Circulating Tumor Cells: The Importance of Single Cell Analysis.

Cancer cells that have shed from the primary tumor are able to invade into surrounding tissues, to intravasate into the bloodstream to become circulating tumor cells (CTCs), at least one part of that cells will be able to generate distant metastases. The discovery of CTCs has improved the study of cancer disease as it represents a non invasive biopsy that can be used as prognostic and prediction biomarkers. Tumour heterogeneity is a concept related to differences in tumor cells within the same tumor or between tumours in terms of genetic and phenotypic profiles, such as morphology, metabolic activity, proliferation rate, migration and metastatic abilities. Characterization of heterogeneity among CTCs at the single cell level may be useful to better understand the causes and progression of disease and for an accurate selection of molecular prognostic/prediction markers. In this chapter we aimed to describe methods for CTC enrichment and isolation as well as current methodologies for single cell analysis at different levels, including RNA, DNA, protein and epigenetic events. Finally we wanted to stress clinical and biological importance of single CTC analysis by reviewing some studies carried out in different cancer subtypes.

Bone Like Arterial Calcification in Femoral Atherosclerotic Lesions: Prevalence and Role of Osteoprotegerin and Pericytes.

OBJECTIVE/BACKGROUND: Arterial calcification, a process that mimics bone formation, is an independent risk factor of cardiovascular morbidity and mortality, and has a significant impact on surgical and endovascular procedures and outcomes. Research efforts have focused mainly on the coronary arteries, while data regarding the femoral territory remain scarce. METHODS: Femoral endarterectomy specimens, clinical data, and plasma from a cohort of patients were collected prospectively. Histological analysis was performed to characterize the cellular populations present in the atherosclerotic lesions, and that were potentially involved in the formation of bone like arterial calcification known as osteoid metaplasia (OM). Enzyme linked immunosorbent assays and cell culture assays were conducted in order to understand the cellular and molecular mechanisms underlying the formation of OM in the lesions. RESULTS: Twenty-eight of the 43 femoral plaques (65%) displayed OM. OM included osteoblast and osteoclast like cells, but very few of the latter exhibited the functional ability to resorb mineral tissue. As in bone, osteoprotegerin (OPG) was significantly associated with the presence of OM (p = .04). Likewise, a high plasma OPG/receptor activator for the nuclear factor kappa B ligand (RANKL) ratio was significantly associated with the presence of OM (p = .03). At the cellular level, there was a greater presence of pericytes in OM+ compared with OM- lesions (5.59 ± 1.09 vs. 2.42 ± 0.58, percentage of area staining [region of interest]; p = .04); in vitro, pericytes were able to inhibit the osteoblastic differentiation of human mesenchymal stem cells, suggesting that they are involved in regulating arterial calcification. CONCLUSION: These results suggest that bone like arterial calcification (OM) is highly prevalent at femoral level. Pericyte cells and the OPG/RANK/RANKL triad seem to be critical to the formation of this ectopic osteoid tissue and represent interesting potential therapeutic targets to reduce the clinical impact of arterial calcification.

Joint bleeding in factor VIII deficient mice causes an acute loss of trabecular bone and calcification of joint soft tissues which is prevented with aggressive factor replacement.

While chronic degenerative arthropathy is the main morbidity of haemophilia, a very high prevalence of low bone density is also seen in men and boys with haemophilia. This study investigates bone degradation in the knee joint of haemophilic mice resulting from haemarthrosis and the efficacy of aggressive treatment with factor VIII in the period surrounding injury to prevent bone pathology. Skeletally mature factor VIII knock-out mice were subjected to knee joint haemorrhage induced by puncture of the left knee joint capsule. Mice received either intravenous factor VIII treatment or placebo immediately prior to injury and at hours 4, 24, 48, 72 and 96 after haemorrhage. Mice were killed 2-weeks after injury and the joint morphology and loss of bone in the proximal tibia was assessed using microCT imaging. Quantitative microCT imaging of the knee joint found acute bone loss at the proximal tibia following injury including loss of trabecular bone volumetric density and bone mineral density, as well as trabecular connectivity density, number and thickness. Unexpectedly, joint injury also resulted in calcification of the joint soft tissues including the tendons, ligaments, menisci and cartilage. Treatment with factor VIII prevented this bone and soft tissue degeneration. Knee joint haemorrhage resulted in acute changes in adjacent bone including loss of bone density and mineralization of joint soft tissues. The rapid calcification and loss of bone has implications for the initiation and progression of osteoarthritic degradation following joint bleeding.

Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients.

OBJECTIVES: Interleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1ß. Wild-type, jnk1(-/-)-jnk2(-/-) and nemo(-/-) murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect. RESULTS: IL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1ß stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway. CONCLUSION: This work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1ß, may contribute to inflammation and bone erosions in RA.

Development of a per-operative procedure for concentrated bone marrow adjunction in postero-lateral lumbar fusion: radiological, biological and clinical assessment.

PURPOSE: Addition of bone marrow to the bone graft in the postero-lateral lumbar arthrodesis is a widely used technique. Bone marrow brings stem cells and growth factors contained in the platelets, favorable for bone growth. Adjunction of concentrated bone marrow should create better conditions and may increase bone growth. METHODS: Simple blind randomized clinical, prospective, monocentric trial was conducted. Fifteen patients underwent lumbar arthrodesis. During surgery, a fraction of the bone marrow harvested was centrifuged. One side received this concentrate with autologous bone and ceramics; the other side received the same graft with unconcentrated bone marrow. A quantitative study, realised with a volume calculating software on CT-scan images, determined the cortical bone volume in the graft post-operatively and at 3 months. The osteoprogenitor cells, nucleated cells and platelet concentrations were determined. RESULTS: The biological study found an average concentration of six times for the nucleated cells, 3.5 times for the platelets and 2.2 times for the osteoprogenitor cells. The comparison of the mean cortical bone volumes post-operatively and at 3 months was not significantly different. CONCLUSIONS: Despite the concentration obtained, there was no increase of bone growth by adding concentrated bone marrow. However, the number of stem cells in bone marrow was low and maybe a stronger concentration is needed to obtain a difference. The 3D reconstruction of the graft and the analysis of the graft's volume using a novel software was efficient according to the similarity of the graft's volume post-operatively in all patients.

Effects of a sulfated exopolysaccharide produced by Altermonas infernus on bone biology.

The growth and differentiation of bone cells is controlled by various factors, which can be modulated by heparan sulfates. Here, we investigated the effects of an oversulfated exopolysaccharide (OS-EPS) on the bone. We compared the effect of this compound with that of a native EPS. Long-term administration of OS-EPS causes cancellous bone loss in mice due, in part, to an increase in the number of osteoclasts lining the trabecular bone surface. No significant difference in cancellous bone volume was found between EPS-treated mice and age-matched control mice, underlying the importance of sulfation in trabecular bone loss. However, the mechanism sustaining this osteoporosis was unclear. To clarify OS-EPS activities, we investigated the effect of OS-EPS on osteogenesis. Our results demonstrated that OS-EPS inhibited osteoclastogenesis in two cell models. Using the surface plasmon resonance technique, we revealed that OS-EPS can form a hetero-molecular complex OS-EPS/receptor activator of NF-κB ligand (RANKL)/RANK and that RANK had a higher affinity for RANKL pre-incubated with OS-EPS than for RANKL alone, which would be in favor of an increase in bone resorption. However, in vitro, OS-EPS inhibited the early steps of osteoclast precursor adhesion and therefore inhibited the cell fusion step. In addition, we showed that OS-EPS reduced proliferation and accelerated osteoblastic differentiation, leading to strong inhibition of mineralized nodule formation, which would be in favor of an increase in bone resorption. Taken together, these data show different levels of bone resorption regulation by EPSs, most of them leading to proresorptive effects.

IL-33 is expressed in human osteoblasts, but has no direct effect on bone remodeling.

The aim of the present study was to investigate the potential role of the recently discovered IL-1 family member IL-33 in bone remodeling. Our results indicate that IL-33 mRNA is expressed in osteocytes in non-inflammatory human bone. Moreover, IL-33 levels are increased by TNF-α and IL-1ß in human bone marrow stromal cells, osteoblasts and adipocytes obtained from three healthy donors. Experiments with the inhibitor GW-9662 suggested that expression of IL-33, in contrast to that of IL-1ß, is not repressed by PPARγ likely explaining why IL-33, but not IL-1ß, is expressed in adipocytes. The IL-33 receptor ST2L is not constitutively expressed in human bone marrow stromal cells, osteoblasts or CD14-positive monocytes, and IL-33 has no effect on these cells. In addition, although ST2L mRNA is induced by TNF-α and IL-1ß in bone marrow stromal cells, IL-33 has the same effects as TNF-α and IL-1ß, and, therefore, the biological activity of IL-33 may be redundant in this system. In agreement with this hypothesis, MC3T3-E1 osteoblast-like cells constitutively express ST2L mRNA, and IL-33 and TNF-α/IL-1ß similarly decrease osteocalcin RNA levels in these cells. In conclusion, our results suggest that IL-33 has no direct effects on normal bone remodeling.

The Bone Niche of Chondrosarcoma: A Sanctuary for Drug Resistance, Tumour Growth and also a Source of New Therapeutic Targets.

Chondrosarcomas are malignant cartilage-forming tumours representing around 20% of malignant primary tumours of bone and affect mainly adults in the third to sixth decade of life. Unfortunately, the molecular pathways controlling the genesis and the growth of chondrosarcoma cells are still not fully defined. It is well admitted that the invasion of bone by tumour cells affects the balance between early bone resorption and formation and induces an "inflammatory-like" environment which establishes a dialogue between tumour cells and their environment. The bone tumour microenvironment is then described as a sanctuary that contributes to the drug resistance patterns and may control at least in part the tumour growth. The concept of "niche" defined as a specialized microenvironment that can promote the emergence of tumour stem cells and provide all the required factors for their development recently emerges in the literature. The present paper aims to summarize the main evidence sustaining the existence of a specific bone niche in the pathogenesis of chondrosarcomas.

Dlx homeobox gene family expression in osteoclasts.

Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts.

Early irradiation of matter in the solar system: magnesium (proton, neutron) scheme.

The occurrence of positive and negative (26)Mg anomalies in inclusions of the Allende meteorite is explained in terms of proton bombardment of a gas of solar composition. A significant fraction of (26)Mg in the irradiated gas is stored temporarily in the form of radioactive (26)Al by the reaction (26)Mg(p,n) (26)Al. Proton fluxes of 10(17) to 10(19) protons per square centimeter per year at l million electron volts are inferred. Aluminum-rich materials condensing from the gas phase have positive (26)Mg anomalies, whereas magnesium-rich materials have negative (26)Mg anomalies. The proton flux required to account for the observed magnesium anomalies is used to investigate possible isotopic anomalies in the elements from oxygen to argon. Detectable isotopic anomalies are predicted only for neon. The anomalous neon is virtually pure (22)Ne from (22)Na decay. The predicted amount of anomalous (22)Ne is about 10(-8) cubic centimeter (at standard temperature and pressure) per milligram of sodium.

Primordial rare gases in unequilibrated ordinary chondrites.

The primordial gases of eight unequilibrated ordinary chondrites are strongly fractionated with respect to" cosmic" proportions. The absolute amounts are roughly proportional to the degree of disequilibration. Apparently, ordinary chondrites originally contained considerably larger amounts of primordial rare gases.

Elements 112 to 119: were they present in meteorites?

Chondrites contain a small fission xenon component of unexplained origin. Evidence on the geochemical behavior of this component suggests that it was not derived from an actinide element (Z = 89 to 103), or from a transition metal between Z = 104 and 111, but from a more volatile progenitor. The most likely candidates are the superheavy elements between Z = 112 and 119, whose lighter congeners (mercury, tellurium, lead, and the like) are known to be strongly fractionated in meteorites.

Inert gases in lunar samples.

Sample 10084,40 (fines, less than 1 millimeter) contains substantial amounts of the inert gases. Their concentrations are inversely proportional to particle size; hence the gases appear to be surface-correlated in the soil fragments. The most likely origin of the gas is solar wind or solar cosmic rays. Glass and feldspar are generally poorer in gas than lithic fragments. Ratios of elements in the sample differ significantly from solar values. Ratios of isotopes in the sample are similar to those in meteorites. Argon-40 appears to consist of a radiogenic and a surface-correlated component. An apparent potassium-argon age of 4.42(+0.24)(-0.28) billion years is calculated.

Fullerenes in the cretaceous-tertiary boundary layer.

High-pressure liquid chromatography with ultraviolet-visible spectral analysis of toluene extracts of samples from two Cretaceous-Tertiary (K-T) boundary sites in New Zealand has revealed the presence of C(60) at concentrations of 0.1 to 0.2 parts per million of the associated soot. This technique verified also that fullerenes are produced in similar amounts in the soots of common flames under ambient atmospheric conditions. Therefore, the C(60) in the K-T boundary layer may have originated in the extensive wildfires that were associated with the cataclysmic impact event that terminated the Mezozoic era about 65 million years ago.

Sensitization of osteosarcoma cells to apoptosis by oncostatin M depends on STAT5 and p53.

Oncostatin M (OSM), a cytokine of the interleukin-6 family, induces growth arrest and differentiation of osteoblastic cells into glial-like/osteocytic cells. Here, we asked whether OSM regulates apoptosis of normal or transformed (osteosarcoma) osteoblasts. We show that OSM sensitizes cells to apoptosis induced by various death inducers such as staurosporine, ultraviolet or tumor necrosis factor-alpha. Apoptosis is mediated by the mitochondrial pathway, with release of cytochrome c from the mitochondria to the cytosol and activation of caspases-9 and -3. DNA micro-arrays revealed that OSM modulates the expression of Bax, Bad, Bnip3, Bcl-2 and Mcl-1. Pharmacological inhibitors, dominant-negative signal transducer and activator of transcriptions (STATs), stable RNA interference and knockout cells indicated that the transcription factors p53 and STAT5, which are activated by OSM, are implicated in the sensitization to apoptosis, being responsible for Bax induction and Bcl-2 reduction, respectively. These results indicate that, in addition to growth arrest and induced differentiation, OSM also sensitizes normal and transformed osteoblasts to apoptosis by a mechanism implicating (i) activation and nuclear translocation of STAT5 and p53 and (ii) an increased Bax/Bcl-2 ratio. Therefore, association of OSM with kinase inhibitors such as Sts represents new therapeutic opportunities for wild-type p53 osteosarcoma.

DU145 human prostate cancer cells express functional receptor activator of NFkappaB: new insights in the prostate cancer bone metastasis process.

Prostate cancer metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer, frequently resulting in significant morbidity and mortality. As the underlying mechanisms are not fully characterized, understanding the biological mechanisms that govern prostate cancer metastases to bone at the molecular level should lead to the determination of new potential therapeutic targets. Receptor activator of NFkappaB ligand (RANKL)/RANK/Osteoprotegerin (OPG) are the key regulators of bone metabolism both in normal and pathological condition, including prostate cancer bone metastases. In the present study, we demonstrated that human prostate cancer cell lines, DU145 and PC3 express biologically functional RANK. Indeed, soluble human RANKL (shRANKL, 100 ng/ml) treatment induced ERK 1/2, p38 and IkappaB phosphorylations in these cells. shRANKL administration also promoted DU145 and PC3 prostate cancer cell invasion in vitro. Whereas human OPG (hOPG) administration alone (100 ng/ml) had no marked effect, combined association of both agents abolished the RANKL-induced DU145 cell invasion. As RANKL had no direct effect on DU145 cell proliferation, the observed effects were indeed related to RANKL-induced cell migration. DU145 human prostate cancer cells promoted osteoclastogenesis of osteoclast precursors generated from mouse bone marrow. Moreover, DU145 cells produced soluble factor(s) that up-regulate the proliferation of MC3T3-E1 pre-osteoblasts through the activation of the ERK 1/2 and STAT3 signal transduction pathways. This stimulation of pre-osteoblast proliferation resulted in an increased local RANKL expression that can activate both osteoclasts/osteoclast precursors and prostate cancer cells, thus facilitating prostate cancer metastasis development in bone. We confirm that RANKL is a factor that facilitates metastasis to bone by acting as an activator of both osteoclasts and RANK-positive prostate cancer cells in our model. Furthermore, the present study provides the evidence that blocking RANKL-RANK interaction offer new therapeutic approach not only at the level of bone resorbing cells, but also by interfering with RANK-positive prostate cancer cells in the prostate cancer bone metastasis development.

Mass vaccination: when and why.

With increased demand for smallpox vaccination during the nineteenth century, vaccination days--early mass vaccination campaigns--were conducted over time-limited periods to rapidly and efficiently protect maximum numbers of susceptible persons. Two centuries later, the challenge to rapidly and efficiently protect populations by mass vaccintion continues, despite the strengthening of routine immunization services in many countries through the Expanded Programme on Immunization strategies and GAVI support. Perhaps the most widely accepted reason for mass vaccination is to rapidly increase population (herd) immunity in the setting of an existing or potential outbreak, thereby limiting the morbidity and mortality that might result, especially when there has been no routine vaccination, or because populations have been displaced and routine immunization services disrupted. A second important use of mass vaccination is to accelerate disease control to rapidly increase coverage with a new vaccine at the time of its introduction into routine immunization programmes, and to attain the herd immunity levels required to meet international targets for eradication and mortality reduction. In the twenty-first century, mass vaccination and routine immunization remain a necessary alliance for attaining both national and international goals in the control of vaccine preventable disease.

[Excimer laser-assisted lamellar endothelial keratoplasty (ExALEK): Technique and results]. / Greffe lamellaire endothéliale assistée au laser Excimer (ExALEK) : technique et résultats.

INTRODUCTION: The purpose of this study is to describe a technique for thin endothelial lamellar keratoplasty and to present the results for endothelial transplant performed at the University Hospital of Nantes. MATERIALS AND METHODS: This paper is a retrospective, single-center descriptive study conducted at the University Hospital of Nantes from September 2010 to May 2014, at first for anatomical or analgesic indications (group 1) and then extended to visual indications (group 2). Patients were followed for 12 months. The preparation of the endothelial graft includes an excimer-laser ablation of the residual stromal bed after lamellar keratectomy by manual deep anterior approach. RESULTS: Seventy surgeries were analyzed. The etiologies were mainly Fuchs Dystrophy, secondary endothelial dystrophy and post-penetrating keratoplasty endothelial failure. Fifty-three patients were integrated in group 1 and seventeen patients in group 2. In group 1, the mean VA at 12 months was 0.70 ± 0.30 Log MAR (0.2 decimal equivalent). In group 2, the mean VA at 12 months was 0.28 ± 0.12 Log MAR (0.5 decimal equivalent). Pachymetry decreased from 740 ± 125.1 µm preoperatively to 613.4 ± 73.4 µm at 12 months. The average central thickness of the graft was 84.1 ± 28.9 µm at 1 month and 80.2 ± 29.4 µm at 12 months. CONCLUSION: The advantage of this new surgical technique is that it is a rapid and repeatable method allowing thin grafts with satisfactory functionality and easy handling. Its performance independent of the scheduled surgery, allows for predictable organization in the operating room.

3D curved multiplanar cone beam CT reconstruction for intracochlear position assessment of straight electrodes array. A temporal bone and clinical study.

A retrospective review of post-op cone beam CT (CBCT) of 8 adult patients and 14 fresh temporal bones that underwent cochlear implantation with straight flexible electrodes array was performed to determine if the position of a long and flexible electrodes array within the cochlear scalae could be reliably assessed with CBCT. An oto-radiologist and two otologists examined the images and assessed the electrodes position. The temporal bone specimens underwent histological analysis for confirm the exact position. The position of the electrodes was rated as scala tympani, scala vestibule, or intermediate position for the electrodes at 180°, 360° and for the apical electrode. In the patient group, for the electrodes at 180° all observers agreed for scala tympani position except for 1 evaluation, while a discrepancy in 3 patients both for the 360° and for the apical electrode assessment were found. In five temporal bones the evaluations were in discrepancy for the 180° electrode, while at 360° a disagreement between raters on the scalar positioning was seen in six temporal bones. A higher discrepancy between was found in assessment of the scalar position of the apical electrode (average pairwise agreement 45.4%, Fleiss k = 0.13). A good concordance was found between the histological results and the consensus between raters for the electrodes in the basal turn, while low agreement (Cohen's k 0.31, pairwise agreement 50%) was found in the identification of the apical electrode position confirming the difficulty to correct identify the electrode position in the second cochlear turn in temporal bones. In conclusion, CBCT is a reliable radiologic exam to correctly evaluate the position of a lateral wall flexible array in implanted patients using the proposed imaging reconstruction method, while some artefacts impede exact evaluation of the position of the apical electrode in temporal bone and other radiological techniques should be preferred in ex vivo studies.