Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

Objectives:  Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Methods:  Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Results:  Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. Conclusions:  These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Topographical body fat distribution links to amino acid and lipid metabolism in healthy non-obese women.

Visceral adiposity is increasingly recognized as a key condition for the development of obesity related disorders, with the ratio between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) reported as the best correlate of cardiometabolic risk. In this study, using a cohort of 40 obese females (age: 25-45 y, BMI: 28-40 kg/m(2)) under healthy clinical conditions and monitored over a 2 weeks period we examined the relationships between different body composition parameters, estimates of visceral adiposity and blood/urine metabolic profiles. Metabonomics and lipidomics analysis of blood plasma and urine were employed in combination with in vivo quantitation of body composition and abdominal fat distribution using iDXA and computerized tomography. Of the various visceral fat estimates, VAT/SAT and VAT/total abdominal fat ratios exhibited significant associations with regio-specific body lean and fat composition. The integration of these visceral fat estimates with metabolic profiles of blood and urine described a distinct amino acid, diacyl and ether phospholipid phenotype in women with higher visceral fat. Metabolites important in predicting visceral fat adiposity as assessed by Random forest analysis highlighted 7 most robust markers, including tyrosine, glutamine, PC-O 44∶6, PC-O 44∶4, PC-O 42∶4, PC-O 40∶4, and PC-O 40∶3 lipid species. Unexpectedly, the visceral fat associated inflammatory profiles were shown to be highly influenced by inter-days and between-subject variations. Nevertheless, the visceral fat associated amino acid and lipid signature is proposed to be further validated for future patient stratification and cardiometabolic health diagnostics.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing - a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites.

BACKGROUND: Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC + K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. RESULTS: Healing time was reduced from 13.9 ± 0.5 days (mean ± SEM) in the control group to 7.2 ± 0.2 days in the PC group (P < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01). CONCLUSION: The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. CLINICAL TRIAL REGISTRY INFORMATION: Protocol Record Identification Number: 132/03Registry URL: http://www.clinicaltrials.gov.

Caution on the storage of waters and aqueous solutions in plastic containers for hydrogen and oxygen stable isotope analysis

RATIONALE The choice of containers for storage of aqueous samples between their collection, transport and water hydrogen (2H) and oxygen (18O) stable isotope analysis is a topic of concern for a wide range of fields in environmental, geological, biomedical, food, and forensic sciences. The transport and separation of water molecules during water vapor or liquid uptake by sorption or solution and the diffusive transport of water molecules through organic polymer material by permeation or pervaporation may entail an isotopic fractionation. An experiment was conducted to evaluate the extent of such fractionation. METHODS Sixteen bottle-like containers of eleven different organic polymers, including low and high density polyethylene (LDPE and HDPE), polypropylene (PP), polycarbonate (PC), polyethylene terephthalate (PET), and perfluoroalkoxy-Teflon (PFA), of different wall thickness and size were completely filled with the same mineral water and stored for 659?days under the same conditions of temperature and humidity. Particular care was exercised to keep the bottles tightly closed and prevent loss of water vapor through the seals. RESULTS Changes of up to +5 parts per thousand for d2H values and +2.0 parts per thousand for d18O values were measured for water after more than 1?year of storage within a plastic container, with the magnitude of change depending mainly on the type of organic polymer, wall thickness, and container size. The most important variations were measured for the PET and PC bottles. Waters stored in glass bottles with Polyseal (TM) cone-lined PP screw caps and thick-walled HDPE or PFA containers with linerless screw caps having an integrally molded inner sealing ring preserved their original d2H and d18O values. The carbon, hydrogen, and oxygen stable isotope compositions of the organic polymeric materials were also determined. CONCLUSIONS The results of this study clearly show that for precise and accurate measurements of the water stable isotope composition in aqueous solutions, rigorous sampling and storage procedures are needed both for laboratory standards and for unknown samples. Copyright (c) 2012 John Wiley & Sons, Ltd.

Primary activity measurements with a 4πβ-4πγ coincidence counting system.

The radioactive concentrations of (166m)Ho, (134)Cs and (133)Ba solutions have been standardised using a 4πβ-4πγ coincidence counting system we have recently set up. The detection in the beta channel is performed using various geometries of a UPS-89 plastic scintillator optically coupled to a selected low-noise 1in. diameter photomultiplier tube. The light-tight thin capsule that encloses this beta detector is housed within the well of a 5in.×5in. NaI(Tl) monocrystal detector. The beta detection efficiency can be varied either by optical filtering or electronic discrimination when the electrons loose all their energy in the plastic scintillator. This 4πβ-4πγ coincidence system improves on our 4πβ(PC)-γ system in that its sample preparation is less labour intensive, it yields larger beta- and gamma-counting efficiencies thus enabling the standardisation of low activity sources with good statistics in reasonable time, and it makes standardising short-lived radionuclides easier. The resulting radioactive concentrations of (166m)Ho, (134)Cs and (133)Ba are found to agree with those measured with other primary measurement methods thus validating our 4πβ-4πγ coincidence counting system.

Expression of neutral endopeptidase, endothelin-1, and nuclear factor kappa B in prostate cancer: interrelations and associations with prostate-specific antigen recurrence after radical prostatectomy.

Objective. To study the impact of the neutral endopeptidase (NEP)/neuropeptides (NPs) axis and nuclear factor kappa B (NFκB) as predictors of prostate-specific antigen (PSA) recurrence after radical prostatectomy (RP). Patients and Methods. 70 patients with early-stage PC were treated with RP and their tumor samples were evaluated for expression of NEP, endothelin-1 (ET-1) and NFκB (p65). Time to PSA recurrence was correlated with the examined parameters and combined with preoperative PSA level, Gleason score, pathological TNM (pT) stage, and surgical margin (SM) assessment. Results and Limitations. Membranous expression of NEP (P < 0.001), cytoplasmic ET-1 (P = 0.002), and cytoplasmic NFκB (P < 0.001) were correlated with time to PSA relapse. NEP was associated with ET-1 (P < 0.001) and NFκB (P < 0.001). ET-1 was also correlated with NFκB (P < 0.001). NEP expression (P = 0.017), pT stage (P = 0.013), and SMs (P = 0.036) were independent predictors of time to PSA recurrence. Conclusions. There seems to be a clinical model of NEP/NPs and NFκB pathways interconnection, with their constituents following inverse patterns of expression in accordance with their biological roles and molecular interrelations.

Changes of beta-amyloid precursor protein splice patterns in brain cell aggregate cultures.

The splice pattern of beta-amyloid precursor protein (beta-APP) has been studied in a variety of neuronal and glial cells and in brain cell aggregate cultures by the polymerase chain reaction (PCR). The brain-typical pattern, in which beta-APP695 is the dominant form, has been found only in aggregate cultures but not in any of the other cell types including neuronal cell lines. Selective elimination of glial cells from aggregates resulted in increased quantities of beta-APP695, whereas removal of neurons led to a reduction of beta-APP695 and to an elevation of beta-APP751 and beta-APP770. This shift of splice pattern was not observed in cocultures of the neuronal cell line PC 12 with primary astrocytes combined in a variety of cellular ratios. Blood serum, which is an essential component of these cultures, tested on aggregates, did not reduce the amount of beta-APP695 or have any marked effects on splice patterns generally. From these results it is concluded that investigations on brain-typical splicing of beta-APP require primary neurons. Neuronal cell lines may be no suitable model systems. Splicing events favoring production of beta-APP695 may mark an important, very early step of amyloid formation in the brain.

Insertion vélamenteuse du cordon et grossesse gémellaire

Problématique : L'insertion vélamenteuse du cordon ombilical est reportée dans environ 1% des grossesses uniques et semble associée à de sévères complications obstétricales. Mais l'étude de cette insertion reste encore lacunaire en ce qui concerne les grossesses gémellaires. Depuis quelques années, ces dernières sont en augmentation et doivent être considérées comme des grossesses à risque. En effet, les jumeaux occupent une place toujours plus importante dans les unités de néonatologie, du fait de la morbidité particulière de ces grossesses. Ces dernières peuvent de plus s'accompagner d'autres anomalies qui peuvent influencer la croissance des jumeaux déjà prétérités par rapport aux bébés uniques. Parmi celles-ci, l'insertion vélamenteuse du cordon. Existe-il une association entre grossesse gémellaire et insertion vélamenteuse ? Quelles seraient les conséquences de ce type d'insertion sur l'évolution de la grossesse ?¦Objectifs : Evaluer l'incidence de la survenue d'une insertion vélamenteuse du cordon ombilical dans les grossesses gémellaires par rapport aux grossesses uniques et la différence de cette incidence entre placentas monochoriaux et bichoriaux. Analyser les conséquences majeures qui peuvent lui être associées.¦Méthode : Analyse rétrospective de tous les placentas de grossesses gémellaires reçus pour examen à l'institut universitaire de pathologie anatomique (IUPA) de Lausanne entre janvier 2000 et septembre 2010, ainsi que du suivi périnatal des enfants issus de ces grossesses.¦Résultats : 722 placentas issus de grossesses gémellaires ont été reçus et analysés à l'IUPA. L'insertion vélamenteuse du cordon est rencontrée dans plus de 17% des grossesses gémellaires. Elle représente plus de 9% de toutes les insertions lors de ces grossesses. Elle est retrouvée dans 35% des cas lorsque le placenta est monochorial et dans 10% des cas lorsque le placenta est bichorial.¦Notre étude a démontré une influence significative de l'insertion vélamenteuse sur l'âge gestationnel, le poids du placenta, la croissance foetale (poids > taille > PC), la durée d'hospitalisation et la mortalité. La monochorialité est le seul facteur de risque à avoir été mis en évidence.¦Conclusion : Avec l'augmentation de l'âge maternel et le recours de plus en plus fréquent aux traitements de fertilité, le nombre de grossesses gémellaires devrait s'accroitre dans les années à venir et, avec elles, le nombre d'insertions vélamenteuses du cordon. Cependant, le diagnostic d'une telle insertion ne peut être que suspecté lors d'un examen ultrasonographique en cours de grossesse. Il est donc difficile de l'identifier avant la naissance et ainsi de prévoir ses conséquences. Mais en étant conscient des complications éventuelles que peut engendrer cette insertion, nous serions plus à même d'y faire face.

CD10 is inversely associated with nuclear factor-kappa B and predicts biochemical recurrence after radical prostatectomy.

INTRODUCTION: The cell surface endopeptidase CD10 (neutral endopeptidase) and nuclear factor-κB (NF-κB) have been independently associated with prostate cancer (PC) progression. We investigated the correlations between these two factors and their prognostic relevance in terms of biochemical (prostate-specific antigen, PSA) relapse after radical prostatectomy (RP) for localized PC. PATIENTS AND METHODS: The immunohistochemical expression of CD10 and NF-κB in samples from 70 patients who underwent RP for localized PC was correlated with the preoperative PSA level, Gleason score, pathological stage and time to PSA failure. RESULTS: CD10 expression was inversely associated with NF-κB expression (p < 0.001), stage (p = 0.03) and grade (p = 0.003), whereas NF-κB was directly related with stage (p = 0.006) and grade (p = 0.002). The median time to PSA failure was 56 months. CD10 and NF-κB were directly (p < 0.001) and inversely (p < 0.001) correlated with biochemical recurrence-free survival, respectively. CD10 expression (p = 0.022) and stage (p = 0.018) were independently associated with time to biochemical recurrence. CONCLUSION: Low CD10 expression is an adverse prognostic factor for biochemical relapse after RP in localized PC, which is also associated with high NF-κB expression. Decreased CD10 expression which would lead to increased neuropeptide signaling and NF-κB activity may be present in a subset of early PCs.

Proteomic analysis of Intercept-treated platelets.

In the past decades, transfusion medicine has been driven by the quest for increased safety against transfusion-transmitted infections, mainly by better donor selection and by the development of improved serological and nucleic-acid-based screening assays. Recently, pathogen reduction technologies became available and started to be implemented in several countries, with the primary goal to fight against bacterial contamination of blood products, a rare but dramatic event against which there was no definitive measure. Though pathogen reduction technologies represent a quantum leap in transfusion safety, the biomedical efficacy of platelet concentrates (PCs) treated with various pathogen reduction techniques has been recently questioned by clinical studies. Here, a gel-based proteomic analysis of PCs (n=5), Intercept-treated or untreated, from pooled buffy-coat (10 donors per PC) at Days 1, 2 and 8, shows that the Intercept process that is the most widespread pathogen reduction technique to date, has relatively low impact on the proteome of treated platelets: the process induces modifications of DJ-1 protein, glutaredoxin 5, and G(i)alpha 2 protein. As for the impact of storage, chloride intracellular channel protein 4 (CLIC4) and actin increased independently of Intercept treatment during storage. Whereas alteration of the DJ-1 protein and glutaredoxin 5 points out an oxidative stress-associated lesion, modification of G(i)alpha2 directly connects a possible Intercept-associated lesion to haemostatic properties of Intercept-treated platelets. This article is part of a Special Issue entitled: Integrated omics.

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases.

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-beta-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

Arenavirus envelope glycoproteins mimic autoprocessing sites of the cellular proprotein convertase subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the arenavirus life cycle is the proteolytic processing of the viral envelope glycoprotein precursor (GPC) by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we conducted a systematic and quantitative analysis of SKI-1/S1P processing of peptides derived from the recognition sites of GPCs of different Old World and New World arenaviruses. We found that SKI-1/S1P showed a strong preference for arenaviral sequences resembling its autoprocessing sites, which are recurrent motifs in arenaviral GPCs. The African arenaviruses Lassa, Mobala, and Mopeia resemble the SKI-1/S1P autoprocessing C-site, whereas sequences derived from Clade B New World viruses Junin and Tacaribe have similarities to the autoprocessing B-site. In contrast, analogous peptides derived from cellular SKI-1/S1P substrates were remarkably poor substrates. The data suggest that arenavirus GPCs evolved to mimic SKI-1/S1P autoprocessing sites, likely ensuring efficient cleavage and perhaps avoiding competition with SKI-1/S1P's cellular substrates.