A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis.

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors1-6. LEPR+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin13,14, distinguishes peri-arteriolar LEPR+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR+osteolectin+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin+ cells depleted osteolectin+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.

Dapsone modulates lipopolysaccharide-activated bone marrow cells by inducing cell death and down-regulating tumor necrosis factor-α production.

Dapsone, an antibiotic, has been used to cure leprosy. It has been reported that dapsone has anti-inflammatory activity in hosts; however, the anti-inflammatory mechanism of dapsone has not been fully elucidated. The present study investigated the anti-inflammatory effects of dapsone on bone marrow cells (BMs), especially upon exposure to lipopolysaccharide (LPS). We treated BMs with LPS and dapsone, and the treated cells underwent cellular activity assay, flow cytometry analysis, cytokine production assessment, and reactive oxygen species assay. LPS distinctly activated BMs with several characteristics including high cellular activity, granulocyte changes, and tumor necrosis factor alpha (TNF-α) production increases. Interestingly, dapsone modulated the inflammatory cells, including granulocytes in LPS-treated BMs, by inducing cell death. While the percentage of Gr-1 positive cells was 57% in control cells, LPS increased that to 75%, and LPS plus dapsone decreased it to 64%. Furthermore, dapsone decreased the mitochondrial membrane potential of LPS-treated BMs. At a low concentration (25 µg/mL), dapsone significantly decreased the production of TNF-α in LPS-treated BMs by 54%. This study confirmed that dapsone has anti-inflammatory effects on LPS-mediated inflammation via modulation of the number and function of inflammatory cells, providing new and useful information for clinicians and researchers.

Bone distribution study of anti leprotic drug clofazimine in rat bone marrow cells by a sensitive reverse phase liquid chromatography method.

The aim of the present study was to investigate the distribution of clofazimine (CLF) in rat bone marrow cells by a validated reverse phase high performance liquid chromatography. CLF and chlorzoxazone (I.S) were extracted by liquid-liquid extraction from plasma and rat bone marrow cells. The chromatographic separation was performed in isocratic mode by the mobile phase consisting of 10mM ammonium formate (pH 3.0 with formic acid) and acetonitrile in a ratio of 50:50 (v/v). The method was accurate and precise in the linear range of 15.6-2000.0 ng/mL with a correlation coefficient (r(2)) of 0.996 and 0.995 in rat plasma and bone marrow cells, respectively. After single oral dose of 20mg/kg, the maximum concentration of CLF in plasma and bone marrow cells were obtained at 12h with the concentrations of 593.2 and 915.4 ng/mL, respectively. The AUC0-t and mean elimination half life (t1/2) of CLF in bone marrow cells were 54339.02 ng h/mL and 52.46 h, respectively, which signified the low body clearance and high distribution of CLF in bone marrow cells. The single oral dose pharmacokinetic investigation was confirmed the CLF endure for a long period in rat due to high distribution in various tissues. The developed method was successfully applied to the estimation of the pharmacokinetic parameters of CLF in plasma and bone marrow cells after administration of single oral dose of 20mg/kg to rats.

The ubiquitin ligase parkin mediates resistance to intracellular pathogens.

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

In vitro and in vivo immunostimulatory activity of Woodfordia fruticosa flowers on non-specific immunity.

CONTEXT: Woodfordia fruticosa Kurz. (Lythraceae), a non-rasayana immunomodulatory Indian medicinal plant, used traditionally as an anthelmintic, in dysentery, leprosy, blood diseases, leucorrhea, and menorrhagia. OBJECTIVE: To investigate the effect of ethanol extract of W. fruticosa flowers on non-specific immune responses in mice. MATERIALS AND METHODS: In vitro immunomodulatory activity of the extract was examined on murine peritoneal macrophage phagocytosis (nitroblue tetrazolium (NBT) dye reduction, lysosomal enzyme activity, nitric oxide and myeloperoxidase) and on proliferation of bone marrow cells by sulforhodamine B (SRB) assay, while the in vivo potential on macrophages and bone marrow cells was evaluated by using carbon clearance test and cyclophosphamide-induced myelosuppression, respectively. RESULTS: Significant increase in the release of myeloperoxidase, nitric oxide lysosomal enzyme and superoxide from macrophages along with significant increase in phagocytic index in carbon clearance test indicate stimulatory activity of the extract on macrophages. The extract also demonstrated 60% increase in bone marrow cell proliferation and offer protection towards cyclophosphamide-induced myelosuppression which represents the stimulation of bone marrow activity. DISCUSSION: Significant increase in mediators released from macrophages and phagocytic index in carbon clearance test suggests the release of cytokines from macrophages and stimulation of reticulo-endothelial system. Proliferation of bone marrow cells indicates the plausible release of colony stimulating factors, which further stimulates the immune system through generation of immune cells. CONCLUSION: The result described here indicates the immunostimulatory activity of ethanol extract of W. fruticosa flowers by stimulating non-specific immune responses, macrophages and bone marrow cells.

Assessment of the in vitro and in vivo genotoxicity of Thalomid (thalidomide).

Thalomid is the FDA-approved commercial formulation of thalidomide currently used in the US to treat erythema nodosum leprosum, a complication of leprosy. The genotoxicity of Thalomid thalidomide was assessed in the Ames reverse mutation, AS52/XPRT mammalian cell forward gene mutation, and mouse bone marrow micronucleus assays. The Ames and AS52 assays were performed with and without S9. In the Ames, Salmonella typhimurium strains TA1535, 1537, 98, 100, and 102 and Escherichia coli strain WP2 uvrA were used. Assays were performed by using plate incorporation and liquid pre-incubation systems at thalidomide doses of 50-10,000 microg/plate. In the AS52 assay, Chinese hamster ovary cells were plated with fortified Ham's F12 medium and incubated overnight. The medium was then incubated with 1-1000 microg/ml thalidomide. After a series of aspirations, washings, reconstitutions, and incubations, mutant AS52 cells were fixed and stained. Colonies were then counted and the relative survival frequencies compared to negative controls. In the mouse micronucleus assay, Crl:CD-1 albino mice were dosed with 500, 2,500, and 5,000 mg/kg thalidomide and sacrificed over 72 h. Femurs were flushed with fetal bovine serum and the suspensions centrifuged. The supernatant was aspirated and the cell pellet resuspended and stained. Polychromatic erythrocytes were scored for micronucleated polychromatic and normochromatic erythrocytes. Thalidomide did not increase revertant frequencies in all bacterial strains. It also did not produce any significant increase in the average mutant frequencies of AS52 cells and mouse micronucleated polychromatic erythrocytes. We conclude that Celgene's Thalomid thalidomide is non-genotoxic.

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages.

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.

Reduction of clastogenic effect of clofazimine, an antileprosy drug, by vitamin A and vitamin C in bone marrow cells of mice.

Clofazimine (CLF), an antileprosy drug, has earlier been proved to be clastogenic in mice in vivo. It is an important constituent of the triple-drug regimen recommended by WHO for the treatment of leprosy. In this study the protective role of vitamins A and C (vit A and vit C) against the clastogenic effect of CLF in mouse bone marrow cells has been evaluated. Two doses (20 and 40 mg/kg) of vit C and two doses (2500 and 5000 IU/kg) of vit A were tested against a dose of 40 mg CLF/kg. The drug alone induced chromosomal aberrations of about 8 times the control value. Neither of the doses of vit C exhibited any clastogenic effect and, when administered simultaneously with CLF, both reduced the effect of CLF very significantly, the higher dose reducing chromosomal aberrations almost to the control value. Conversely, both doses of vit A, when administered alone, brought about significant increases in chromosome aberrations over the control value; the higher, but not the lower dose, given simultaneously with CLF, minimized the effect of CLF significantly but not as greatly as vit C. A scavenging effect of the vitamins, removing free radicals produced by CLF, is assumed to be responsible for modulation of the clastogenic effect of CLF.

Mycobacterium leprae-specific Lyt-2+ T lymphocytes with cytolytic activity.

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.

Susceptibility of Mycobacterium leprae to the bactericidal activity of mouse peritoneal macrophages and to hydrogen peroxide.

Macrophages from athymic nude mice were infected in vitro with Mycobacterium leprae to study the intracellular fate of this organism. Using the proportional bactericidal test, we have shown that the viability of M. leprae declines rapidly within these macrophages, although results of clearance experiments demonstrate that live and killed organisms are cleared at comparable rates. We have also shown that M. leprae is susceptible to the bactericidal effects of hydrogen peroxide and we suggest that hydrogen peroxide generated by macrophages is responsible for the killing of intracellular M. leprae.

Exchange of material between the extracellular medium and macrophage phagosomes containing different species of bacteria including mycobacteria.

Pathogenic mycobacteria survive and multiply once they have infected macrophages. The aim of the present work was to determine whether the persistence of pathogenic bacteria such as M. avium inside the host cell phagosomes had any effect on the exchanges that normally occur between the extracellular medium and the macrophage vacuolar compartment. Our results indicate that fusions between phagosomes and lysosomes or/and incoming pinosomes appear to be slowed down by the presence of mycobacteria and even partially inhibited when phagosomes contain viable pathogenic mycobacteria.

Growth of macrophages obtained from various sources.

Techniques to obtain macrophages from various sources of the mouse were reported. The following sources were included: peritoneal exudate, alveolar lavage, blood leucocytes, bone marrow, spleen, liver, lungs, lymph nodes, thymus, thyroid, heart muscle, kidney, and subcutaneous cover glass implants. Human blood macrophages were also included. Long-term cinemicrographic studies revealed sustained good growth of these macrophages. Cell multiplication was detected in all of these cultures except those obtained from the peritoneal exudate. Pure cultures of macrophages were obtained from blood of the mouse and human. Macrophages obtained from other sources were accompanied by some growth of fibroblasts. Methods to eliminate the fibroblasts in cultures were discussed.