Myeloperoxidase is a potential molecular imaging and therapeutic target for the identification and stabilization of high-risk atherosclerotic plaque.

Aims: As the inflammatory enzyme myeloperoxidase (MPO) is abundant in ruptured human atherosclerotic plaques, we aimed to investigate the role of MPO as a potential diagnostic and therapeutic target for high-risk plaque. Methods and results: We employed the tandem stenosis model of atherosclerotic plaque instability in apolipoprotein E gene knockout (Apoe-/-) mice. To test the role of MPO, we used Mpo-/-Apoe-/- mice and the 2-thioxanthine MPO inhibitor AZM198. In vivo MPO activity was assessed by liquid chromatography-tandem mass spectrometry detection of 2-chloroethidium generation from hydroethidine and by bis-5HT-DTPA-Gd (MPO-Gd) molecular magnetic resonance imaging (MRI), while plaque phenotype was verified histologically. Myeloperoxidase activity was two-fold greater in plaque with unstable compared with stable phenotype. Genetic deletion of MPO significantly increased fibrous cap thickness, and decreased plaque fibrin and haemosiderin content in plaque with unstable phenotype. AZM198 inhibited MPO activity and it also increased fibrous cap thickness and decreased fibrin and haemosiderin in plaque with unstable phenotype, without affecting lesion monocytes and red blood cell markers or circulating leukocytes and lipids. MPO-Gd MRI demonstrated sustained enhancement of plaque with unstable phenotype on T1-weighted imaging that was two-fold greater than stable plaque and was significantly attenuated by both AZM198 treatment and deletion of the Mpo gene. Conclusion: Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

Microscopic diffusion properties of fixed breast tissue: Preliminary findings.

PURPOSE: To investigate the microscopic diffusion properties of formalin-fixed breast tissue. METHODS: Diffusion microimaging was performed at 16.4T with 40-µm isotropic voxels on two normal and two cancer tissue samples from four patients. Results were correlated with histology of the samples. RESULTS: Diffusion-weighted images and mean diffusivity maps demonstrated distinct diffusivity differences between breast tissue components. Mean diffusivity (MD) in normal tissue was 0.59 ± 0.24 µm(2) /ms for gland lobule (voxels containing epithelium and intralobular stroma) and 1.23 ± 0.34 µm(2) /ms for interlobular fibrous stroma. In the cancer samples, MD = 0.45 ± 0.23 µm(2) /ms for invasive ductal carcinoma (voxels contain epithelium and intralobular stroma) and 0.61 ± 0.35 µm(2) /ms for ductal carcinoma in situ. There were significant MD differences between all tissue components (P < 0.005), except between gland lobule and ductal carcinoma in situ (P = 0.71). The low diffusivity of epithelium-rich cancer tissue and of normal epithelium relative to its supporting fibrous stroma was similar to that reported for prostate tissue and the esophageal wall. CONCLUSION: Diffusion microimaging demonstrates distinct diffusivity differences between breast tissue glandular structures. Low diffusivity may be a distinctive feature of mammalian epithelia.

Humanised xenograft models of bone metastasis revisited: novel insights into species-specific mechanisms of cancer cell osteotropism.

The determinants and key mechanisms of cancer cell osteotropism have not been identified, mainly due to the lack of reproducible animal models representing the biological, genetic and clinical features seen in humans. An ideal model should be capable of recapitulating as many steps of the metastatic cascade as possible, thus facilitating the development of prognostic markers and novel therapeutic strategies. Most animal models of bone metastasis still have to be derived experimentally as most syngeneic and transgeneic approaches do not provide a robust skeletal phenotype and do not recapitulate the biological processes seen in humans. The xenotransplantation of human cancer cells or tumour tissue into immunocompromised murine hosts provides the possibility to simulate early and late stages of the human disease. Human bone or tissue-engineered human bone constructs can be implanted into the animal to recapitulate more subtle, species-specific aspects of the mutual interaction between human cancer cells and the human bone microenvironment. Moreover, the replication of the entire "organ" bone makes it possible to analyse the interaction between cancer cells and the haematopoietic niche and to confer at least a partial human immunity to the murine host. This process of humanisation is facilitated by novel immunocompromised mouse strains that allow a high engraftment rate of human cells or tissue. These humanised xenograft models provide an important research tool to study human biological processes of bone metastasis.

Effect of formalin fixation on biexponential modeling of diffusion decay in prostate tissue.

PURPOSE: To evaluate the effect of formalin fixation on biexponential modeling of diffusion decay in prostate tissue. METHODS: Three whole prostate specimens were imaged unfixed immediately postsurgery, and again after formalin fixation. Diffusion-weighted imaging was performed over an extended range of b-values and a biexponential model fitted to the signal decay curves. RESULTS: Tissue fixation resulted in a 35%, 20%, and 20% reduction in mean apparent diffusion coefficient of the higher diffusivity fit component for the three organs, respectively, and a 64%, 57%, and 45% reduction in mean apparent diffusion coefficient of the lower diffusivity component. The mean signal fraction of the higher diffusivity component was increased by 23%, 5%, and 1%, respectively. The effect of fixation did not appear to vary according to tissue type or glandular zone. CONCLUSION: Formalin fixed tissue appears to provide a stable model for detailed investigation of the microscopic biophysical basis of diffusion phenomena observed in vivo. Diffusivity changes that result from fixation may provide information about the microscopic environments of the biexponential components.

The teratogenic effect of dofetilide during rat limb development and association with drug-induced bradycardia and hypoxia in the embryo.

BACKGROUND: Dofetilide is an antiarrhythmic drug that blocks the cardiac repolarizing current IKr ((IKr, rapid component of the delayed rectifying potassium current). Previous studies have shown that (a) IKr is essential for normal cardiac function of the embryonic heart and (b) dofetilide is teratogenic in rodents. This study was undertaken to examine the mechanism by which dofetilide causes limb defects on gestational day 13 (GD 13) in the rat. METHODS: Rats were treated with dofetilide (single oral dose, 5 mg/kg) on GD 13 and embryonic heart rates assessed by ultrasound (Vevo770, VisualSonics, Toronto, Ontario, Canada) 2 hr later. Fetuses were examined for malformations on GD 20. In a separate experiment, dofetilide treatment of GD 13 rats was followed 2, 4, 12, or 24 hr with iv dosing with the hypoxia marker, pimonidazole (60 mg/kg). Embryos were collected and heart rates were assessed in vitro and hypoxia in embryo limbs analyzed. RESULTS: A teratogenic dose of dofetilide at a susceptible stage of development (GD 13) resulted in a period of bradycardia and arrhythmia of the embryonic heart and hypoxia in the developing limbs (GD 13) resulting in limb malformations (GD 20). CONCLUSIONS: Drugs that induce periods of bradycardia and/or arrhythmia of the embryonic heart and cause the embryo to become hypoxic are potential human teratogens.

Will cell reprogramming resolve the embryonic stem cell controversy? A narrative review.

In the past few years, relatively straightforward laboratory techniques have been developed to reprogram normal body cells to enter an embryonic stem cell-like state. Not only do these induced pluripotent stem cells hold great medical promise--perhaps greater than that of embryonic stem cells--but they also have escaped the ethical controversy in which the latter is mired. This article examines how cell reprogramming is likely to transform regenerative and reproductive medicine and highlights some of the medical, moral, and political hurdles that it faces. It also argues that induced pluripotent stem cells are more ethically problematic than most people believe and that cell reprogramming will not solve the stem cell controversy but complicate it further.

Promises and challenges of stem cell research for regenerative medicine.

In recent years, stem cells have generated increasing excitement, with frequent claims that they are revolutionizing medicine. For those not directly involved in stem cell research, however, it can be difficult to separate fact from fiction or realistic expectation from wishful thinking. This article aims to provide internists with a clear and concise introduction to the field. While recounting some scientific and medical milestones, the authors discuss the 3 main varieties of stem cells-adult, embryonic, and induced pluripotent-comparing their advantages and disadvantages for clinical medicine. The authors have sought to avoid the moral and political debates surrounding stem cell research, focusing instead on scientific and medical issues.

Therapeutic inhibition of MPO stabilizes pre-existing high risk atherosclerotic plaque.

Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2+- and CD68+-cells and MPO protein. These findings demonstrate that inhibition of arterial MPO activity converts unstable into stable atherosclerotic lesions in a preclinical model of plaque instability and highlight the potential therapeutic potency of MPO inhibition for the management of high-risk patients and the development of novel protective strategies against cardiovascular diseases.

Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta.

Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta-gonad-mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5-E11.5 aorta-gonad-mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta-gonad-mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

Moving with the Times: The Health Science Alliance (HSA) Biobank, Pathway to Sustainability.

Human biobanks are recognised as vital components of translational research infrastructure. With the growth in personalised and precision medicine, and the associated expansion of biomarkers and novel therapeutics under development, it is critical that researchers can access a strong collection of patient biospecimens, annotated with clinical data. Biobanks globally are undertaking transformation of their operating models in response to changing research needs; transition from a 'classic' model representing a largely retrospective collection of pre-defined specimens to a more targeted, prospective collection model, although there remains a research need for both models to co-exist. Here we introduce the Health Science Alliance (HSA) Biobank, established in 2012 as a classic biobank, now transitioning to a hybrid operational model. Some of the past and current challenges encountered are discussed including clinical annotation, specimen utilisation and biobank sustainability, along with the measures the HSA Biobank is taking to address these challenges. We describe new directions being explored, going beyond traditional specimen collection into areas involving bioimages, microbiota and live cell culture. The HSA Biobank is working in collaboration with clinicians, pathologists and researchers, piloting a sustainable, robust platform with the potential to integrate future needs.

Synthesis and pharmacological evaluation of [18F]PBR316: a novel PET ligand targeting the translocator protein 18 kDa (TSPO) with low binding sensitivity to human single nucleotide polymorphism rs6971.

Radiopharmaceuticals that target the translocator protein 18 kDa (TSPO) have been investigated with positron emission tomography (PET) to study neuroinflammation, neurodegeneration and cancer. We have developed the novel, achiral, 2-phenylimidazo[1,2-a]pyridine, PBR316 that targets the translocator protein 18 kDa (TSPO) that addresses some of the limitations inherent in current TSPO ligands; namely specificity in binding, blood brain barrier permeability, metabolism and insensitivity to TSPO binding in subjects as a result of rs6971 polymorphism. PBR316 has high nanomolar affinity (4.7-6.0 nM) for the TSPO, >5000 nM for the central benzodiazepine receptor (CBR) and low sensitivity to rs6971 polymorphism with a low affinity binders (LABs) to high affinity binders (HABs) ratio of 1.5. [18F]PBR316 was prepared in 20 ± 5% radiochemical yield, >99% radiochemical purity and a molar activity of 160-400 GBq µmol-1. Biodistribution in rats showed high uptake of [18F]PBR316 in organs known to express TSPO such as heart (3.9%) and adrenal glands (7.5% ID per g) at 1 h. [18F]PBR316 entered the brain and accumulated in TSPO-expressing regions with an olfactory bulb to brain ratio of 3 at 15 min and 7 at 4 h. Radioactivity was blocked by PK11195 and Ro 5-4864 but not Flumazenil. Metabolite analysis showed that radioactivity in adrenal glands and the brain was predominantly due to the intact radiotracer. PET-CT studies in mouse-bearing prostate tumour xenografts indicated biodistribution similar to rats with radioactivity in the tumour increasing with time. [18F]PBR316 shows in vitro binding that is insensitive to human polymorphism and has specific and selective in vivo binding to the TSPO. [18F]PBR316 is suitable for further biological and clinical studies.

Induction of muscle-regenerative multipotent stem cells from human adipocytes by PDGF-AB and 5-azacytidine.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.

Development of Th1 imprints to rBCG expressing a foreign protein: implications for vaccination against HIV-1 and diverse influenza strains.

We demonstrate here that immunizing naïve mice with low numbers of recombinant Bacille Calmette-Guérin (rBCG) expressing beta-galactosidase (beta-gal) generates predominant Th1 responses to both BCG and beta-gal whereas infection with high numbers generates a mixed Th1/Th2 response to both BCG and beta-gal. Furthermore, the Th1 response to both BCG and beta-gal is stable when mice, pre-exposed to low numbers of rBCG, are challenged four months later with high numbers of rBCG. Thus the Th1/Th2 phenotypes of the immune responses to beta-gal and to BCG are "coherently" regulated. Such rBCG vectors, encoding antigens of pathogens preferentially susceptible to cell-mediated attack, may be useful in vaccinating against such pathogens. We discuss vaccination strategies employing rBCG vectors that are designed to provide protection against diverse influenza strains or numerous variants of HIV-1 and consider what further experiments are essential to explore the possibility of realizing such strategies.

Inhibition of micrometastatic prostate cancer cell spread in animal models by 213Bilabeled multiple targeted alpha radioimmunoconjugates.

PURPOSE: To investigate the therapeutic potential of 213Bilabeled multiple targeted alpha-radioimmunoconjugates for treating prostate cancer (CaP) micrometastases in mouse models. EXPERIMENTAL DESIGN: PC-3 CaP cells were implanted s.c., in the prostate, and intratibially in NODSCID mice. The expression of multiple tumor-associated antigens on tumor xenografts and micrometastases was detected by immunohistochemistry. Targeting vectors were two monoclonal antibodies, and a plasminogen activator inhibitor type 2 that binds to cell surface urokinase plasminogen activator, labeled with 213Bi using standard methodology. In vivo efficacy of multiple alpha conjugates (MTAT) at different activities was evaluated in these mouse models. Tumor growth was monitored during observations and local regional lymph node metastases were assessed at the end of experiments. RESULTS: The take rate of PC-3 cells was 100% for each route of injection. The tumor-associated antigens (MUC1, urokinase plasminogen activator, and BLCA-38) were heterogeneously expressed on primary tumors and metastatic cancer clusters at transit. A single i.p. injection of MTAT (test) at high and low doses caused regression of the growth of primary tumors and prevented local lymph node metastases in a concentration-dependent fashion; it also caused cancer cells to undergo necrosis and apoptosis. CONCLUSIONS: Our results suggest that MTAT can impede primary PC-3 CaP growth at three different sites in vivo through induction of apoptosis, and can prevent the spread of cancer cells and target lymph node micrometastases in a concentration-dependent manner. MTAT, by targeting multiple antigens, can overcome heterogeneous antigen expression to kill small CaP cell clusters, thus providing a potent therapy for micrometastases.

A novel model of bone-metastatic prostate cancer in immunocompetent mice.

BACKGROUND: Bone metastasis is a frequent and catastrophic consequence of prostate cancer for which only palliative treatment is available. Animal models of bone metastatic prostate cancer are necessary for understanding disease mechanisms but few models exist. METHODS: We have used the murine prostate carcinoma cell line RM1 to generate a bone metastatic model of prostate cancer. Repeated intracardiac injection of RM1 cells followed by isolation of cells from bone tumors has yielded a cell line with strong bone-metastatic potential, RM1 bone metastatic (BM). RESULTS: This cell line metastasizes to multiple bony sites in over 95% of injected C57BL/6 mice and is far less tropic to soft tissues. Bone tumors produced by the RM1(BM) cell line show no preference for particular skeletal sites as most bones are affected. Histology, and micro-computed tomography show that RM1(BM) cells form osteolytic tumors, but with evidence of osteoblastic changes. In vitro the RM1 cells express E-cadherin but not vimentin, do not form colonies in soft agar, are non-invasive but are more motile than the parent cell line. CONCLUSIONS: This model provides a novel means for identifying cellular and molecular mechanisms that contribute to bone metastasis and allow for preclinical testing of therapies to prevent and treat tumor metastasis to bone. Finally as the syngeneic tumor cells are injected into immunocompetent mice, this model will provide a means to study interactions between the immune system, tumors and bone, and therapies that target such interactions.

Whither prometheus' liver? Greek myth and the science of regeneration.

Stem-cell biologists and those involved in regenerative medicine are fascinated by the story of Prometheus, the Greek god whose immortal liver was feasted on day after day by Zeus' eagle. This myth invariably provokes the question: Did the ancient Greeks know about the liver's amazing capacity for self-repair? The authors address this question by exploring the origins of Greek myth and medicine, adopting a 2-fold strategy. First, the authors consider what opportunities the ancient Greeks had to learn about the liver's structure and function. This involves a discussion of early battlefield surgery, the beginnings of anatomical research, and the ancient art of liver augury. In addition, the authors consider how the Greeks understood Prometheus' immortal liver. Not only do the authors examine the general theme of regeneration in Greek mythology, they survey several scholarly interpretations of Prometheus' torture.