Novel characterization of monocyte-derived cell populations in the meninges and choroid plexus and their rates of replenishment in bone marrow chimeric mice

The mouse dura mater, pia mater, and choroid plexus contain resident macrophages and dendritic cells (DCs). These cells participate in immune surveillance, phagocytosis of cellular debris, uptake of antigens from the surrounding cerebrospinal fluid and immune regulation in many pathologic processes. We used Cx3cr1 knock-in, CD11c-eYFP transgenic and bone marrow chimeric mice to characterize the phenotype, density and replenishment rate of monocyte-derived cells in the meninges and choroid plexus and to assess the role of the chemokine receptor CX3CR1 on their number and tissue distribution. Iba-1 major histocompatibility complex (MHC) Class II CD169 CD68 macrophages and CD11c putative DCs were identified in meningeal and choroid plexus whole mounts. Comparison of homozygous and heterozygous Cx3cr1 mice did not reveal CX3CR1-dependancy on density, distribution or phenotype of monocyte-derived cells. In turnover studies, wild type lethally irradiated mice were reconstituted with Cx3cr1/-positive bone marrow and were analyzed at 3 days, 1, 2, 4 and 8 weeks after transplantation. There was a rapid replenishment of CX3CR1-positive cells in the dura mater (at 4 weeks) and the choroid plexus was fully reconstituted by 8 weeks. These data provide the foundation for future studies on the role of resident macrophages and DCs in conditions such as meningitis, autoimmune inflammatory disease and in therapies involving irradiation and hematopoietic or stem cell transplantation.

Bone marrow chimeras and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Turnover of bone marrow-derived cells in the irradiated mouse cornea

In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation.

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears

BACKGROUND: Acute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides. FINDINGS: SOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature. CONCLUSION: Our dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer.

Autologous bone marrow transplantation in a dog with lymphoma: a clinical study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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.

Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of Centrifuged Bone Marrow on Bone Regeneration Around Implants in Rabbit Tibia

Objective: To evaluate the bone regeneration of cervical defects produced around titanium implants filled with blood clot and filled with centrifuged bone marrow (CBM) by means of histomorphometric analysis.Materials and Methods: Twelve rabbits received 2 titanium implants in each right tibia, with the upper cortical prepared with a 5-mm drill and the lower cortex with a 3-mm-diameter drill. Euthanasia was performed to allow analysis at 7, 21, and 60 days after operation. The samples were embedded in light curing resin, cut and stained with alizarin red and Stevenel blue for a histomorphometric analysis of the bone-to-implant contact (BIC) and the bone area around implant (BA). The values obtained were statistically analyzed using the nonparametric Kruskal-Wallis test (P = 0.05).Results: At 60 days postoperation, the groups had their cervical defects completely filled by neo-formed bone tissue. There was no statistically significant difference between the groups regarding BIC and BA during the analyzed periods.Conclusion: There was no difference in the bone repair of periimplant cervical defects with or without the use of CBM. (Implant Dent 2012;21:481-485)

MKK3/6-p38 MAPK signaling is required for IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells

Coupled bone turnover is directed by the expression of receptor-activated NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1 beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1 beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1 beta treatment and subsequently reduced similar to 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1 beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1 beta or TNF-alpha treatment. IL-1 beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1 beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

Microscopic Evidences That Bone Marrow Mononuclear Cell Treatment Improves Sciatic Nerve Regeneration After Neurorrhaphy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)