Improved joint health in subjects with severe haemophilia A treated prophylactically with recombinant factor VIII Fc fusion protein.

INTRODUCTION: Joint arthropathy is the long-term consequence of joint bleeding in people with severe haemophilia. AIM: This study assessed change in joint health over time in subjects receiving recombinant factor VIII Fc fusion protein (rFVIIIFc) prophylaxis. METHODS: ALONG is the phase 3 pivotal study in which the benefit of rFVIIIFc as a prophylactic treatment for bleeding control was shown in previously treated severe haemophilia patients ≥12 years of age (arm 1: 25-65 IU/kg every 3-5 days, arm 2: 65 IU/kg weekly and arm 3: episodic). After completing ALONG, subjects had the option to enrol into the extension study (ASPIRE). This interim, post hoc analysis assessed changes in joint health over ~2.8 years in these patients. RESULTS: Forty-seven subjects had modified Haemophilia Joint Health Score (mHJHS) data at A-LONG baseline, ASPIRE baseline and ASPIRE Year 1 and Year 2. Compared with A-LONG baseline (23.4), mean improvement at ASPIRE Year 2 was -4.1 (95% confidence interval [CI], -6.5, -1.8; P = .001). Regardless of prestudy treatment regimen, subjects showed continuous improvement in mHJHS from A-LONG baseline through ASPIRE Year 2 (prestudy prophylaxis: -2.4, P = .09; prestudy episodic treatment: -7.2, P = .003). Benefits were seen in subjects with target joints (-5.6, P = .005) as well as those with severe arthropathy (-8.8, P = .02). The mHJHS components with the greatest improvement at ASPIRE Year 2 were swelling (-1.4, P = .008), range of motion (-1.1, P = .03) and strength (-0.8, P = .04). CONCLUSIONS: Prophylaxis with rFVIIIFc may improve joint health over time regardless of prestudy prophylaxis or episodic treatment regimens.

Measurement of tumor load and distribution in a model of cancer-induced osteolysis: a necessary precaution when testing novel anti-resorptive therapies.

The Arguello model of cancer metastasis to bone has been used extensively to study breast cancer-induced osteolytic disease. The effects of therapy on skeletal disease and on tumour burden in soft organs are traditionally measured using radiography and/or time-consuming histomorphometry, respectively. The purpose of this study was to develop a sensitive and efficient method for evaluating tumour burden in vivo using MDA-231 cells transduced with the E. coli lacZ gene (MDA-231BAG). Osteolysis was measured by radiography and tumour burden was measured histomorphometrically or biochemically. In untreated mice, measurements of tumour burden in bone extracts using human cytokeratin-associated tissue polypeptide antigen (TPA) ELISA or E. coli beta-galactosidase (beta-gal) activity immunoassay reflected the extent of osteolytic disease as measured by radiography; however, tumour load could be detected before onset of osteolysis. When monitoring the effect of therapy (0.2 mg/kg ibandronate/day), radiography alone proved to be insufficient. Mice treated with the bisphosphonate ibandronate from time of inoculation with cancer cells had no radiologically visible signs of osteolysis but significant tumour load was measured in the bone extracts using these assays. Furthermore, beta-gal activity could be used as a measurement of tumour load in soft organs, and unlike other human breast cancer markers expressed by the MDA-231 cells in vitro, beta-gal activity was detected in the serum of mice with progressive disease. In conclusion, we describe an efficient model of breast cancer-induced osteolysis to quantify the effect of therapy on disease load and distribution, which could be beneficial in evaluating novel therapies for the treatment of the disease.

Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions.

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3-4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.

Human breast cancer cells induced angiogenesis, recruitment, and activation of osteoclasts in osteolytic metastasis.

PURPOSE: The purpose of this study was to elucidate the potential of human breast cancer cells (BCC) to induce matrix degradation and neo-vascularization, essential for continued tumor growth, in osteolytic lesions. METHODS: BCC were inoculated into the left cardiac ventricle of female athymic mice and osteolytic lesions were radiologically visualized within 4 weeks from inoculation. RESULTS: Histomorphometric analysis of bone sections revealed a significant increase in the number and maturity of osteoclasts (OCl) lining the bone surfaces next to tumor tissue when compared to corresponding bone surfaces in healthy mice. In addition, a large number of newly formed blood vessels could be visualized by immunohistochemistry at the periphery of and within tumor tissue. When bone marrow (BM) cells were cultured in the presence of BCC the OCl formation was increased threefold. These OCl were also found to be more mature and to have greater resorptive activity. Moreover, BCC were found to stimulate proliferation, migration, and differentiation of BM-derived endothelial cells. CONCLUSIONS: Matrix destruction and neo-vascularization are accomplished by BCC arrested in the BM cavity by increasing recruitment and activity of OCl and by induction of angiogenesis within or in proximity to the tumor tissue.

Proteinases in bone resorption: obvious and less obvious roles.

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Identification of breast cancer cell line-derived paracrine factors that stimulate osteoclast activity.

Metastatic breast cancer causes destruction of significant amounts of bone, and, although bone is the most likely site of breast cancer metastasis, little is understood about interactions between tumor cells and bone-resorbing osteoclasts. We have investigated the paracrine factors produced by breast cancer cells that are involved in increasing osteoclast activity. We have determined by immunoassay that the human breast cancer cell line MDA MB 231 (231) cultured in serum-free medium secretes transforming growth factors type beta(TGF-beta) 1 and 2, macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL) -1 and -6, tumor necrosis factor alpha (TNF-alpha), insulin-like growth factor II (IGF II), and parathyroid hormone-related peptide. To determine which of these are involved in increased bone destruction, we have fractionated serum-free 231-conditioned media and measured these fractions for effects on osteoclast resorption activity using multiple activity assays. The pattern of responses was complex. Several fractions stimulated osteoclast resorption either by increasing the number of osteoclasts binding to the bone or by elevating the resorption activity of the individual osteoclasts. Other fractions inhibited osteoclast activity. Analysis of active fractions for the factors identified in the 231-conditioned medium revealed that the presence of TNF-alpha and IGF-II was restricted to separate fractions that stimulated osteoclast resorption activity. The fractions that inhibited osteoclast resorption activity contained M-CSF, IL-6, TGF-beta2, and GM-CSF. No TGF-beta1 or IL-1 was detected in any of the active fractions. Our data support the hypothesis that breast cancer cells modulate osteoclast activity using multiple regulatory factors that increase both the number of mature osteoclasts attached to the bone and the bone resorption activity of these individual osteoclasts. Once it is understood how metastatic breast cancer elevates osteoclast-mediated bone loss, effective therapies to slow the progression and/or prevent this bone loss will become possible.

Pituitary adenylyl cyclase-activating polypeptides and vasoactive intestinal peptide inhibit bone resorption by isolated rabbit osteoclasts.

Nerve fibres present in the periosteum, cortical bone and bone marrow are proposed to be involved in the regulation of bone metabolism by the release of neuropeptides acting locally on bone cells. The present study describes the effects of vasoactive intestinal polypeptide (VIP), shown to be present in the vicinity of bone, and the two related neuropeptides pituitary adenylyl cyclase-activating polypeptide(1-38) (PACAP-38) and PACAP-27 on bone resorption in vitro induced by osteoclasts isolated from 10-day-old rabbits. Bone resorption was measured as the number and total area of pits formed by tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNCs) cultured for 3 days on devitalized slices of bovine cortical bone. All three neuropeptides had significant inhibitory effects on both the number and area of pits formed. At a high concentration (10(-7) M) the mean +/- S.E.M. reductions in the total area resorbed compared with controls were 70.3 +/- 8.2, 45.2 +/- 7.3 and 63.4 +/- 7.2% for PACAP-38, PACAP-27 and VIP, respectively. The corresponding values for the apparent dissociation constants were 0.93 +/- 0.30, 3.2 +/- 1.6 and 0.35 +/- 0.14 nM, respectively. The number of TRAP + MNCs was unaffected by application of neuropeptides. Autoradiography showed the presence of 125I-VIP binding sites on some stromal cells, whereas osteoclasts had no binding sites for 125I-VIP. A high number of 125I-calcitonin binding sites was demonstrated on osteoclasts in the same preparation. The signal transduction pathway remains to be fully elucidated but seems to be partly dependent on changes in intracellular calcium concentrations, since a number of stromal cells responded to application of 10(-8) M PACAP-38 or VIP, and at least partly independent of cAMP accumulation. Trifluoperazine and mellitin, two selective calmodulin inhibitors, failed to block the VIP-induced inhibition of bone resorption. Our results demonstrate a non-toxic and probably stromal cell-derived effect of PACAP-38, PACAP-27 and VIP on isolated rabbit osteoclasts, resulting in a potent inhibition of bone resorption in vitro. The signal transduction pathway for inhibition induced by PACAP-38, PACAP-27 and VIP may be mediated partly by changes in intracellular Ca2+ in stromal cells.

Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsiveness.

Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels.

Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay.

Difficulties in the geometrical definition and measurement of resorption pits is a major problem for the quantitative analysis of bone resorption by isolated osteoclasts cultured on bone or dentin substrates. In this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically quantifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or dentin. A consistently high correlation between the formation of resorption pits and the release of antigenic collagen fragments was observed for isolated rabbit osteoclasts seeded at various densities and cultured for various periods on bovine, elephant, and human substrates. In a further support of the osteoclastic nature of the collagenolytic effects, a high consistency between pit formation and collagenolysis was also observed when the rabbit bone cells were cultured in the presence of very differently acting but typical inhibitors of pit formation, i.e., the carbonic anhydrase inhibitor acetazolamide, the cysteine proteinase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisphosphonate ibandronate (BM 21.0955). In conclusion, the ELISA represents a simple, precise, and objective way to dynamically monitor bone resorption in vitro through quantification of the collagenolytic activity of isolated osteoclasts.

Desensitization and reactivation of ACh-regulated exocrine secretion in hen tracheal epithelium.

We have studied acetylcholine (ACh) desensitization of Cl- secretion and its reactivation by atropine in the hen tracheal epithelium employing voltage-clamp technique. Adding 4 microM ACh to the serosal side induces a maximal bumetanide-sensitive Cl- secretion. The ACh-induced Cl- secretion is desensitized nearly 100% at 512 microM ACh. The desensitization of Cl- secretion may be reduced 30-40% by 0.32 microM atropine (reactivation). From a kinetic model for desensitization and reactivation with two muscarinic receptors (mAChR), we have estimated the maximal secretory response, the apparent dissociation coefficients, and the apparent Hill coefficients for ACh and atropine at the two receptors. Based on the kinetic model we can predict an optimal fixed concentration ratio (Ofcor) between ACh and atropine that will maintain Cl- secretion above 40% of maximum over a wide concentration range, ACh 1-4,100 microM, and we have confirmed the predicted Ofcor experimentally. A cellular model is proposed with two mAChR, one stimulatory and another inhibitory, that accounts for an observed dissociation between desensitization in short-circuit current and conductance.

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea.

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl- equal to approximately 60-90 microA/cm2. 2. Radio-ligand-displacement for IP3, cAMP and cGMP and ion channel selective drugs in voltage clamp set-ups were employed to characterize second messengers and Cl-, K+ and Ca2+ channels involved in the ACh response. 3. ACh induced a significant rise in IP3 in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 microM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold. 4. Around 35% of ACh Cl- secretion depends on Ca2+ mobilization from internal stores and about 65% on Ca2+ influx over basolateral membrane. The activated Ca2+ channel is insensitive to class I, II, III and IV Ca2+ antagonists. 5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase. 6. Two K+ channels are involved in ACh secretion, one sensitive to Ba2+ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA. 7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl- channel is NPPB-sensitive.

Characterization of a muscarinic receptor controlling Cl- secretion in hen trachea.

We have attempted to characterize a muscarinic receptor subtype involved in Cl- secretion in isolated epithelium of hen trachea, taking advantage of drugs developed in the last 15 yr. Hen trachea can be stimulated to secrete Cl- equal to approximately 80-90 microA/cm2 by application of acetylcholine (ACh) to the serosal side. The process has an apparent dissociation constant (Kd) for ACh of 740 nM. The Cl- secretion is completely inhibited by 20 microM bumetanide at the serosal side. Of five selective antagonists for muscarinic receptors, pirenzepine, hexahydrosiladifenidol, dicyclomine, 11-([2-[(diethylamino)-methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, and 4 diphenyl acetoxy-N-methylpiperidine methobromide, only the latter had a high affinity for the functional receptor with an apparent Kd of 3 nM. The receptor may be classified as an M4-muscarinic receptor subtype and probably belongs to a group of muscarinic receptors on exocrine glands and mucosal cells involved in ion transport. All the functional responses caused by muscarinic agonists and antagonists tested exhibited exponents (apparent Hill coefficients) in the range from 1.3 to 2.4, indicating a gain in the stimulus secretion coupling mechanism, an aspect of muscarinic receptor function that is not revealed in radioligand binding studies.