularly performed to estimate bound water (BW) and pore water (PW) in the quantification of bone matrix and porosity, respectively. Human cortical bone possesses a considerable amount of fat, which appears as MRI T2* signal oscillation and can subsequently lead to BW overestimation when using a bicomponent model. Tricomponent T2* fitting model has been developed to improve BW and PW estimations by accounting for fat contribution in the MRI signal. This study aimed to investigate the correlations of microstructural and mechanical properties of human cortical bone with water pool fractions obtained from a tricomponent T2* model. 135 cortical bone strips (~4 × 2 × 40 mm³) from tibial and femoral midshafts of 37 donors (61 ± 24 years old) were scanned using ten sets of dual‐echo 3D‐UTE‐Cones sequences (TE = 0.032–24.0 ms) on a 3 T MRI scanner for T2* fitting analyses. Average bone porosity and pore size were measured using microcomputed tomography (μCT) at 9 μm voxel size. Bone mechanical properties were measured using 4‐point bending tests. Using a tricomponent model, bound water fraction (Frac_BW) showed significant strong (R = 0.70, P < 0.01) and moderate (R = 0.58–0.62, P < 0.01) correlations with porosity and mechanical properties, respectively. Correlations of bone microstructural and mechanical properties with water pool fractions were higher for tricomponent model results compared with the bicomponent model. The tricomponent T2* fitting model is suggested as a useful technique for cortical bone evaluation where the MRI contribution of bone fat is accounted for.     

Studies of human bone marrow treated with soybean lectin and sheep erythrocytes: stepwise analysis of cell morphology, phenotype and function.

Morphological, phenotypic and functional analyses were made of cells obtained at each step after successive treatments of 23 separate human bone marrow suspensions with soybean lectin and sheep erythrocytes (SRBC). The average total number of nucleated cells harvested was 1.9 X 10(10) and the final cell suspensions contained a mean of 1.9 X 10(9) nucleated cells or 9.2 +/- 4.8% of the initial counts. Monoclonal antibody analyses revealed that both T and B lymphocytes were present in every cell fraction in percentages similar to those found initially until after the first SRBC rosette-depletion. Moreover, both soy lectin agglutinated and non-agglutinated cells exhibited vigorous proliferative responses to phytohaemagglutinin and allogeneic cells. Following the SRBC depletions, no cells having T lymphocyte phenotypes or functions could be detected, whereas 5% of the cells reacted with a monoclonal antibody to B lymphocytes. The final fraction was composed predominantly of immature myeloid cells and blasts and was depleted of erythroid elements, lymphocytes and essentially all mature cells. It contained cells reactive with monoclonal antibodies recognizing undifferentiated T cell precursors (3A1), the transferrin receptor (5E9), and a human progenitor cell antigen (My-10). The final fraction was also enriched 10-100-fold for CFU-C and 5-10-fold for CFU-GEMN colonies. These studies fail to demonstrate selective removal of T lymphocytes from human bone marrow cells by soybean lectin agglutination.     

Expression of low-density lipoprotein receptors in peripheral blood and tonsil B lymphocytes

B lymphocytes, purified from peripheral leucocytes from young normolipaemic humans, expressed and internalized low-density lipoprotein receptors (LDLR). The expression was assessed by a monoclonal anti-LDLR. The internalization of LDL was assessed by LDL labelled with ¹²⁵I (¹²⁵I-LDL) and 1,1′-dioctadecyl-3,3,3′,3′ tetramethyl-indocarboxycyanine perchlorate (LDL-DiI). The expression of LDLR, assessed by anti-LDLR, was: 38 ± 8% (n = 5) for fresh purified cells, 60 ± 10% (n = 12) for non-stimulated cells, 79 ± 5% (n = 10) for IL-2 (100 U/ml)-stimulated cells and 95 ± 5% (n = 8) for pokeweed mitogen (PWM) (1:200 dilution)-stimulated cells. The optimal concentrations of agonist were 100 U/ml of IL-2, and 1:200 dilution of PWM. IL-2 and PWM increased the internalization of LDL-DiI by 1.5-fold. The internalization of LDL-DiI was maximal at 60 μg of protein/ml (48 ± 8%). Scatchard analysis revealed a Kd of 3.2 ± 0.22 × 10^?8 M and 2180 ± 190 binding sites in non-stimulated cells, a Kd of 7.73 ± 0.36 × 10^?9 M and 12 500 ± 430 binding sites for IL-2 (100 U/ml)-stimulated cells, and a Kd of 7.2 ± 0.43 × 10^?9 M and 13 250 ± 450 binding sites for PWM (1:200 dilution)-stimulated cells. Lineweaver–Burk analysis of LDL binding (LDL-DiI) revealed that the apparent Kd for non-stimulated cells was 1.3 ± 0.11 × 10^?8 M , and 9.2 ± 0.2 × 10^?9 M and 7.5 ± 0.25 × 10^?9 M for IL-2- and PWM-stimulated cells, respectively. B lymphocytes from tonsils also showed a high expression of LDLR assessed with anti-LDLR (70 ± 6%). The high expression of LDLR and the avid internalization of LDL suggest that LDL may be important for B cell physiological responses.     

腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞

背景：骨髓间充质干细胞作为骨、软骨创伤缺损及退变修复的种子细胞越来越受到关注。 目的：分析人骨形态发生蛋白2基因转染对白色封闭群大鼠(SD大鼠)骨髓间充质干细胞的影响。 方法：分离纯化SD大鼠骨髓间充质干细胞并体外扩增,通过腺病毒载体介导人骨形态发生蛋白2基因转染骨髓间充质干细胞,分别通过荧光显微镜观察荧光表达情况及蛋白质水平来测定转染后人骨形态发生蛋白2的表达,碱性磷酸酶定量测定鉴定成骨活性及MTT法评估人骨形态发生蛋白2转染对骨髓间充质干细胞的影响。 结果与结论：从SD大鼠骨髓提取物中分离培养的细胞形态为梭形,呈铺路石状、漩涡状生长,经流式细胞仪检测及多项分化能力鉴定符合骨髓间充质干细胞的特征;经转染人骨形态发生蛋白2基因后,骨髓间充质干细胞表达人骨形态发生蛋白2、碱性磷酸酶;MTT法检测转染人骨形态发生蛋白2基因后,骨髓间充质干细胞增殖能力明显增强(P < 0.05)。说明人骨形态发生蛋白2基因转染骨髓间充质干细胞后可以持续、高效表达人骨形态发生蛋白2和碱性磷酸酶,在体外明显促进骨髓间充质干细胞的增殖。     

Thymopoietic and bone marrow response to murine Pneumocystis pneumonia

CD4⁺ T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4⁺ T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9⁺ multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4⁺ T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4⁺ cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4⁺ T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4⁺ T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9⁺ MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9⁺ MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4⁺ T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection.