Sostdc1: A soluble BMP and Wnt antagonist that is induced by the interaction between myeloma cells and osteoblast lineage cells.

Multiple myeloma (MM) is characterised by destructive lytic bone disease, caused by induction of bone resorption and impaired bone formation. Our understanding of the molecular mechanisms responsible for osteoblast suppression, are limited. Using the 5T2MM murine model of MM we have previously shown that suppression of the activity of a known inhibitor of bone formation Dikkopf-1 (Dkk1) prevents the development of lytic bone disease. Here we have demonstrated that another potential inhibitor of bone formation, sclerostin domain containing 1 (Sostdc1) is expressed at low levels in MM and osteoblast lineage cells when these cells are grown separately in cell culture but its expression is significantly induced in both cell types when these cells are in contact. The distribution of Sostdc1 staining in bones infiltrated with 5TGM1 myeloma cells in vivo suggested its presence in both myeloma and osteoblast lineage populations when in close proximity. We have also shown that recombinant Sostdc1 inhibits both bone morphogenic proteins (BMP2 and 7) and Wnt signalling in primary osteoblasts and suppresses differentiation of these cells. Together, these findings suggest that Sostdc1 expression in 5TGM1-infiltrated bones as a result of the interaction between myeloma and osteoblast lineage populations, could result in suppression of osteoblast differentiation.

Osteolytica: An automated image analysis software package that rapidly measures cancer-induced osteolytic lesions in in vivo models with greater reproducibility compared to other commonly used methods.

Methods currently used to analyse osteolytic lesions caused by malignancies such as multiple myeloma and metastatic breast cancer vary from basic 2-D X-ray analysis to 2-D images of micro-CT datasets analysed with non-specialised image software such as ImageJ. However, these methods have significant limitations. They do not capture 3-D data, they are time-consuming and they often suffer from inter-user variability. We therefore sought to develop a rapid and reproducible method to analyse 3-D osteolytic lesions in mice with cancer-induced bone disease. To this end, we have developed Osteolytica, an image analysis software method featuring an easy to use, step-by-step interface to measure lytic bone lesions. Osteolytica utilises novel graphics card acceleration (parallel computing) and 3-D rendering to provide rapid reconstruction and analysis of osteolytic lesions. To evaluate the use of Osteolytica we analysed tibial micro-CT datasets from murine models of cancer-induced bone disease and compared the results to those obtained using a standard ImageJ analysis method. Firstly, to assess inter-user variability we deployed four independent researchers to analyse tibial datasets from the U266-NSG murine model of myeloma. Using ImageJ, inter-user variability between the bones was substantial (±19.6%), in contrast to using Osteolytica, which demonstrated minimal variability (±0.5%). Secondly, tibial datasets from U266-bearing NSG mice or BALB/c mice injected with the metastatic breast cancer cell line 4T1 were compared to tibial datasets from aged and sex-matched non-tumour control mice. Analyses by both Osteolytica and ImageJ showed significant increases in bone lesion area in tumour-bearing mice compared to control mice. These results confirm that Osteolytica performs as well as the current 2-D ImageJ osteolytic lesion analysis method. However, Osteolytica is advantageous in that it analyses over the entirety of the bone volume (as opposed to selected 2-D images), it is a more rapid method and it has less user variability.

A review of current murine models of multiple myeloma used to assess the efficacy of therapeutic agents on tumour growth and bone disease.

Pre-clinical in vivo models of multiple myeloma are essential tools for investigating the pathophysiology of multiple myeloma and for testing new therapeutic agents and strategies prior to their potential use in clinical trials. Over the last five decades, several different types of murine models of multiple myeloma have been developed ranging from immunocompetent syngeneic models, e.g. the 5 T series of myeloma cells, to immunocompromised models including the SCID xenograft models, which use human myeloma cell lines or patient-derived cells. Other models include hybrid models featuring the implantation of SCID mice with bone chips (SCID-hu or SCID-rab) or 3-D bone scaffolds (SCID-synth-hu), and mice that have been genetically engineered to develop myeloma. Bearing in mind the differences in these models, it is not surprising that they reflect to varying degrees different aspects of myeloma. Here we review the past and present murine models of myeloma, with particular emphasis on their advantages and limitations, characteristics, and their use in testing therapeutic agents to treat myeloma tumour burden and bone disease.

Bisphosphonate therapy in the treatment of multiple myeloma.

Multiple myeloma is an incurable B cell neoplasm caused by the monoclonal expansion of malignant plasma cells in the bone marrow, often resulting in devastating bone disease. For over 2 decades bisphosphonates have been successfully used to treat the tumour-induced bone disease associated with multiple myeloma. This review will focus on preclinical studies and investigations in patients with multiple myeloma that have led to our current understanding of the mechanisms of action of bisphosphonates in myeloma bone disease. Major advances in the use of bisphosphonates, including findings that they may have additional benefits such as anti-tumour effects and promoting patient survival will be discussed.

Targeting RANK/RANKL in the treatment of solid tumours and myeloma.

Cancers which damage the human skeleton include multiple myeloma, where the primary tumour colonises bone directly, or breast and prostate cancer, where malignant cells travel from the primary tumour to form clonal outgrowths within the bone. Owing to the interaction of tumour cells with those normally found in the bone microenvironment, such as osteoclasts and osteoblasts, these cancers affect the closely linked processes of bone formation and resorption. As a result, these twin processes contribute to the clinical manifestations of cancer metastasis, including bone pain and pathological fractures. A critical component of physiologically normal bone remodelling, the RANK/RANKL/OPG pathway, has been implicated in the formation of osteolytic, and possibly osteoblastic, lesions, which characterise the bone disease associated with these malignancies. In these cancers that affect the skeleton in this way the abnormally regulated RANK/RANKL system appears to be the final effector pathway. As a result, there has been much research focused upon targeting these molecules using OPG constructs, peptidomimetics, soluble receptor constructs and antibodies to RANKL, in pre-clinical studies. The success of these studies has paved the way for a clinical programme, the success of which is likely to lead to a new therapeutic approach to treating cancers that develop in the skeleton.

Differences between bisphosphonates in binding affinities for hydroxyapatite.

Bisphosphonates (BPs) inhibit bone resorption and are widely used for the treatment of bone diseases, including osteoporosis. BPs are also being studied for their effects on hydroxyapatite (HAP)-containing biomaterials. There is a growing appreciation that there are hitherto unexpected differences among BPs in their mineral binding affinities that affect their pharmacological and biological properties. To study these differences, we have developed a method based on fast performance liquid chromatography using columns of HAP to which BPs and other phosphate-containing compounds can adsorb and be eluted by using phosphate buffer gradients at pH 6.8. The individual compounds emerge as discrete and reproducible peaks for a range of compounds with different affinities. For example, the peak retention times (min; mean +/- SEM) were 22.0 +/- 0.3 for zoledronate, 16.16 +/- 0.44 for risedronate, and 9.0 +/- 0.28 for its phosphonocarboxylate analog, NE10790. These results suggest that there are substantial differences among BPs in their binding to HAP. These differences may be exploited in the development of biomaterials and may also partly explain the extent of their relative skeletal retention and persistence of biological effects observed in both animal and clinical studies.

CSF concentrations of brain tryptophan and kynurenines during immune stimulation with IFN-alpha: relationship to CNS immune responses and depression.

Cytokine-induced activation of indoleamine 2,3-dioxygenase (IDO) catabolizes L-tryptophan (TRP) into L-kynurenine (KYN), which is metabolized to quinolinic acid (QUIN) and kynurenic acid (KA). QUIN and KA are neuroactive and may contribute to the behavioral changes experienced by some patients during exposure to inflammatory stimuli such as interferon (IFN)-alpha. A relationship between depressive symptoms and peripheral blood TRP, KYN and KA during treatment with IFN-alpha has been described. However, whether peripheral blood changes in these IDO catabolites are manifest in the brain and whether they are related to central nervous system cytokine responses and/or behavior is unknown. Accordingly, TRP, KYN, QUIN and KA were measured in cerebrospinal fluid (CSF) and blood along with CSF concentrations of relevant cytokines, chemokines and soluble cytokine receptors in 27 patients with hepatitis C after approximately 12 weeks of either treatment with IFN-alpha (n=16) or no treatment (n=11). Depressive symptoms were assessed using the Montgomery-Asberg Depression Rating Scale. IFN-alpha significantly increased peripheral blood KYN, which was accompanied by marked increases in CSF KYN. Increased CSF KYN was in turn associated with significant increases in CSF QUIN and KA. Despite significant decreases in peripheral blood TRP, IFN-alpha had no effect on CSF TRP concentrations. Increases in CSF KYN and QUIN were correlated with increased CSF IFN-alpha, soluble tumor necrosis factor-alpha receptor 2 and monocyte chemoattractant protein-1 as well as increased depressive symptoms. In conclusion, peripheral administration of IFN-alpha activated IDO in concert with central cytokine responses, resulting in increased brain KYN and QUIN, which correlated with depressive symptoms.

Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice.

Although elevated activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) has been proposed to mediate comorbid depression in inflammatory disorders, its causative role has never been tested. We report that peripheral administration of lipopolysaccharide (LPS) activates IDO and culminates in a distinct depressive-like behavioral syndrome, measured by increased duration of immobility in both the forced-swim and tail suspension tests. Blockade of IDO activation either indirectly with the anti-inflammatory tetracycline derivative minocycline, that attenuates LPS-induced expression of proinflammatory cytokines, or directly with the IDO antagonist 1-methyltryptophan (1-MT), prevents development of depressive-like behavior. Both minocycline and 1-MT normalize the kynurenine/tryptophan ratio in the plasma and brain of LPS-treated mice without changing the LPS-induced increase in turnover of brain serotonin. Administration of L-kynurenine, a metabolite of tryptophan that is generated by IDO, to naive mice dose dependently induces depressive-like behavior. These results implicate IDO as a critical molecular mediator of inflammation-induced depressive-like behavior, probably through the catabolism of tryptophan along the kynurenine pathway.

Transmission of two Australian strains of murine cytomegalovirus (MCMV) in enclosure populations of house mice (Mus domesticus).

To control plagues of free-living mice (Mus domesticus) in Australia, a recombinant murine cytomegalovirus (MCMV) expressing fertility proteins is being developed as an immunocontraceptive agent. Real-time quantitative PCR was used to monitor the transmission of two genetically variable field strains of MCMV through mouse populations after 25% of founding mice were infected with the N1 strain, followed by the G4 strain 6 weeks later. Pathogen-free wild-derived mice were released into outdoor enclosures located in northwestern Victoria (Australia). Of those mice not originally inoculated with virus, N1 DNA was detected in more than 80% of founder mice and a third of their offspring and similarly, G4 DNA was detected in 13% of founder mice and in 3% of their offspring. Thus, prior immunity to N1 did not prevent transmission of G4. This result is promising for successful transmission of an immunocontraceptive vaccine through Australian mouse populations where MCMV infection is endemic.

Species-specificity of a murine immunocontraceptive utilising murine cytomegalovirus as a gene delivery vector.

Cytomegaloviruses are species-specific DNA viruses. Recombinant murine cytomegaloviruse (MCMV) expressing the mouse egg-coat protein zona pellucida 3 (mZP3) has been shown to sterilise female mice by breaking self-tolerance and inducing an immune response against the host ZP3. This virus has the potential to be used for mouse population control, however the effect of this recombinant immunocontraceptive virus in non-host species must be determined. Recombinant MCMV-mZP3, based on both laboratory and wild strains of virus, induced long-lived antibody responses against structural viral proteins and mZP3 when inoculated into laboratory rats, although no viral DNA or replicating virus was identified. The anti-mZP3 antibodies were specific for mouse ZP3, did not cross-react with rat ZP3, and had no effect on the fertility of the rats.

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels.

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.

Isolates of cytomegalovirus (CMV) from the black rat Rattus rattus form a distinct group of rat CMV.

Two different betaherpesviruses, the English and Maastricht species of rat cytomegalovirus (CMV), have previously been isolated from Rattus norvegicus. CMVs were isolated from both the brown rat, R. norvegicus, and the black rat, R. rattus, within Australia. The viruses isolated from R. norvegicus appeared to be genetically related to the English species of rat CMV by PCR, RFLP, and sequencing, but the viruses isolated from R. rattus were distinct from both prototype virus species, although more closely genetically related to the Maastricht virus. This is the first genetic characterization of cytomegaloviruses from R. rattus, and the first isolation of CMVs from Australian rats.

Effect of added µ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture.

The effects of µ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, µ-calpain became evenly distributed throughout the fibre. µ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that µ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2 °C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 µM Ca(2+) were less stiff than fibres incubated in 200 µM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.

Pathogens of house mice on arid Boullanger Island and subantarctic Macquarie Island, Australia.

Studies on island populations of house mice (Mus domesticus) and their viruses reveal insights into viral persistence in isolated communities. We surveyed the ectoparasites, endoparasites, and antiviral antibodies for 11 murine viruses and two bacteria of house mice inhabiting two islands off Australia. House mice on Boullanger Island were seropositive to two viruses, murine cytomegalovirus and epizootic diarrhea of infant mice. On subantarctic Macquarie Island, house mice were seropositive for five viruses: murine cytomegalovirus, lymphocytic choriomeningitis virus, mouse parvovirus, epizootic diarrhea of infant mice, and Theiler's murine encephalomyelitis virus. The diversity of antiviral antibodies was lower among populations of house mice on islands than those inhabiting mainland Australia. The decreased diversity of viruses in island populations of house mice may be a function of which agent the founder mice transfer to the island and related to the low densities which the host population may periodically reach over time.

Ecological basis for fertility control in the house mouse (Mus domesticus) using immunocontraceptive vaccines.

Laboratory studies confirm the potential for fertility control in the house mouse Mus domesticus using mouse cytomegalovirus (MCMV) as a vector for an immunocontraceptive vaccine. This article presents an overview of key results from research in Australia on enclosed and field populations of mice and the associated epidemiology of MCMV. The virus is geographically widespread in Australia. It also persists in low population densities of mice, although if population densities are low for at least a year, transmission of the virus is sporadic until a population threshold of approximately 40 mice ha(-1) is reached. The serological prevalence of MCMV was high early in the breeding season of four field populations. Enclosure studies confirm that MCMV has minimal impact on the survival and breeding performance of mice and that it can be transmitted to most adults within 10-12 weeks. Other enclosure studies indicate that about two-thirds of females would need to be sterilized to provide effective control of the rate of growth of mouse populations. If this level is not maintained for 20-25 weeks after the commencement of breeding, the mouse population can compensate through increased recruitment per breeding female. The findings from this series of descriptive and manipulative population studies of mice support the contention that MCMV would be a good carrier for an immunocontraceptive vaccine required to sustain female sterility levels at or above 65%.

Androgen responsiveness of the pituitary gonadotrope cell line LbetaT2.

Androgens have a profound effect on the hypothalamic-pituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, LbetaT2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the LbetaT2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7alpha-methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in LbetaT2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the LbetaT2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.

Development of components of the extracellular matrix, basal lamina and sarcomere in chick quadriceps and pectoralis muscles.

1. The purpose of this study was to investigate differences in the development of components of the cell/matrix linkage in two functionally different muscle types: the pectoralis muscle, a major locomotory muscle in birds but not particularly functional in chickens, and the quadriceps muscle, a smaller and more functionally active muscle in the chicken. 2. The development of the extracellular matrix, basal lamina and sarcomere in the pectoralis and quadriceps muscles in chick embryos was examined biochemically to determine differences in the rate of development between these two muscles. Samples of these muscle types were dissected out from chick embryos from embryonic day 10 until 8 weeks post hatch. 3. Using SDS-PAGE electrophoresis and western blotting with antibodies against sarcomeric actin, laminin and collagens I, III and IV, it was apparent that muscle development begins earlier in the quadriceps muscle than in the pectoralis, and that late in the developmental process (d 18) both muscle types were well differentiated. The final concentration of collagens in the mature muscle remained higher in the quadriceps than in the pectoralis muscle. 4. The onset of development of the extracellular matrix, basal lamina and sarcomere was earlier in the quadriceps than the pectoralis, which could have functional implications for these muscles as a whole.

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone.

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.