Morten Hanefeld Dziegiel

Acidification of the Osteoclastic Resorption Compartment Provides Insight into the Coupling of Bone Formation to Bone Resorption

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.

Primary structure and localization of a conserved immunogenicPlasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle

A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.

Characterization of Osteoclasts from Patients Harboring a G215R Mutation in ClC-7 Causing Autosomal Dominant Osteopetrosis Type II

Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption.

Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis

BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D− women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening.

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14− population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.

A Novel Antibody-Dependent Cellular Cytotoxicity Mechanism Involved in Defense against Malaria Requires Costimulation of Monocytes FcγRII and FcγRIII

Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic activity. Using particulate Ag as pseudomerozoites, we show that only Ags, and no other parasite-derived factor, are required to trigger MN activation and that a single Ag is as potent as the complex combination of Ags constituting the merozoite surface. Moreover, we found that soluble Ags binding at least two Abs are as effective as the parasite at stimulating MN and that nonmalarial Ags are as efficient provided they are targeted by cytophilic Abs. Indeed, only cytophilic IgGs are potent and, in agreement with immunoepidemiological findings, IgG3 is superior to IgG1. Very low Ab concentrations (>700 pM), i.e., in the range of molecules having a hormonal effect, are effective, in contrast to Abs having a direct, neutralizing effect. Finally, Ab-dependent cell-mediated inhibition proved to require the synergistic activation of both FcγRIIa and FcγRIIIa which both distinguish it from other Ab-dependent cellular cytotoxicity and implies that all MN are not equally effective. These findings have both fundamental and practical implications, particularly for vaccine discovery.

Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin

Platelet‐rich fibrin (PRF®) is an autologous fibrin sealant (FS) enriched with a platelet concentrate (>1,000,000 platelets/μL) produced by the automated Vivostat® system and used to enhance wound healing. The effects of PRF were compared with supernatant from thrombin‐activated platelet concentrate, recombinant human platelet‐derived growth factor (rhPDGF) isoforms, and a homologous FS in cultured normal human dermal fibroblasts. Also, the release of selected endogenous growth factors from PRF and their stability against proteolytic degradation were studied. The proliferative effect of PRF exceeded that of FS and rhPDGF‐BB, although it was lower than thrombin‐activated platelet concentrate possibly due to sustained growth factor release from platelets in PRF. Anti‐PDGF antibody blocked the mitogenic effect of rhPDGF‐BB but not that of PRF in growth‐arrested fibroblasts. PRF promoted secretion of carboxyterminal propeptide of type I collagen into conditioned medium while rhPDGF‐AB had no significant effect on collagen biosynthesis. Limited proteolysis of PDGF‐AB and no proteolysis of transforming growth factor‐β1 (TGF‐β1) in PRF were observed with trypsin treatment, whereas rhPDGF‐AB and rhTGF‐β1 in bovine serum albumin, matching the total protein concentration of PRF, were almost completely degraded after 24 hours at 37 °C. To conclude, PRF provides sustained release and protection against proteolytic degradation of endogenous fibrogenic factors important for wound healing.

FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS

The defective generation or function of regulatory T (Treg) cells in autoimmune disease contributes to chronic inflammation and tissue injury. We report the identification of FoxA1 as a transcription factor in T cells that, after ectopic expression, confers suppressive properties in a newly identified Treg cell population, herein called FoxA1+ Treg cells. FoxA1 bound to the Pdl1 promoter, inducing programmed cell death ligand 1 (Pd-l1) expression, which was essential for the FoxA1+ Treg cells to kill activated T cells. FoxA1+ Treg cells develop primarily in the central nervous system in response to autoimmune inflammation, have a distinct transcriptional profile and are CD4+FoxA1+CD47+CD69+PD-L1hiFoxP3−. Adoptive transfer of stable FoxA1+ Treg cells inhibited experimental autoimmune encephalomyelitis in a FoxA1–and Pd-l1–dependent manner. The development of FoxA1+ Treg cells is induced by interferon-β (IFN-β) and requires T cell–intrinsic IFN-α/β receptor (Ifnar) signaling, as the frequency of FoxA1+ Treg cells was reduced in Ifnb−/− and Ifnar−/− mice. In individuals with relapsing-remitting multiple sclerosis, clinical response to treatment with IFN-β was associated with an increased frequency of suppressive FoxA1+ Treg cells in the blood. These findings suggest that FoxA1 is a lineage-specification factor that is induced by IFN-β and supports the differentiation and suppressive function of FoxA1+ Treg cells.

Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification

Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K.

Quality assessment of a placental perfusion protocol

Validation of in vitro test systems using the modular approach with steps addressing reliability and relevance is an important aim when developing in vitro tests in e.g. reproductive toxicology. The ex vivo human placental perfusion system may be used for such validation, here presenting the placental perfusion model in Copenhagen including control substances. The positive control substance antipyrine shows no difference in transport regardless of perfusion media used or of terms of delivery (n=59, p<0.05). Negative control studies with FITC marked dextran correspond with leakage criteria (<3 ml h(-1) from the fetal reservoir) when adding 2 (n=7) and 20mg (n=9) FITC-dextran/100 ml fetal perfusion media. Success rate of the Copenhagen placental perfusions is provided in this study, including considerations and quality control parameters. Three checkpoints suggested to determine success rate revealed that 15% of the cannulated placentae received in one year (n=202) were successfully perfused.