Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.

To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression in vivo. Murine bone marrow cells were transduced by each of the recombinant viruses and subsequently used to reconstitute the hematopoietic system of lethally irradiated recipients. Five to seven months after transplantation, analysis of the peripheral blood of animals transplanted with cells transduced by vectors which employ viral long terminal repeats (LTRs) for gene expression indicated that in 83% (77/93) of these animals, the level of human enzyme was equal to or greater than the level of endogenous murine enzyme. Even in bone marrow transplant recipients reconstituted for over 1 year, significant levels of gene expression were observed for each of the vectors in their bone marrow, spleen, macrophages, and B and T lymphocytes. However, derivatives of the parental MFG-ADA vector which possess either a single base mutation (termed B2 mutation) or myeloproliferative sarcoma virus LTRs rather than the Moloney murine leukemia virus LTRs led to significantly improved gene expression in all lineages. These studies indicate that retroviral vectors which employ viral LTRs for the expression of inserted sequences make it possible to obtain high levels of a desired gene product in most hematopoietic cell lineages for close to the lifetime of bone marrow transplant recipients.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells

Background context Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. Purpose To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Study design/setting Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. Methods We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. Results As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. Conclusions These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states. © 2014 Elsevier Inc. All rights reserved.

Study of lymphocyte subpopulations in bone marrow in a model of protein-energy malnutrition

Objective: Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. Hematopoietic tissue requires a high nutrient supply, and a reduction in leukocytes, especially lymphocytes, suggests that some nutritional deficiencies might be altering bone marrow function and decreasing its ability to produce lymphocytes. In this study, we evaluated the effect that PEM has on lymphocyte subtypes and the cell cycle of CD5(+) cells. Methods: Swiss mice were subjected to PEM using a low-protein diet containing 4% protein. When the experimental group had lost about 20% of their original body weight, we collected blood and bone marrow cells and evaluated the hemogram, the myelogram, bone marrow lymphoid markers using flow cytometry, and the cell cycle in CD5(+) bone marrow. Results: Malnourished animals presented anemia, reticulocytopenia, and leukopenia with lymphopenia. The bone marrow was hypocellular, and flow cytometric analyses of bone marrow cells showed cells that were CD45(+) (91.2%), CD2(+) (84.9%), CD5(+) (37.3%), CD3(+) (23.5%), CD19(+) (43.3%), CD22(+) (34.7%), CD19(+)/CD2(+) (51.2%), CD19(+)/CD3(+)(24.0%), CD19(+)/CD5(+) (13.2%), CD22(+)/CD2(+) (40.1%), CD22(+)/CD3(+) (30.3%), and CD22(+)/CD5(+) (1.1%) in malnourished animals and CD45(+) (97.5%), CD2(+) (42.9%), CD5(+) (91.5%), CD3(+) (92.0%), CD19(+) (52.0%), CD22(+) (75.6%), CD19(+)/CD2(+) (62.0%), CD19(+)/CD3(+) (55.4%), CD19(+)/CO5(+) (6.7%), CD22(+)/CD2(+) (70.3%), CD22(+)/CD3(+) (55.9%), and CD22(+)/ CD5(+) (8.4%) in control animals. Malnourished animals also presented more CD5(+) cells in the G0 phase of cell cycle development. Conclusion: Malnourished animals presented bone marrow hypoplasia, maturation interruption, prominent lymphopenia with depletion in the lymphoid lineage, and changes in cellular development. We suggest that these changes are some of the primary causes of lymphopenia in cases of PEM and partly explain the increase in susceptibility to infections found in malnourished individuals. Published by Elsevier Inc.

EVALUATION OF CENTRIFUGED OSTEOGENIC BONE MARROW IN FRACTURE CONSOLIDATION IN RABBITS

Objective The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone marrow aspirate to stimulate healing in rabbit fibular osteotomies Methods Ten white New Zealand rabbits were used A transverse medial diaphyseal fibular osteotomy was performed on the right fibula where an absorbable collagen sponge embedded in osteogenic centrifuged bone marrow aspirate obtained from the ipsilateral iliac bone was inserted The left fibula was used as the control group where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate The rabbits were sacrificed four weeks after surgery to evaluate bone callus formation Analyses of results were performed with DEXA bone densitometry to evaluate callus mineral mass multislice computed tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation Results The employment of centrifuged osteogenic bone marrow aspirate resulted in a 40 3% increase of callus bone mineral mass and increased relative quantity of bone tissue formation by 9 4% without a significant increase in the relative quantities of cartilage fibrous tissue or in callus volume Conclusions This study shows that the centrifuged osteogenic bone marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Effect of the bone marrow cell transplantation on elevated plus-maze performance in hippocampal-injured mice

Several reports have shown that the hippocampus plays an important role in different aspects of the emotional control. There is evidence that lesions in this structure cause behavioral disinhibition, with reduction of reactions expressing fear and anxiety. Thus, to portray the aptitude of cell therapy to abrogate injuries of hippocampal tissue, we examined the behavioral effects of bone marrow mononuclear cells (BMMCs) transplantation on C57BL/6 mice that had the hippocampus damaged by electrolytic lesion. For this purpose, mice received, seven days after bilateral electrolytic lesion in the dorsal hippocampus, culture medium or BMMCs expressing the enhanced green fluorescent protein (EGFP) transgene. One week after transplantation, animals were tested in the elevated plus-maze (EPM). On the whole, three assessment sessions in the EPM were carried out, with seven days separating each trial. Thirty-five days after the induction of injury, mice were sacrificed and their brains removed for immunohistochemistry. The behavioral evaluation showed that the hippocampal lesion caused disinhibition, an effect which was slightly lessened, from the second EPM test, in transplanted subjects. On the other hand, immunohistochemical data revealed an insignificant presence of EGFP+ cells inside the brains of injured mice. In view of such scenario, we hypothesized that the subtle rehabilitation of the altered behavior might be a result from a paracrine effect from the transplanted cells. This might have been caused by the release of bioactive factors capable of boosting endogenous recuperative mechanisms for a partial regaining of the hippocampal functions. © 2013 Elsevier B.V.

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Plasma levels of endothelin-1 after a pulmonary embolism of bone marrow fat

BACKGROUND: During orthopedic surgery, embolization of bone marrow fat can lead to potentially fatal, intra-operative cardiovascular deterioration. Vasoactive mediators may also be released from the bone marrow and contribute to these changes. Increased plasma levels of endothelin-1 (ET-1) have been observed after pulmonary air and thrombo-embolism. The role of ET-1 in the development of acute cardiovascular deterioration as a result of bone marrow fat embolization during vertebroplasty was therefore investigated. METHODS: Bone cement was injected into three lumbar vertebrae of six sheep in order to force bone marrow fat into the circulation. Invasive blood pressures and heart rate were recorded continuously until 60 min after the last injection. Cardiac output, arterial and mixed venous blood gas parameters and plasma ET-1 concentrations were measured at selected time points. Post-mortem, lung biopsies were taken for analysis of intravascular fat. RESULTS: Cement injections resulted in a sudden (within 1 min) and severe increase in pulmonary arterial pressure (>100%). Plasma concentrations of ET-1 started to increase after the second injection, but no significant changes were observed. Intravascular fat and bone marrow cells were present in all lung lobes. CONCLUSION: Cement injections into vertebral bodies elicited fat embolism resulting in subsequent cardiovascular changes that were characterized by an increase in pulmonary arterial pressure. Cardiovascular complications as a result of bone marrow fat embolism should thus be considered in patients undergoing vertebroplasty. No significant changes in ET-1 plasma values were observed. Thus, ET-1 did not contribute to the acute cardiovascular changes after fat embolism.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

AUGMENTATION OF RECONSTITUTION OF GUT ASSOCIATED LYMPHOID TISSUE IN THE SYNGENEIC MURINE BONE MARROW TRANSPLANTATION MODEL

Gut was studied as a prototypical mucosal membrane in the murine BDF-1 syngeneic bone marrow transplant model. Measures of jejunal intraepithelial lymphocytes (IELs) and crypt cells were obtained by standard techniques and a method of quantifying gut lamina propria plasma cells (PCs) was developed. The degree of ablation of gut PCs and IELs after 900 rads total body irradiation with ('60)Co, and their repopulation effected by transplantation with 2.0 x 10('5) or 1.0 x 10('6) bone marrow cells demonstrated a prolonged period of profound depression in population levels of these cells which was not reflected by the extent of damage sustained to the epithelium. Differences in the depopulation and recovery of gut PCs and IELs revealed a tendency towards initial differentiation of effector cells. A positive dose response to high bone marrow cell innocula was obtained. Subsequent studies determined that gut IEL and PC repopulation was potentiated by the addition of IELs or buffy coat cells (BCs) to the bone marrow transplant. A method of isolating 1.4 - 4.0 x 10('7) viable IELs per gram of murine small bowel was devised employing intralumenal hyaluronidase digestion of the epithelial layer and centrifugation of the resulting suspension through discontinuous Percoll gradients. Irradiated mice received 2.0 x 10('5) bone marrow cells along with an equal number of IELs or BCs. The extent and duration of depression of numbers of IELs and PCs was markedly reduced by the addition of the IEL isolate to the transplantation innocula, and to a lesser degree by the addition of BCs. The augmentation of repopuation far exceeded that expected by simple lodging of cells suggesting that the additionally transplanted cells contained a subpopulation of mucosal membrane lymphoid stem cells or helper cells. Correlation analysis of PC versus IEL levels suggests a possible feedback mechanism governing the relative size of their populations. Normal ratios of IgA, IgM, and IgG bearing PCs was maintained post transplantation with all of the regimens. ^

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity

Background: The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated. Methodology: Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water. Results: ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells. Conclusions: The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.