Acute hypoxia and osteoclast activity: a balance between enhanced resorption and increased apoptosis

Osteoclasts are the primary mediators of pathological bone resorption in many conditions in which micro‐environmental hypoxia is associated with disease progression. However, effects of hypoxia on human osteoclast activity have not been reported. Mature human osteoclasts were differentiated from peripheral blood or obtained from giant cell tumour of bone. Osteoclasts were exposed to a constant hypoxic environment and then assessed for parameters including resorption (toluidine blue staining of dentine slices), membrane integrity (trypan blue exclusion), apoptosis (TUNEL, DAPI), and osteolysis‐associated enzyme activity (TRAP, cathepsin K). 24 h exposure to 2% O₂ produced a 2.5‐fold increase in resorption associated with increased TRAP and cathepsin K enzyme activity. Hypoxia‐Inducible Factor‐1α (HIF‐1α) siRNA completely ablated the hypoxic increase in osteoclast resorption. 24 h at 2% O₂ also increased the number of osteoclasts with compromised membrane integrity from 6% to 21%, with no change in the total osteoclast number or the proportion of late‐stage apoptotic cells. Transient reoxygenation returned the percentage of trypan blue‐positive cells to normoxic levels, suggesting that osteoclasts can recover from the early stages of cell death. Repeated over an extended period, hypoxia/reoxygenation enhanced osteoclast differentiation at this pO₂. These data suggest that in diseased bone, where the pO₂ may fall to ≤2% O₂, a delicate balance between hypoxia‐induced osteoclast activation and hypoxia‐induced osteoclast apoptosis mediates pathological bone resorption. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Interleukin‐15‐activated natural killer cells kill autologous osteoclasts via LFA‐1, DNAM‐1 and TRAIL,and inhibit osteoclast‐mediated bone erosion in vitro

Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone‐marrow‐derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte‐derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co‐culture experiments revealed that interleukin‐15‐activated, but not resting, NK cells trigger osteoclast apoptosis in a dose‐dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function‐associated antigen‐1, DNAX accessory molecule‐1 or tumour necrosis factor‐related apoptosis‐inducing ligand enhance osteoclast survival when co‐cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin‐15‐activated NK cells may directly affect bone erosion under physiological and pathological conditions.     

Immunolocalization of vacuolar‐type H+‐ATPase,cathepsin K,matrix metalloproteinase‐9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth

To investigate the cellular mechanisms of physiological root resorption in human deciduous teeth, the authors examined the immunocytochemical localization of vacuolar‐type H⁺‐ATPase, a lysosomal cysteine proteinase, cathepsin K, matrix metalloproteinase‐9 (MMP‐9), and receptor activator of NFKB ligand (RANKL) in odontoclasts. H⁺‐ATPase, cathepsin K, and MMP‐9 are the most important enzymes for decalcification of apatite crystals and degradation of type‐I collagen. In addition, RANKL is one of the key regulatory molecules in osteoclast formation and functions. Odontoclasts developed extensive ruffled borders and clear zones apposed to the resorbing root dentine surfaces. On immunoelectron microscopy, the expression of vacuolar‐type H⁺‐ATPase was detected along the limiting membranes of pale vacuoles and the ruffled border membranes of odontoclasts. Cathepsin K in odontoclasts was localized within pale vacuoles, lysosomes, the extracellular canals of ruffled borders, and the underlying resorbing dentine surfaces. MMP‐9 localization in odontoclasts was similar to those of cathepsin K. RANKL was detected in both mononuclear stromal cells and odontoclasts located on resorbing dentine surfaces. These results suggest that (1) odontoclasts are directly involved in decalcification of apatite crystals by active extrusion of proton ions mediated by H⁺‐ATPase and (2) extracellular degradation of dentine type‐I collagen by both cathepsin K and MMP‐9, and (3) odontoclast differentiation and activity are regulated, at least in part, by RANKL, possibly produced by mononuclear stromal cells and odontoclasts themselves in the resorbing tissues. Thus, the cellular mechanisms of physiological root resorption appear to be quite similar to those of osteoclastic bone resorption. Anat Rec 264:305–311, 2001. © 2001 Wiley‐Liss, Inc.     

Bisphosphonate administration alters subcellular localization of vacuolar‐type H+‐ATPase and cathepsin K in osteoclasts during experimental movement of rat molars

This study was designed to clarify the effects of bisphosphonate (BP) administration on structure and functions of osteoclasts in alveolar bone resorption during experimental movement of rat molars. To produce orthodontic force, elastic band was inserted between the upper first and second molars for 4 days, and dissected maxillae were then examined by means of light and electron microscopic immunocytochemistry for vacuolar‐type H⁺‐ATPase and lysosomal cystein proteinase, cathepsin K in osteoclasts. Vacuolar‐type H⁺‐ATPase and cathepsin K in osteoclasts are the most important enzymes for demineralization of apatite crystals and degradation of bone type‐I collagen, respectively. At 1 day before elastic band insertion, BP was administered intraperitoneally. Control rats received the same volume of physiologic saline. In BP‐administered rats, most osteoclasts exhibited either irregularly‐formed ruffled borders and clear zones or only clear zones of various degrees of extension. Subcellular localization and expression of both vacuolar‐type H⁺‐ATPase and cathepsin K was significantly decreased in such osteoclasts with impaired ruffled borders and/or only clear zones by BP administration. In particular, cathepsin K secretion by osteoclasts towards resorption lacunae was markedly inhibited by BP administration. Our results indicate for the first time that BP administration significantly impair the osteoclast structure and reduces expression of both vacuolar‐type H⁺‐ATPase and cathepsin K in osteoclasts during tooth movement. Anat Rec 260:72–80, 2000. © 2000 Wiley‐Liss, Inc.     

Bone loss in survival motor neuron (Smn−/− SMN2) genetic mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is characterized by degenerating lower motor neurons and an increased incidence of congenital bone fractures. Survival motor neuron (SMN) levels are significantly reduced due to deletions/mutations in the telomeric SMN1 gene in these patients. We utilized the Smn^?/? SMN2 mouse model of SMA to determine the functional role for SMN in bone remodelling. µCT analysis of lumber vertebrae, tibia and femur bones from SMA mice revealed an osteoporotic bone phenotype. Histological analysis demonstrated a thin porous cortex of cortical bone and thin trabeculae at the proximal end of the growth plate in the vertebrae of SMA mice compared to wild‐type mice. Histochemical staining of the vertebrae showed the presence of abundant activated osteoclasts on the sparse trabeculae and on the endosteal surface of the thin cortex in SMA mice. Histomorphometric analysis of vertebrae from SMA mice showed an increased number of osteoclasts. Serum TRAcP5b and urinary NTx levels were elevated, consistent with increased bone resorption in these mice. SMA mice showed a significant decrease in the levels of osteoblast differentiation markers, osteocalcin, osteopontin and osterix mRNA expression; however, there were no change in the levels of alkaline phosphatase expression compared to WT mice. SMA mouse bone marrow cultures revealed an increased rate of osteoclast formation (54%) and bone resorption capacity (46%) compared to WT mice. Pre‐osteoclast cells from SMA mice showed constitutive up‐regulation of RANK receptor signalling molecules critical for osteoclast differentiation. Our results implicate SMN function in bone remodelling and skeletal pathogenesis in SMA. Understanding basic mechanisms of SMN action in bone remodelling may uncover new therapeutic targets for preventing bone loss/fracture risk in SMA. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Paeoniflorin ameliorates collagen‐induced arthritis via suppressing nuclear factor‐κB signalling pathway in osteoclast differentiation

Paeoniflorin (PF), extracted from the root of Paeonia lactiflora Pall, exhibits anti‐inflammatory properties in several autoimmune diseases. Osteoclast, the only somatic cell with bone resorbing capacity, was the direct cause of bone destruction in rheumatoid arthritis (RA) and its mouse model, collagen‐induced arthritis (CIA). The objective of this study was to estimate the effect of PF on CIA mice, and explore the mechanism of PF in bone destruction. We demonstrated that PF treatment significantly ameliorated CIA through inflammatory response inhibition and bone destruction suppression. Furthermore, PF treatment markedly decreased osteoclast number through the altered RANKL/RANK/OPG ratio and inflammatory cytokines profile. Consistently, we found that osteoclast differentiation was significantly inhibited by PF through down‐regulation of nuclear factor‐κB activation in vitro. Moreover, we found that PF suppressed nuclear factor‐κB activation by decreasing its translocation to the nucleus in osteoclast precursor cells. Taken together, our new findings provide insights into a novel function of PF in osteoclastogenesis and demonstrate that PF would be a new therapeutic modality as a natural agent for RA treatment and other autoimmune conditions with bone erosion.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.     

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.     

Apparent diffusion coefficients in prostate cancer: correlation with molecular markers Ki‐67, HIF‐1α and VEGF

Prostate cancer (PCa) is the second most common cancer in men. The Gleason score (GS) and biomarkers play important roles in the diagnosis and treatment of patients with PCa. The purpose of this study was to investigate the relationship between the apparent diffusion coefficient (ADC) and the molecular markers Ki‐67, hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) in PCa. Thirty‐nine patients with 39 lesions, who had been diagnosed with PCa, were enrolled in this study. All patients underwent diffusion‐weighted magnetic resonance imaging (DW‐MRI) (b = 800 s/mm²). The expression of Ki‐67, HIF‐1α and VEGF was assessed by immunohistochemistry. Statistical analysis was applied to analyze the association between ADC and prostate‐specific antigen (PSA), GS and the expression of Ki‐67, HIF‐1α and VEGF. The group differences in ADC among different grades of Ki‐67, HIF‐1α and VEGF were also analyzed. The mean ± standard deviation of ADC was (0.76 ± 0.27) × 10^?3 mm²/s. ADC correlated negatively with PSA and GS (p < 0.05). The Ki‐67 staining index (SI), HIF‐1α expression and VEGF expression in PCa were correlated inversely with ADC, controlling for age (r = –0.332, p < 0.05; r = ?0.662, p < 0.0005; and r = ?0.714, p < 0.0005, respectively). ADC showed a significant difference among different grades of Ki‐67 (F = 9.164, p = 0.005), HIF‐1α (F = 40.333, p < 0.0005) and VEGF (F = 22.048, p < 0.0005). In conclusion, ADC was correlated with PSA, GS, and Ki‐67, HIF‐1α and VEGF expression in patients with PCa. ADC may be used to evaluate tumor proliferation, hypoxia and angiogenesis in PCa.     

HIF‐1α triggers long‐lasting glutamate excitotoxicity via system xc− in cerebral ischaemia–reperfusion

Hypoxia‐inducible factor 1α (HIF ‐1α) controls many genes involved in physiological and pathological processes. However, its roles in glutamatergic transmission and excitotoxicity are unclear. Here, we proposed that HIF ‐1α might contribute to glutamate‐mediated excitotoxicity during cerebral ischaemia–reperfusion (CIR ) and investigated its molecular mechanism. We showed that an HIF ‐1α conditional knockout mouse displayed an inhibition in CIR ‐induced elevation of extracellular glutamate and N ‐methyl‐d ‐aspartate receptor (NMDAR ) activation. By gene screening for glutamate transporters in cortical cells, we found that HIF ‐1α mainly regulates the cystine–glutamate transporter (system x_c^?) subunit xCT by directly binding to its promoter; xCT and its function are up‐regulated in the ischaemic brains of rodents and humans, and the effects lasted for several days. Genetic deletion of xCT in cortical cells of mice inhibits either oxygen glucose deprivation/reoxygenation (OGDR ) or CIR ‐mediated glutamate excitotoxicity in vitro and in vivo . Pharmaceutical inhibition of system x_c^? by a clinically approved anti‐cancer drug, sorafenib, improves infarct volume and functional outcome in rodents with CIR and its therapeutic window is at least 3 days. Taken together, these findings reveal that HIF ‐1α plays a role in CIR ‐induced glutamate excitotoxicity via the long‐lasting activation of system x_c^?‐dependent glutamate outflow and suggest that system x_c^? is a promising therapeutic target with an extended therapeutic window in stroke. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Effects of soluble cobalt and cobalt incorporated into calcium phosphate layers on osteoclast differentiation and activation

Metal ions originating from mechanical debris and corrosive wear of prosthetic implant alloys accumulate in peri-implant soft tissues, bone mineral, and body fluids. Eventually, metal ions such as cobalt (II) (Co²⁺), which is a major component of cobalt–chromium-based implant alloys and a known activator of osteolysis, are incorporated into the mineral phase of bone. We hypothesize that the accumulation of Co²⁺ in the mineral could directly activate osteolysis by targeting osteoclasts. To test this hypothesis, we coated tissue culture plastic with a thin layer of calcium phosphate (CaP) containing added traces of Co²⁺, thereby mimicking the bone mineral accumulation of Co²⁺. Murine bone marrow osteoclasts formed in the presence of M-CSF and RANKL were cultured on these surfaces to examine the effects of Co²⁺ on osteoclast formation and resorptive activity. Treatment conditions with Co²⁺ involved incorporation into the CaP layer, adsorption to the mineral surface, or addition to culture media. Micromolar concentrations of Co²⁺ delivered to developing osteoclast precursors by all 3 routes increased both osteoclast differentiation and resorptive function. Compared to CaP layers without Co²⁺, we observed a maximal 75% increase in osteoclast numbers and a 2.3- to 2.7-fold increase in mineral resorption from the tissue culture wells containing 0.1 μm Co²⁺ and 0.1–10 μm Co²⁺, respectively. These concentrations are well within the range found in peri-implant tissues in vivo. This direct effect of Co²⁺ on osteoclasts appears to act independently of the particulate phagocytosis/inflammation-mediated pathways, thus enhancing osteolysis and aseptic implant loosening.     

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine‐treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H₁‐ and H₂‐receptors. Cimetidine is a H₂‐receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100 mg kg^?1 of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate‐resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling) method and immunohistochemical reactions for detecting caspase‐3 and RANKL were performed. The number of TRAP‐positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL‐positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP‐positive osteoclasts with TUNEL‐positive nuclei and caspase‐3‐immunolabeled osteoclasts were found. A significant reduction in the number of TRAP‐positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL‐positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine‐induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.     

Multiple increased osteoclast functions in individuals with neurofibromatosis type 1

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human‐derived osteoclasts and compare the in vitro findings with the clinical phenotype. Functional characteristics (e.g., osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g., F‐actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N = 75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data. NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1‐derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes. While NF1 haploinsufficiency produces a generalized osteoclast gain‐in‐function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with NF1, additional and perhaps local modifiers are likely required for the development of skeletal abnormalities in NF1. © 2011 Wiley‐Liss, Inc.     

Human tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

Macrophages are commonly found within osteolytic secondary carcinomas in bone, but the manner in which these cells contribute to malignant bone resorption is uncertain. Macrophages isolated from primary breast carcinomas were co-cultured for up to 21 days with UMR 106 rat osteoblast-like cells on bone slices and glass coverslips in the presence and absence of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and human macrophage colony stimulating factor (M-CSF). Cell cultures were then assessed for the presence of phenotypic markers of macrophage and osteoclast differentiation. Isolated cells were negative for osteoclast markers including tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and the ability to carry our lacunar bone resorption, but were positive for CD11b and CD14, macrophage markers which are not present on osteoclasts. In 21-day co-cultures of breast carcinoma tumour-associated macrophages (TAMs) and UMR 106 cells, incubated in the presence of 1,25(OH)₂D₃ and M-CSF, numerous TRAP- and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed. Contact with UMR 106 cells and the presence of 1,25(OH)₂D₃ and M-CSF were absolute requirements for differentiation of human breast carcinoma TAMs into mature functional osteoclasts. TAM–osteoclast differentiation may represent an important cellular mechanism of osteolysis in metastatic skeletal carcinomas. © 1998 John Wiley & Sons, Ltd.     

Interleukin‐34 is expressed by giant cell tumours of bone and plays a key role in RANKL‐induced osteoclastogenesis

Interleukin‐34 (IL‐34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage‐colony stimulating factor receptor (M‐CSF receptor, c‐fms). M‐CSF, the main ligand for c‐fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M‐CSF in osteoclastogenesis and GCTs, the expression of IL‐34 in human GCTs was first assessed. Quantitative analysis of IL‐34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M‐CSF and c‐fms. Immunohistochemistry demonstrated that osteoclast‐like cells exhibited a huge immunostaining for IL‐34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone‐resorbing osteoclasts showed very strong staining for IL‐34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL‐34 in osteoclastogenesis was then studied in murine and human models. IL‐34 was able to support RANKL‐induced osteoclastogenesis in the absence of M‐CSF in all models. Multinucleated cells generated in the presence of IL‐34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL‐34 induced phosphorylation of ERK 1/2 and Akt through the activation of c‐fms, as revealed by the inhibition of signalling by a specific c‐fms tyrosine kinase inhibitor. Furthermore, IL‐34 stimulated RANKL‐induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL‐34 can be entirely substituted for M‐CSF in RANKL‐induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.