Epizootics of Streptococcus agalactiae infection in captive rays from Queensland, Australia.

The aim of this study was to describe two epizootics of high mortalities from infection with Streptococcus agalactiae, occurring in captive rays held in a marine display aquarium in south-east Queensland, Australia, in 2009 and 2010. Five different species of rays were affected, including mangrove whiprays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white-spotted eagle rays (Aetobatus narinari) and blue-spotted mask rays (Neotrygon kuhlii). This report describes the history of both epizootics including collection, quarantine and husbandry of rays, the disease epizootics, clinico-pathological features of the disease, antimicrobial therapy, autogenous vaccine production, and laboratory studies including clinical and histopathology, bacteriology, PCR, molecular serotyping and sequencing of the bacterium S. agalactiae.

Interactions between Cooccurring Lactic Acid Bacteria in Honey Bee Hives.

In contrast to the honey bee gut, which is colonized by a few characteristic bacterial clades, the hive of the honey bee is home to a diverse array of microbes, including many lactic acid bacteria (LAB). In this study, we used culture, combined with sequencing, to sample the LAB communities found across hive environments. Specifically, we sought to use network analysis to identify microbial hubs sharing nearly identical operational taxonomic units, evidence which may indicate cooccurrence of bacteria between environments. In the process, we identified interactions between noncore bacterial members (Fructobacillus and Lactobacillaceae) and honey bee-specific "core" members. Both Fructobacillus and Lactobacillaceae colonize brood cells, bee bread, and nectar and may serve the role of pioneering species, establishing an environment conducive to the inoculation by honey bee core bacteria. Coculture assays showed that these noncore bacterial members promote the growth of honey bee-specific bacterial species. Specifically, Fructobacillus by-products in spent medium supported the growth of the Firm-5 honey bee-specific clade in vitro. Metabolic characterization of Fructobacillus using carbohydrate utilization assays revealed that this strain is capable of utilizing the simple sugars fructose and glucose, as well as the complex plant carbohydrate lignin. We tested Fructobacillus for antibiotic sensitivity and found that this bacterium, which may be important for establishment of the microbiome, is sensitive to the commonly used antibiotic tetracycline. Our results point to the possible significance of "noncore" and environmental microbial community members in the modulation of honey bee microbiome dynamics and suggest that tetracycline use by beekeepers should be limited.

Focused ion beam milling and ultramicrotomy of mineralised ivory dentine for analytical transmission electron microscopy.

The use of focused ion beam (FIB) milling for preparation of sections of mineralised ivory dentine for transmission electron microscopy (TEM) is investigated. Ivory dentine is essentially composed of fibrillar type-I collagen and apatite crystals. The aim of this project is to gain a clearer understanding of the relationship between the organic and inorganic components of ivory dentine using analytical TEM, in order to utilise these analytical techniques in the context of common skeletal diseases such as osteoporosis and arthritis. TEM sections were prepared in both single and dual beam FIB instruments, using two standard lift-out techniques, in situ and ex situ. The FIB sections were systematically compared with sections prepared by ultramicrotomy, the traditional preparation route in biological systems, in terms of structural and chemical differences. A clear advantage of FIB milling over ultramicrotomy is that dehydration, embedding and section flotation can be eliminated, so that partial mineral loss due to dissolution is avoided. The characteristic banding of collagen fibrils was clearly seen in FIB milled sections without the need for any chemical staining, as is commonly employed in ultramicrotomy. The FIB milling technique was able to produce high-quality TEM sections of ivory dentine, which are suitable for further investigation using electron energy-loss spectroscopy (EELS) and energy-filtering TEM (EFTEM) to probe the collagen/apatite interface.

Identifying early osteoclastic resorptive lesions in feline teeth: a model for understanding the origin of multiple idiopathic root resorption.

BACKGROUND AND OBJECTIVE: Domestic cats commonly suffer from external osteoclastic tooth resorption, a disease with many similarities to human multiple idiopathic root resorption. In both diseases, it is unclear whether anatomical features of the tooth surface are associated with a predisposition for resorptive lesions. The aim of the present study was to investigate the origin and progression of early feline osteoclastic resorptive lesions in teeth exhibiting no clinical signs of disease. MATERIAL AND METHODS: The entire surfaces of 138 teeth from 13 adult cats were analysed using back-scattered electron microscopy. The distribution of lesions was assessed by tooth type, location and between individuals. RESULTS: Seventy-three (53%) teeth showed at least one resorptive lesion. Eleven (85%) cats had lesions, and there was a significant association between increasing age and incidence of resorptive lesions. The highest frequency occurred in mandibular molars (82%). On average, there were 3.5 lesions per tooth. Fifty-two (38%) teeth featured resorptive lesions at the cemento-enamel junction. Twenty-three per cent of teeth with resorptive lesions showed evidence of repair of lesions that was limited to the root surface. There was no evidence of repair of resorptive lesions at the cemento-enamel junction. CONCLUSION: Resorption is prevalent without evidence of clinical disease, and occurred at younger ages than previously reported. It can initiate anywhere on the root surface, but lack of repair of lesions at the cemento-enamel junction indicates that mechanisms of replacement are absent or compromised in this region. Whereas resorption of the root may undergo repair, resorption at the cervix may progress to clinically evident lesions.

Nanoscale scraping and dissection of collagen fibrils.

The main function of collagen is mechanical, hence there is a fundamental scientific interest in experimentally investigating the mechanical and structural properties of collagen fibrils on the nanometre scale. Here, we present a novel atomic force microscopy (AFM) based scraping technique that can dissect the outer layer of a biological specimen. Applied to individual collagen fibrils, the technique was successfully used to expose the fibril core and reveal the presence of a D-banding-like structure. AFM nanoindentation measurements of fibril shell and core indicated no significant differences in mechanical properties such as stiffness (reduced modulus), hardness, adhesion and adhesion work. This suggests that collagen fibrils are mechanically homogeneous structures. The scraping technique can be applied to other biological specimens, as demonstrated on the example of bacteria.

Integrated confocal and scanning probe microscopy for biomedical research.

Atomic force microscopy (AFM) continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM) and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.

Skeletal tissues as nanomaterials.

Collagen is the most abundant protein in the body and, though the fibre-forming collagens have a 'common' structure, it is adapted to perform a large range of functions-from the differing mechanical needs of tendon versus bone to forming a transparent support structure in the cornea. This perfidy also suggests that collagen could form a generic basis for a range of scaffold needs for tissue engineering or medical device coating applications. We at the London Centre for Nanotechnology--a joint venture between University College London and Imperial College--are taking a bottom-up approach having decided that many of the 'accepted dogmas' of collagen biology may not be quite as soundly based as currently held. We are using several of the tools of 'hard' nanotechnology--such as atomic force microscopy--to re-examine collagen structure with the longer term aim of using such information to design materials with appropriate physical attributes. Examples of our current research on mineralised and soft tissue collagens are presented.

Analysis of the surface characteristics and mineralization status of feline teeth using scanning electron microscopy.

External resorption of teeth by odontoclasts is a common condition of unknown origin affecting domestic cats. Odontoclastic resorptive lesions involve the enamel cementum junction (ECJ, cervix) and root surface, leading to extensive loss of enamel, dentine and cementum. This study was undertaken in order to determine whether features of the surface anatomy and mineralization of feline teeth could explain why odontoclastic resorptive lesions are so prevalent in this species. Backscattered electron scanning electron microscopy was used to study enamel, cementum and dentine in non-resorbed, undemineralized teeth from adult cats. Analysis of the ECJ revealed thin enamel and cementum and exposed dentine at this site. Furthermore, enamel mineralization decreased from the crown tip to the ECJ, and dentine mineralization was lowest at the ECJ and cervical root. Analysis of cementum revealed variations in the organization and composition of fibres between the cervical, mid- and apical root although no significant differences in mineralization of cementum were detected between different regions of the root. Reparative patches associated with resorption of cementum by odontoclasts and repair by cementoblasts were present on the root surface. In conclusion, results suggest that the ECJ and cervical dentine could be at a greater risk of destruction by odontoclasts compared with other regions of the tooth. The relationship of these features to the development and progression of resorption now requires further examination.

Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat.

Dental disease due to osteoclast (OC) overactivity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline odontoclastic resorptive lesions (FORL) involve extensive resorption of the tooth, leaving it liable to root fracture and subsequent loss. The etiopathogenesis of FORL remains unclear. Here, we explore the hypothesis that FORL is associated with hypoxia in the oral microenvironment, leading to increased OC activity. To investigate this, we developed a method of generating OCs from cat blood. Reducing O2 from 20% to 2% increased the mean area of OC eightfold from 0.01 to 0.08 mm2. In hypoxic cultures, very large OCs containing several hundred nuclei were evident (reaching a maximum size of approximately 14 mm2). Cultures exposed to 2% O2 exhibited an increase of approximately 13-fold in the area of bone slices covered by resorption lacunae. In line with this finding, there was a significant increase in cells differentiating under hypoxic conditions, reflected in increased expression of cathepsin K and proton pump enzymes. In conclusion, these results demonstrate that oxygen tension is a major regulator of OC formation in the cat. However, in this species, hypoxia induces the formation of "giant" OCs, which can be so large as to be visible with the naked eye and yet also actively resorb. This suggests that local hypoxia is likely to play a key role in the pathogenesis of FORL and other inflammatory conditions that are associated with bone resorption in cats.

Mineralised tissues as nanomaterials: analysis by atomic force microscopy.

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225 nm up to 1.4 microm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69 nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis.

Recapitulation of the parathyroid hormone-related peptide-Indian hedgehog pathway in the regenerating deer antler.

Parathyroid hormone (PTH)-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR) play an essential role in controlling growth plate development. The aim of the present study was to use the deer antler as a model to determine whether PTHrP and PPR may also have a function in regulating cartilage and bone regeneration in an adult mammal. Antlers are the only mammalian appendages that are able to undergo repeated cycles of regeneration, and their growth from a blastema involves a modified endochondral process. Immunohistochemistry was used to establish sites of localization of PTHrP and PPR in antlers at different stages of development. The pattern of Indian Hedgehog (IHH) and transforming growth factor-beta1 (TGF beta1) distribution was also investigated, because PTHrP expression in the developing limb is regulated by IHH and during embryonic growth plate formation TGF beta1 acts upstream of PTHrP to regulate the rate of chondrocyte differentiation. In the antler blastema (<10 days of development), PTHrP, PPR, and TGF beta1 were localized in epidermis, dermis, regenerating epithelium, and in mesenchymal cells but IHH expression was not detected. In the rapidly growing antler (weeks 4-8 of development), PTHrP, PPR, and TGF beta1 were localized in skin, perichondrium, undifferentiated mesenchyme, recently differentiated chondrocytes, and in perivascular cells in cartilage but not in fully differentiated hyperytrophic chondrocytes. IHH was restricted to recently differentiated chondrocytes and to perivascular cells in cartilage. In mineralized cartilage and bone, PTHrP, PPR, IHH, and TGF beta1 were immunolocalized in perivascular cells and differentiated osteoblasts. PTHrP and PPR were also present in the periosteum. TGF beta1 in vitro stimulated PTHrP synthesis by cells from blastema, perichondrium, and cartilage. The findings of this study suggest that molecules which regulate embryonic skeletal development and postnatal epiphyseal growth may also control blastema formation, chondrogenesis, and bone formation in the regenerating deer antler. This finding is further evidence that developmental signaling pathways are recapitulated during adult mammalian bone regeneration.

A comparison of methods for measuring serum and urinary markers of bone metabolism in cats.

Biochemical markers of bone cell activity have recently been shown to be useful for monitoring skeletal health in domestic animals, including dogs and horses. The aim of this study was to evaluate a number of biochemical assays, originally developed for use in humans, for their ability to measure indicators of bone cell activity in serum and urine of normal cats over a range of ages. Bone alkaline phosphatase (BAP), a marker of bone formation, was measured in serum using wheatgerm lectin precipitation (WGL) and by ELISA. The curve derived from serial dilution of feline serum was parallel with the ELISA standard curve, indicating species cross-reactivity, and there was a significant relationship between assays (rs = 0.97, P < 0.001). Deoxypyridinoline (DPD), a marker of bone resorption, was measured in its total form in urine by HPLC and ELISA, and in its free form in serum and urine by ELISA. The dilution curve for free DPD in urine showed parallelism with the assay standard curve; however, the curves for total DPD in urine and serum did not. A significant relationship was established between total urinary DPD (HPLC) with total serum DPD (rs = 0.69, P < 0.001), and with free urinary DPD (rs = 0.95, P < 0.001) concentrations. Carboxy-terminal telopeptide of type I collagen (CTX) concentration, another marker of bone resorption, was measured in serum and urine by ELISA, and there was a significant relationship between assays (rs = 0.82, P < 0.001). CTX could not be measured reliably using an auto-analysis method. A significant relationship was established between total urinary DPD (HPLC) with serum CTX (rs = 0.59, P < 0.05), and urinary CTX (rs = 0.65, P < 0.001) concentrations. BAP (ELISA and WGL), total urinary DPD (HPLC), urinary CTX (ELISA), and serum CTX (ELISA) concentrations were significantly inversely correlated with age (rs = -0.66, -0.88, -0.61, -0.70, and -0.51, P < 0.05 respectively). Cats under two years of age had significantly higher BAP, total urinary DPD (HPLC), and urinary CTX concentrations compared to older cats. In conclusion, this study has shown that a number of commercially available assays provide reliable methods for non-invasively monitoring bone cell activity in cats and has shown that bone turnover decreases within the first two years of life, until complete skeletal maturity is attained. Future studies can now be directed at evaluating the potential clinical application of these methods.

A feline assay using osteoclasts generated in vitro from peripheral blood for screening anti-resorptive agents.

Musculo-skeletal diseases are a major cause of pain and suffering in cats and several conditions involve increased bone resorption by osteoclasts. However, little is known about the biology of these cells in the cat. In this study we established a method to generate feline osteoclasts from blood mononuclear cells stimulated by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Cultured osteoclasts are multinucleated, express tartrate resistant acid phosphatase (TRAP), form F-actin rings and resorb bone. They express alpha(v)beta3 vitronectin receptor and osteoclast enzymes, cathepsin K and MMP9; the myeloid antigen, CD18, and the megakaryocyte/platelet integrin, CD41, are absent. This phenotype is typical of osteoclasts from other species. Three resorption inhibitors were examined for activity against feline osteoclasts. Calcitonin, bisphosphonate and RGD integrin inhibitory peptide all reduced bone resorption at doses similar to those efficacious in rabbit or human. We conclude that blood-derived osteoclast cultures are a suitable in vitro system for assessing the ability of drugs to inhibit bone resorption in domestic cats.

Cytokine expression in feline osteoclastic resorptive lesions.

Feline osteoclastic resorptive lesions (FORL) of the teeth are common in cats, and lead to pain, destruction of the periodontal ligament, and tooth loss. The expression of interleukin (IL)-1 beta and IL-6 mRNA was higher in teeth with FORL than in normal teeth (P<0.01 and P<0.001, respectively), but no such differences were found between pathological and normal gingival tissue samples. There were no differences between teeth affected with FORL and normal teeth in respect of the expression of receptor activator of NF kappa B ligand (RANKL) mRNA or osteoprotegerin (OPG) mRNA. However, OPG mRNA expression was higher in gingival tissue associated with teeth affected with FORL than in normal gingival tissue (P<0.05), whereas the reverse was true of RANKL mRNA expression (P<0.05). OPG mRNA expression was significantly higher in teeth than in femoral and alveolar bone (P<0.001). RANKL and OPG mRNAs were detected in all tissues examined. The data suggest that the elevated expression of IL-l beta and IL-6 mRNA plays a role in the mediation of osteoclast activity in advanced FORL. In contrast, OPG and RANKL do not appear to regulate osteoclasts in advanced disease. The results also suggest that OPG and RANKL mRNA play a role in mediating inflammatory responses in gingival cells, and that OPG has an inhibiting effect on tooth resorption.

Nuclear localization of type I parathyroid hormone/parathyroid hormone-related protein receptors in deer antler osteoclasts: evidence for parathyroid hormone-related protein and receptor activator of NF-kappaB-dependent effects on osteoclast formation in regenerating mammalian bone.

Parathyroid hormone-related protein (PTHrP) is not required for osteoclastogenesis during embryonic development; however, after birth it has been shown to regulate osteoclast formation during tooth eruption. Our study explores the hypothesis that PTHrP also may regulate osteoclast differentiation in the regenerating skeletal tissues of deer antlers, bones capable of complete regeneration. Osteoclast-like multinucleated cells (MNCs) formed spontaneously in micromass cultures derived from antler cartilage and these cells had the phenotypic characteristics of osteoclasts. PTHrP and receptor activator of NF-kappaB ligand (RANKL) stimulated antler osteoclast formation although the effect of RANKL was less marked than that of PTHrP. The addition of osteoprotegerin (OPG) only partially decreased (by approximately 65%) the number of osteoclasts in PTHrP-treated cultures. To determine whether PTHrP also potentially could have direct effects on antler osteoclasts, we studied, by confocal microscopy, the expression of the type I PTH/PTHrP receptor (PTH1R) in MNCs cultured on glass and found the receptor protein to have a nuclear localization. In situ hybridization showed that antler MNCs also expressed PTH1R and PTHrP messenger RNAs (mRNAs). PTHrP was immunolocalized in MNCs cultured on glass but was undetectable in cells resorbing a dentine substrate. In tissue sections of antler cartilage, PTHrP and PTH1R were expressed in vitronectin receptor-positive (VNR+) osteoclast-like cells localized in the perivascular stroma. Thus, these data show that PTHrP plays a role in the regulation of osteoclast differentiation in regenerating skeletal tissues and that PTHrP can have effects on osteoclastogenesis that are independent of RANKL synthesis. Ours is the first study to describe the expression of the type I PTH/PTHrP receptor in mammalian osteoclasts at a protein and mRNA level, which indicates that PTHrP also may have a direct effect on osteoclasts. This also is the first study to show a nuclear localization of the PTHIR in cells of the osteoclast lineage, although the functional significance of this observation has yet to be established.

Macrophage colony-stimulating factor and receptor activator NF-kappaB ligand fail to rescue osteoclast-poor human malignant infantile osteopetrosis in vitro.

Malignant infantile osteopetrosis (MIOP) is a disease characterized by failure in bone resorption, leading to dense fragile bones with a severely reduced bone marrow cavity. Normal or increased numbers of osteoclasts are present in the common variant of this disease; in such cases, the defect is likely to be inherent to the mature osteoclast and can be cured by bone marrow transplantation. However, MIOP also results from failure of osteoclast formation (osteoclast-poor MIOP). We report on two infants diagnosed with osteoclast-poor MIOP and utilize modern cell culture techniques to investigate the pathogenesis of disease. Peripheral blood mononuclear cells (PBMNCs) from these children were cultured in the presence of recombinant macrophage colony-stimulating factor and receptor activator NF-kappaB ligand for up to 3 weeks. Control cultures included PBMNCs from age-matched children, one of whom had an osteoclast-rich form of MIOP. Formation of osteoclasts (cells coexpressing vitronectin receptor and F-actin rings) occurred in all the control cultures. Significant bone resorption occurred in cultures from PBMNCs of the healthy individuals, whereas almost no bone resorption occurred in the osteoclast-rich MIOP cultures. In contrast, PBMNC cultures from the osteoclast-poor MIOP child formed only very occasional small F-actin ring-positive osteoclasts, which coexpressed vitronectin receptor and cathepsin K, and extremely rare foci of resorption. Because neither macrophage colony-stimulating factor nor receptor activator NF-kappaB ligand rescued the defect in osteoclast differentiation in the two cases of osteoclast-poor MIOP in vitro, there would be little benefit in treating these children with either of these recombinant proteins. Finally, these results demonstrate that this experimental culture model replicates the human osteopetrosis phenotype observed in vivo and should prove useful in analyzing the pathogenesis of the various forms of MIOP.

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Integrin antagonists as inhibitors of bone resorption: implications for treatment.

Currently 'accepted' treatments for bone disease utilise drugs that inhibit osteoclastic bone resorption; these lead to a reduction in subsequent bone loss and thence, indirectly, to an increase in bone mass and fewer fractures. Three classes of compounds currently form the mainstay of therapy for osteoporosis: oestrogens (hormone-replacement therapy), 'selective oestrogen receptor modulators' and the bisphosphonates. Problems of patient compliance, real or theoretical long-term toxicological risks and the lack of bone anabolic agents of clinical utility suggest that there is a need for the development of further novel osteoclast resorption inhibitors. Recent biological and genetic findings in the area of bone cell function have led to the identification of new drug targets. These drugs include agents that (directly or indirectly): inhibit osteoclast adhesion to bone matrix; modify osteoclast differentiation; act on the proton pump and hence affect extracellullar acidification; antagonise extracellular enzymes that are involved in bone matrix protein degradation. Particular emphasis is placed in the present review on the evaluation of antagonists of alphavbeta3 integrin-mediated cell adhesion for use in bone disease. The wealth of new agents being developed suggests that resorption inhibition will be the best treatment for osteoporosis in the short to medium term, with the long-term aim still being toward developing anabolic drugs or cell therapeutics.

Interleukin-4 and interleukin-13: bidirectional effects on human osteoclast formation.

Osteoclasts are cells that resorb bone; they derive from macrophage colony-stimulating factor (M-CSF)-dependent hematopoietic precursors in the presence of soluble activator of NFkappaB ligand (sRANKL). Because transforming growth factor (TGF)-beta, a macrophage deactivator, enhances osteoclast formation we hypothesized that interleukin (IL)-4 and IL-13, also macrophage deactivators, should exert a similar effect. However, IL-4 and IL-13 have been reported as suppressors of murine osteoclast formation. In contrast to the effect of these molecules on murine osteoclast formation, IL-4 and IL-13 were found to be powerful inducers of osteoclast formation and bone resorption when added to human peripheral blood mononuclear cell (PBMC) cultures for 4 days. This stimulatory effect was only observed in cultures containing nonadherent PBMCs. In contrast, both molecules significantly suppressed osteoclast formation in lymphocyte-depleted cultures. These data demonstrate that the cytokine milieu and/or state of cell activation determines how cells of the osteoclast precursor respond to IL-4 and IL-13.