Robot-Assisted Bone Cement Injection.

In this article, assistance to bone cement injection is studied, with a focus on vertebroplasty, a procedure dedicated to the treatment of vertebral compression fractures. A robotic system that can remotely be operated at pressures up to 140 bar is presented. It improves cement polymerization control, combining a cold passive exchanger that slows down the cement curing in the syringe and an active exchanger that controls the injected cement temperature. The cement remote injection uses a rate control teleoperation strategy with force feedback to help monitoring the cement state. In addition to laboratory assessments, cadaver experiments were performed to illustrate the satisfactory operation of the whole system.

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

STUDY HYPOTHESIS: Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING: We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY: Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS, REASONS FOR CAUTION: All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.

Design considerations for a novel MRI compatible manipulator for prostate cryoablation.

PURPOSE: Prostate carcinoma is a commonly diagnosed cancer in men. Nonsurgical treatment of early stage prostate cancer is an important alternative. The use of MRI for tumor cryoablation is of particular interest: it offers lower morbidity compared with other localized techniques. However, the current manual procedure is very time-consuming and has limited accuracy. A novel robotic assistant is therefore designed for prostate cancer cryotherapy treatment under MRI guidance to improve efficiency and accuracy. METHODS: Gesture definition was achieved based on actions of interventional radiologists at University Hospital of Strasbourg. A transperineal approach with a semiautonomous prostatic cryoprobe localization procedure was developed where the needle axis is automatically positioned before manual insertion. The workflow was developed simultaneously with the robotic assistant used for needle positioning. RESULTS: The design and the associated workflow of an original wire-driven manipulator were developed. The device is compact and has a low weight: its overall dimensions in the scanner are 100 × 100 × 40 mm with a weight of 120 g. Very good MRI compatibility was demonstrated. CONCLUSIONS: A novel cryoablation procedure based on the use of a robotic assistant is proposed. The device design was presented with demonstration of MRI compatibility. Further developments include automatic registration and in vivo experimental testing.

Mechanical properties of alginate beads hosting hepatocytes in a fluidized bed bioreactor.

Fluidized bed bioartificial liver has been proposed as a temporary support to bridge patients suffering from acute liver failure to transplantation. In such a bioreactor, alginate beads hosting hepatocytes are in continuous motion during at least six hours. After having shown in vitro the functionality of such a device, the present study aims at analyzing the potential mechanical alterations of the beads in the bioreactor, perfused by different surrounding media. Compression experiments are performed and coupled for analysis with Hertz theory. They provide qualitative and quantitative data. The average value of the shear modulus, calculated for the different cases studied varied from 2.4 to 10.4 kPa, and could therefore be considered as a quantitative measure of the beads mechanical properties. From the compression experiments and the estimated values of the shear modulus, we could now evaluate the effect of different operating conditions (fluidization, presence of cells, surrounding medium) on the mechanical behavior of alginate beads. On the one hand, the motion during six hours in the bioreactor does not alter the beads significantly. On the other hand, the presence of different substances in the fluid phase might change their mechanical strength. These results can be considered as new encouragements to use such a device as a bioartificial organ.

Alteration of the spinal modulation of nociceptive processing in patients with irritable bowel syndrome.

BACKGROUND: Visceral hypersensitivity has been evidenced in patients with irritable bowel syndrome (IBS) but its mechanisms remain poorly elucidated. We investigated the spinal transmission of nociceptive signals in IBS patients by analysing the effects of rectal distensions on electromyographic recordings of the somatic nociceptive flexion (RIII) reflex, an objective index of spinal nociceptive processes. METHODS: Fourteen IBS and 10 healthy volunteers were included in the study. Slow ramp (40 ml/min) and rapid phasic (900 ml/min, 10, 20, 30, and 40 mm Hg) rectal distensions were randomly performed while the RIII reflex evoked by electrical stimulation of the sural nerve at the ankle was continuously recorded from the ipsilateral biceps femoris. RESULTS: In healthy volunteers, significant progressive inhibition of the RIII reflex was observed during slow ramp distension (61 (13)% of control values) while biphasic effects (facilitation and inhibition) were observed during rapid distensions. In contrast, in IBS patients, the RIII reflex was significantly facilitated during slow ramp distension (139 (15)% of control values) and inhibitions induced by rapid distensions were significantly reduced. Volumes of distension and rectal compliance were similar in both groups. CONCLUSIONS: Our results provide direct evidence that a hyperexcitability of spinal nociceptive processes is present in a large subgroup of IBS patients.

Regulation of intracellular chloride concentration in rat lactotrope cells and its relation to the membrane resting potential.

Rat lactotrope cells in primary culture exhibit physiological properties closely associated with chloride ions (Cl-) homeostasis. In this work, we studied the regulation of intracellular Cl- concentrations ([Cl-]i) and its relation to the membrane resting potential, using a combination of electrophysiology and spectrofluorimetry. Variations in [Cl-]i resulting from the patch clamp technique, pHi, antagonists of Cl(-)-Ca(2+)-dependent channels, an anion exchanger antagonist, and an antagonist of K(+)-Cl- cotransport were considered with respect to their involvement in membrane potential. We show that: (i) The patch-pipette does not always impose its Cl- concentration. (ii) In rat lactotrope cells, membrane resting potential is partially determined by [Cl-]i. (iii) Besides ion channel activity, electroneutral ion transports (cotransports such as K(+)-Cl- and Na(+)-K(+)-2Cl-) participate actively in maintaining a high [Cl-]i. (iv) Finally, Cl- homeostasis is probably linked to cell energetics.

An in vitro system for testing leucocyte and leukaemic cell line adhesion to synthetic fibres.

Leucocyte adhesion is an important phenomenon in antimicrobial defence, inflammation and immunological mechanisms and has been shown to be dependent upon specialized adhesion molecules. To prevent side-effects related to blood transfusion (e.g. anti-human leucocyte antigen immunization and transmission of infectious agents) leucocyte reduction of blood products is now systematically performed in various countries. The most common system used for leucoreduction is blood filtration. For further understanding of the mechanisms responsible for the interaction between leucocytes and the fibres present in filters we used a flow chamber to study the adhesion of leucocytes and leukaemic cell lines to different types of fibre. Adhesion was quantified using video-microscopy and computer image analysis. Our results demonstrate that adhesion to filter fibres was dependent on the expression of beta2-integrins CD11--CD18 and was inhibited by anti-CD18. The amount of fibres present, their spatial arrangement and the physicochemical characteristics of the fibres were important factors in leucocyte adhesion. Leucocyte adhesion was the highest to polyethylene terephthalate (PET) and polyimide fibres. Lymphocytes or lymphocytic cell lines were poorly adherent to PET fibres. The retaining capacity of leucocyte filters can be improved by taking into account the different parameters for the design of new filters

Human promyelocytic cell line: a convenient tool for studying the molecular basis of WBC filtration.

BACKGROUND: Blood filtration is a technique widely used to reduce the levels of WBCs in blood components. Several studies have been conducted to define the factors that are involved in WBC reduction, but the various mechanisms are not clearly delineated. This study explored the role of WBC adhesion molecules in WBC reduction during filtration. STUDY DESIGN AND METHODS: A minifilter has been developed that has properties similar to those of the standard filter (Sepacell, Asahi Medical) but that allows a smaller volume of blood to be used (15 mL). WBC reduction was achieved to a similar extent in the standard filter and the minifilter (4.15 log and 4.18 log, respectively). Samples of human promyelocytic cell line (HL60) were filtered before and after differentiation induced by vitamin D3 (D3-HL60). Flow cytometry was used to characterize the D3-HL60 filtrates and to count the WBCs after filtration. RESULTS: HL60 was retained in the filter to the same extent as all other WBCs. A higher level of integrin receptors (CD11b/CD18; CD11c/CD18) was expressed by D3-HL60 than by HL60. When the blood was incubated with anti-CD11b, anti-CD11c, or anti-CD18, fewer D3-HL60 cells were trapped by the filter, while only anti-CD11b alters HL60 retention in the filter. CONCLUSION: The receptors CD11b/CD18 and CD11c/CD18 appear to bind to the filter fibers and to be one of the mechanisms responsible for WBC retention.

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line.

Our aim was to determine whether the expression of K(+) currents is related to the cell cycle in the excitable GH3 pituitary cell line. K(+) currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cell cycle. We show that the peak density of the transient outward K(+) current (I(to)) was 33% lower in cells in S phase (BrdU+) than in cells in other phases of the cell cycle (BrdU-). The voltage-dependence of I(to) was not modified. However, of the two kinetic components of I(to) inactivation, the characteristics of the fast component differed significantly between BrdU+ and BrdU- cells. Recovery from inactivation of I(to) showed biexponential and monoexponential function in BrdU- and BrdU+ cells, respectively. This suggests that the molecular basis of this current varies during the cell cycle. We further demonstrated that 4-aminopyridine, which blocks I(to), inhibited GH3 cell proliferation without altering the membrane potential. These data suggest that I(to) may play a role in GH3 cell proliferation processes.

Calcium store depletion induced by mitochondrial uncoupling in prostatic cells.

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.

Effects of Cl(-) substitution on electrophysiological properties, Ca(2+) influx and prolactin secretion of rat lactotropes in vitro.

In this study, we compared the effects of different chloride (Cl(-)) substitutes - methane sulfonate (CH(3)SO(-)(3)), bromide (Br(-)), nitrate (NO(-)(3)), thiocyanate (SCN(-)) and perchlorate (ClO(-)(4)) - on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH(3)SO(-)(3) decreased basal prolactin (PRL) secretion. Br(-) had no effect, whereas the more lyotropic anions, such as NO(-3), SCN(-) and C1O(-4), increased basal PRL secretion. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH(3)SO(-3) was less permeant than Cl(-), whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN(-) > ClO(-4) > NO(-3) > Br(-). In conclusion, deprivation of Cl(-) ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca(2+) ions. This process involves voltage-dependent Ca(2+) channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO(-3), ClO(-4) and SCN(-), on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.

Characterization of Ca(2+)-inhibited potassium channels in the LNCaP human prostate cancer cell line.

Potassium plasma membrane channels have been studied in the LNCaP androgen-sensitive human prostate cancer cell line, derived from a lymph node of a subject with metastatic carcinoma of the prostate. Membrane currents were recorded by the patchclamp technique, using the cell-attached, cell-free and whole-cell mode. A voltage-dependent, non-inactivating potassium channel (delayed rectifier) was the most commonly observed ion channel in LNCaP cells. The slope conductance of K+ channels in a symmetrical 140 mM K+ gradient was 78 pS. In excised inside-out patches, the channel was inhibited by increasing the cytoplasmic Ca2+ concentration (with half-block at 0.5 microM Ca2+) over a wide range of membrane potentials. The K+ channel had a high sensitivity to tetraethylammonium (TEA), that reduced the single channel conductance with Kd of 280 +/- 27 microM. The K+ channel open probability was inhibited by alpha-dendrotoxin (DTX) (with a half-blocking concentration of approximately 5 nM) and mast cell degranulating peptide (MCDP) (with half-blocking concentration of approximately 70 nM) at all membrane potentials and with very slow reversibility. In view of the biophysical and pharmacological properties of K+ channels in LNCaP cells, it is not possible to classify these channels as one of the previously characterized types of voltage- or ligand-gated K+ channels in other cell lines.

[Cystic and mucinous lesion in an antral ectopic pancreas]. / Lésion kystique et mucineuse développée sur un pancréas aberrant antral.

We report a case of heterotopic pancreas located in the gastric antrum. The cystic formation contained mucus. Tumoral markers in the cyst fluid were within the range pancreatic cystic mucinous tumors. Pathology examination of the resected specimen did not evidence any proliferative lesions but showed papillary hyperplasia probably due to duct occlusion.

Potassium channel inhibition reduces cell proliferation in the GH3 pituitary cell line.

Potassium (K+) conductances are known to be involved in cell proliferation of a number of nonexcitable cell types. The nature of the mechanism by which K+ channel inhibition reduces cell proliferation has remained elusive despite intensive search. We investigated whether such a phenomenon could be demonstrated in excitable cells, using the GH3 pituitary cell line as a cell model. Our aims were: 1) to study the effect of K+ channel inhibition on the proliferation of GH3 cells; and 2) to investigate the putative intracellular signals involved in this inhibition. Tetraethylammonium chloride (TEA), a blocker of the calcium (Ca2+)-dependent K+ conductances of GH3, was found to reversibly inhibit cell proliferation, as measured by 3H-thymidine incorporation. Cell cycle block specifically occurred at the G1/S phase of the cell cycle. This inhibition of proliferation was observed for 1-4 mM TEA, which suppressed most of the Ca2+-activated K+ current and part of the inward rectifying K+ current, as shown by electrophysiological experiments. Increasing extracellular K+ concentrations with KCI also inhibited cell proliferation in a dose-dependent manner. Both TEA and KCl depolarized the cells and increased intracellular Ca2+ levels ([Ca2+]i), showing that, in this type of excitable cell, inhibition of cell proliferation can be associated with elevated Ca2+ levels. Ca2+ and membrane resting potential (MRP) were considered as possible messengers of this inhibition. Our results suggest that cell cycle arrest of GH3 cells by K+ channel block probably involves an additional pathway, distinct from those of Ca2+ and MRP.

[Benign intraductal papillary-mucinous tumors of the pancreas with a 30-year follow-up]. / Tumeur intra-canalaire papillaire mucineuse pancréatique bénigne, évoluant depuis 30 années.

Intraductal papillary mucinous tumors of the pancreas are rare and characterised by a malignant potential. Their natural history is unknown. We report a case of intraductal papillary mucinous tumor of the pancreas, that was still benign although the first symptom was appeared 30 years before the diagnosis. This case report demonstrate the possible slow course of these tumors, for which malignant degeneration is unpredictable.

Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP: involvement in cell proliferation.

BACKGROUND: Very little is known about the expression of ion channels in prostate cells (both normal and malignant), and their possible role in physiological and pathological functions. We therefore studied ion conductances and their role in the proliferation of LNCaP cells, an androgen-sensitive human prostate cancer cell line. METHODS: We applied patch-clamp recording techniques for electrophysiological studies, and 3H-thymidine incorporation and protein content assays for cell growth studies. RESULTS: Only one type of voltage-dependent ion conductance, a potassium K+ conductance, was identified. This current, which was depressed by a rise in intracellular Ca2+, had a high sensitivity to tetraethylammonium (TEA) (with half-block at 2 mM) and was also inhibited by 2 nM alpha-dendrotoxin (DTX) and 20 nM mast-cell degranulating peptide (MCDP). K+ channel inhibitors inhibited [3H]thymidine incorporation and protein content, in a dose-dependent fashion, indicating that K+ channels are involved in cell growth. CONCLUSIONS: We conclude from our findings that the human cancer prostate cell line LNCaP has a new type of K+ channel, likely to play an essential role in the physiology of these cells and, more specifically, in cell proliferation.

[Intraductal papillary mucinous tumors of the pancreas. Clinical and morphological aspects in 30 patients]. / Tumeurs intra-canalaires papillaires mucineuses pancréatiques. Aspects cliniques et morphologiques chez 30 malades.

AIM: Intraductal papillary-mucinous tumors of the pancreas are rare and characterized by a malignant potential. The aim of this study was to clarify their clinical presentation and the performance of different imaging procedures to determine malignancy and tumor extent. METHODS: Medical records and radiographs of 30 patients with histologically confirmed intraductal papillary-mucinous tumor of the pancreas were reviewed retrospectively. Imaging procedures were compared with pathological data of resected pancreas to evaluate their performances. RESULTS: The most frequent symptom was acute pancreatitis (37%). The onset of symptoms preceded the diagnosis by 2.5 years. Diabetes mellitus and diarrhea were respectively detected in 33 and 23% of the cases. The combination of CT scan, endoscopic retrograde cholangiopancreatography and endosonography allowed correct diagnosis of intraductal papillary-mucinous tumor of the pancreas in 100% of the cases. Tumor extent could be accurately determined considering the location of cystic dilatation of the pancreatic ducts, the presence of intraductal material or parietal irregularity. Actuarial 2-year survival rate was 43% in patients with malignant tumors. Radiological factors predicting malignancy were: vascular invasion, common bile duct dilatation, stricture of the main pancreatic duct and the presence of solid component in the tumor. CONCLUSION: The combination of CT scan, ERCP and endosonography provide accurate diagnosis of intraductal papillary-mucinous tumor of the pancreas as well as assessment of tumor extent and malignancy.

GHRP6-stimulated hormone secretion in somatotrophs: involvement of intracellular and extracellular calcium sources.

GHRP6 is a synthetic hexapeptide which stimulates growth hormone (GH) secretion from the pituitary in vivo and in vitro. We have previously shown that in identified somatotrophs, GHRP6 induces a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i) consisting of an abrupt increase (first phase) followed by a sustained plateau of elevated [Ca2+]i (second phase). The first phase corresponds to mobilization of intracellular Ca2+ pools and the second phase to influx of extracellular Ca2+ ions through voltage-sensitive Ca2+ channels. In these experiments, we investigated the specific role of each of these two phases in the hormone response to GHRP6. We found that inhibition by thapsigargin of the intracellular Ca2+ mobilization phase significantly inhibited the hormone response to the peptide during 30 min incubations. Inhibition of the extracellular Ca2+ influx phase by nifedipine, a blocker of voltage-sensitive Ca2+ channels, resulted in a 53 percent reduction of the secretory response to 10(-5)M GHRP6. Antagonism of PKC by phloretin, a flavonoid which prevents PKC activation, and PKC depletion induced by a 24 h treatment with 10(-6)M PMA, completely inhibited the response to GHRP6. Somatostatin, which also inhibits the second phase of the Ca2+ response, suppressed the secretory response to GHRP6. We conclude that, Ca2+ is the main second messenger and both Ca2+ mobilization and Ca2+ entry play a role in the response to GHRP6. However, experiments with PKC depletion and SRIF suggest that other messengers are implicated in GHRP6 signalling in somatotrophs.

[Effects of preoperative artificial nutrition in intestinal resections for Crohn disease]. / Effets de la nutrition artificielle préopératoire dans les résections intestinales pour maladie de Crohn.

UNLABELLED: Artificial nutrition prior to bowel resection has not been evaluated fully. The aim of the present study was to assess the effects of preoperative artificial nutrition upon postoperative complications, length of resected bowel and relapses of Crohn disease. RESULTS: Between 1990 and 1994, 108 consecutive patients underwent bowel resection for Crohn disease. Thirty nine patients had received exclusive enteral nutrition (n = 14) or parenteral nutrition (n = 25) for 19 +/- 10 days. Patients who had received artificial nutrition were more malnourished and had complicated Crohn disease (fistulae, abscesses) more often than patients operated without artificial nutrition. After 19 days of artificial nutrition, the nutritional state of patients was not significantly improved. Postoperative complication rate was higher in patients operated after artificial nutrition (33 vs. 16%; P = 0.03). Using multivariate prognosis analysis, the extent of colic resection was significantly associated with postoperative complications (P = 0.0003). Length of resected bowel and relapse rates were similar in patients with or without preoperative nutrition. CONCLUSION: Artificial nutrition prior to bowel resection for Crohn's disease is indicated in patients with the most severe form of the disease. A preoperative nutrition of 19 days does not seem to reduce postoperative complications nor the length of resected bowel.