Bisphosphonates inhibit bone resorption in vivo and in vitro by unknown mechanisms. The effect of bisphosphonates on the formation of osteoclasts from their mononuclear hematopoietic precursors was investigated using human long-term marrow cultures in which multinucleated cells form that express most of the known features of the osteoclast phenotype (e.g., bone resorption, tartrate-resistant acid phosphatase, calcitonin responsiveness, and reactivity with specific MAbs). The five bisphosphonates that were tested strongly inhibited 1,25-dihydroxyvitamin D3-stimulated formation of these cells with the same relative pqtencies as they inhibit ione resorption in vivo. Two representative compounds (3-amino-1-hydroxypropylidene-1,1-bisphosphonate and dichloromethylene bisphosphonate) failed to inhibit the proliferation of precursors of the osteoclast-like cells. However, these compounds decreased the proportion of mononuclear and multinucleated cells expressing an osteoclast antigen, thus suggesting a degree of specificity for cells of the osteoclast lineage. We conclude that bisphosphonates are potent inhibitors of osteoclast-like cell formation in long-term human mprrow cultures, #nd that this may be related to their ability to inhibit bone resorption in vivo.

Key Points• Investigation of the ironrestrictive effect of minihepcidin peptides in the treatment of b-thalassemia and polycythemia vera.In b-thalassemia and polycythemia vera (PV), disordered erythropoiesis triggers severe pathophysiological manifestations. b-Thalassemia is characterized by ineffective erythropoiesis, reduced production of erythrocytes, anemia, and iron overload and PV by erythrocytosis and thrombosis. Minihepcidins are hepcidin agonists that have been previously shown to prevent iron overload in murine models of hemochromatosis and induce iron-restricted erythropoiesis at higher doses. Here, we show that in young Hbb th3/1 mice, which serve as a model of untransfused b-thalassemia, minihepcidin ameliorates ineffective erythropoiesis, anemia, and iron overload. In older mice with untransfused b-thalassemia, minihepcidin improves erythropoiesis and does not alter the beneficial effect of the iron chelator deferiprone on iron overload. In PV mice that express the orthologous JAK2 mutation causing human PV, administration of minihepcidin significantly reduces splenomegaly and normalizes hematocrit levels. These studies indicate that drug-like minihepcidins have a potential as future therapeutics for untransfused b-thalassemia and PV. (Blood. 2016;128(2):265-276)

The osteoclast is thought to be derived from immature marrow cells of the monocyte/macrophage lineage (1-3). However, the osteoclast has been difficult to study directly because ofits relative inaccessibility. Therefore investigators have used other cells of the monocyte/macrophage series, such as peritoneal macrophages (4), alveolar macrophages (5), HL-60 cells (6), and U937 cells (7), as models for osteoclasts or their precursors. Recently, it has been shown that the most potent biologically active metabolite of vitamin D, la,25-dihydroxyvitamin D3, causes fusion of alveolar macrophages (5) and differentiation of mouse leukemic cells and HL-60 and U937 human leukemic cells in culture into macrophages (6)(7)(8). Since the normal osteoclast precursor is believed to be present in the marrow mononuclear cell population, we cultured normal primate marrow mononuclear cells with 1,25-dihydroxyvitamin D3. We utilized the long-term culture system of Testa et al. (9), which they and we have used to examine formation of multinucleated cells with biologic and morphologic characteristics of osteoclasts in cultures of normal feline marrow (9, 10). We found that in the presence of 1,25-dihydroxyvitamin D3, a subpopulation of primate marrow mononuclear cells formed multinucleated cells with some of the characteristics of osteoclasts. MATERIALS AND METHODSCollection and Processing of Baboon Marrow Cells. Bone marrow was aspirated from the sternum or posterior iliac crest of adult baboons anesthetized with ketamine (10 mg/kg). Marrow was collected in syringes containing minimal essential medium alpha (a-MEM, GIBCO) supplemented with 5% (vol/vol) fetal bovine serum (Sterile Systems, Logan, UT) and preservative-free heparin (100 units/ml; Sigma). Marrow mononuclear cells were harvested from the interface of Ficoll/Hypaque density gradients (11) that had been centrifuged at 400 x g for 30 min at 12TC. The marrow mononuclear cells were washed twice with medium and then cultured in 24-well plates (Linbro) at 106 cells per ml, in a-MEM with 20o horse serum. In some experiments marrow mononuclear cells were incubated for 1 hr in a-MEM containing 20% fetal calf serum in 35-mm plastic tissue culture dishes, and the nonadherent cells were collected. The nonadherent cells were cultured in a similar fashion as the unfractionated marrow mononuclear cells. All cultures were maintained in a humidified 4% CO2 atmosphere at 370C. In selected experiments, various concentrations of osteotropic hormones were added at the initiation ofthe cultures and with each feeding. Cultures were fed weekly by removing half of the medium and nonadherent cells and replacing it with an equal volume of fresh medium. No attempt was made to recover the nonadherent cells. After culture, cells were fixed with 5% (vol/vol) glutaraldehyde (Sigma) and then stained with Wright stain. Cells were examined with an inverted phase-contrast microscope; those cells containing more than three nuclei were counted as multinucleated.Assay of Acid Phosphatase Activity. Marrow cells w...

Studies of osteoclasts and their precursors in normal and pathological states have been severely hampered by the lack of an in vitro system for forming osteoclasts. We developed a human marrow culture system in which multinucleated cells with several characteristics of osteoclasts form. Multinucleated cells began to form during the first week of culture, with maximum numbers formed after 3 weeks. PTH (25-50 ng/ml) and 1,25-dihydroxyvitamin D3 (10(-10)-10(-8) M) increased formation of these cells, and these effects were inhibited by calcitonin. These multinucleated cells contained nonspecific esterase and tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts, and had several ultrastructural features of osteoclasts. We used this marrow cell culture technique to study a patient with hyperparathyroidism and markedly increased osteoclasts on bone marrow biopsy. The marrow from this patient formed increased numbers of multinucleated cells in vitro. After parathyroidectomy both multinucleated cell formation in vitro and osteoclast numbers on bone biopsy decreased significantly. This long term marrow culture system represents the first demonstration of human osteoclast-like cell formation in vitro. This system should permit studies to evaluate factors controlling formation of cells with certain osteoclast characteristics in vitro and their precursors as well as to evaluate abnormalities in osteoclast formation in patients with metabolic bone disease.

No abstract

Transforming growth factor-alpha (TGF-a) is synthesized by a variety of tumor cell lines and stimulates osteoclastic bone resorption in vitro. The mechanism by which TGF-a increases osteoclast activity is unknown. We used a human marrow culture system that forms osteoclast-like multinucleated cells (MNCs) to determine the effects of recombinant human TGF-a on MNC formation. Addition of 0.01 ng/ml TGF-a for the 1st week followed by 1,25-dihydroxyvitamin D3 I1,25(0H)2D31 for the subsequent 2 wk significantly increased MNCs. Treatment of these cultures with TGF-a without later addition of 1,25(0H)2D3 did not increase MNC formation. Autoradiographic studies revealed that TGF-a stimulated proliferation of precursors for MNCs, and 1,25(OH)2D3 increased their rate of fusion into MNCs. Addition of murine epidermal growth factor (EGF) (0.1 ng/ml) followed by 1,25(OH)2D3 also significantly stimulated MNC formation. These data suggest that TGF-a and EGF may stimulate bone resorption by increasing the proliferation of osteoclast precursors, which leads to increased numbers of osteoclasts.

Evidence is presented that the site of cell division in Salmonella typhimurium is flanked by two circumferential zones of cell envelope differentiation, the periseptal annuli, which separate the division site from the remainder of the cell envelope. Each annulus is composed of a continuous structure in which the membranous elements of the cell envelope are closely associated with, the murein cytoskeleton. The paired annuli appear early in the division process and the region between them defines a new cellular domain, the.periseptal compartment, within which the division septum is formed.The process of bacterial cell division occurs by ingrowth-of the cell envelope from a narrow circumferential zone at the midpoint of the cell. This leads to segregation of the cytoplasm into two compartments and finally to separation of the daughter cells. Ingrowth of the new septum at the proper location requires that the molecular organization of the division site differ from the organization of the envelope over-the remainder of the cell. The nature of this local differentiation and the cellular mechanisms that initiate and maintain it at the proper location are not known.In this paper we describe an organelle that is associated with early stages of the division process in Gram-negative bacteria and that is likely to play a role in these events. Fixation (1.5 hr) and subsequent washing and postfixation with 2.0% OS04, were carried out in the presence of the respective plasmolyzing solutions. In the experiment described in Fig. 3, the cells were subsequently washed twice more with distilled water and resuspended for 20 min in a saturated aqueous solution of thiocarbohydrazide (Sigma) to enhance contrast in the final sections; excess thiocarbohydrazide was removed by two washes with distilled water and the cells were again exposed to 2% OsO4 prior to embedding. Embedding, sectioning, and counterstaining with lead citrate and uranyl acetate were performed as described (2). Serial. sections approximately 60 nm thick were mounted on carbon-backed Formvar-coated slotted (2 X 1 mm) copper grids and examined in a Hitachi HUIIE electron microscope at 75 kV accelerating voltage. RESULTS The cell envelopes of Gram-negative bacteria contain two membranes which completely surround the cell. Between the inner (cytoplasmic) and outer membranes lies the continuous murein layer, a rigid crosslinked peptidoglycan that provides the only known cytoskeletal structure of these and other bacteria (3). During normal cell division the three layers invaginate coordinately to form the new septum (Fig. la). METHODSWhen cells are plasmolyzed by brief exposure to hypertonic solutions, the resulting decrease in cytoplasmic volume causes the inner membrane to shrink away from the rigid murein/ outer membrane layer. As originally shown by Bayer (4, 5), the plasmolysis procedure reveals the presence of small zones where the cytoplasmic membrane fails -to pull away from the murein/outer membrane layer. These sites of membrane adhesion are thought to re...

Using cultured human osteoblast-like cells, we studied the effects of tumor necrosis factor (TNF) and recombinant human y-interferon ( y-IFN) on osteoblast growth and function, and demonstrated that TNF stimulated bone cell proliferation and prostaglandin production while inhibiting 1 ,25-(OH),D,-stimulated alkaline phosphatase activity and osteocalcin release. In contrast, y-IFN inhibited proliferation and stimulated alkaline phosphatase activity of the cells, while inhibiting 1,25-(OH),D3-stimulated osteocalcin production and having variable effects on the release of prostaglandins, depending on the presence of other factors. Our results suggest that TNF and y-IFN can act directly on boneforming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.Localized subchondral bone loss is one of the most painful and debilitating aspects of chronic inflammatory diseases such as rheumatoid arthritis (RA).