Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture.

BACKGROUND: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. METHODS: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. RESULTS: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. CONCLUSIONS: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy.

Correction to: Antimicrobial-coated sutures to decrease surgical site infections: a systematic review and meta-analysis.

In the original version of this article, reference citations found in Tables 2, 3, and 4 contain errors in linking. The correct tables are reproduced below.

Antimicrobial-coated sutures to decrease surgical site infections: a systematic review and meta-analysis.

To investigate the effectiveness of antimicrobial-coated sutures compared with non-coated sutures in reducing surgical site infection (SSI) and develop recommendations for World Health Organization (WHO) SSI prevention guidelines. We searched Medline, Embase, Cinahl, Cochrane Central Register of Controlled Trials, and WHO Global Health from 1990-16/02/2015 with language restricted to English, Spanish, and French. Meta-analysis was performed with a random-effects model. Meta-regression analysis assessed whether the effect of antimicrobial coating changed according to the type of suture and surgery. Subgroup analyses were based on types of sutures. Quality of the retrieved evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation. Thirteen randomized controlled trials (RCTs) and five observational studies (OBSs) met the inclusion criteria. Antimicrobial sutures significantly reduced SSI risk (for RCTs: OR 0.72, 95 % CI 0.59-0.88, p = 0.001, I2 = 14 %; for OBSs: OR 0.58, 95 % CI 0.40-0.83, p = 0.003, I2 = 22 %). Only Vicryl Plus vs Vicryl revealed consistent results in favor of antimicrobial sutures (for seven RCTs: OR 0.62, 95 % CI 0.44-0.88, p = 0.007, I2 = 3 %; for four OBSs: OR 0.58, 95 % CI 0.37-0.92, p = 0.02, I2 = 41 %). The effect of antimicrobial coating was similar between different suture, wound, and procedure types. Quality of RCT evidence was moderate, and OBS evidence was very low quality. Triclosan-coated sutures may reduce SSI risk. However, the available evidence is of moderate/low quality, and many studies had conflicts of interest.

The at-wavelength metrology facility for UV- and XUV-reflection and diffraction optics at BESSY-II.

A technology center for the production of high-precision reflection gratings has been established. Within this project a new optics beamline and a versatile reflectometer for at-wavelength characterization of UV- and XUV-reflection gratings and other (nano-) optical elements has been set up at BESSY-II. The Plane Grating Monochromator beamline operated in collimated light (c-PGM) is equipped with an SX700 monochromator, of which the blazed gratings (600 and 1200 lines mm(-1)) have been recently exchanged for new ones of improved performance produced in-house. Over the operating range from 10 to 2000 eV this beamline has very high spectral purity achieved by (i) a four-mirror arrangement of different coatings which can be inserted into the beam at different angles and (ii) by absorber filters for high-order suppression. Stray light and scattered radiation is removed efficiently by double sets of in situ exchangeable apertures and slits. By use of in- and off-plane bending-magnet radiation the beamline can be adjusted to either linear or elliptical polarization. One of the main features of a novel 11-axes reflectometer is the possibility to incorporate real life-sized gratings. The samples are adjustable within six degrees of freedom by a newly developed UHV-tripod system carrying a load up to 4 kg, and the reflectivity can be measured between 0 and 90° incidence angle for both s- and p-polarization geometry. This novel powerful metrology facility has gone into operation recently and is now open for external users. First results on optical performance and measurements on multilayer gratings will be presented here.

[Undesired awareness phenomena during general anesthesia: Evidence-based state of knowledge, current discussions and strategies for prevention and management]. / Unerwünschte Wachphänomene ("Awareness") während Allgemeinanästhesie: Evidenzbasierter Kenntnisstand, aktuelle Diskussionen und Strategien zu Prävention und Management.

Patient awareness during general anesthesia and the later recall of what happened during surgery can be experienced by patients as horrifying events that leave lasting mental trauma in the form of posttraumatic stress syndrome (PTSS). Awareness is related to a temporary insufficient depth of anesthesia. This phenomenon has been discussed for more than 20 years. According to randomized controlled studies, in the absence of risk factors awareness phenomena occur in 1-2 per 1000 operations involving general anesthesia (0.1-0.2%) and are classified as occasionally occurring critical events. An astonishing twist occurred elicited by the recent data from the 5th National Audit Project from Great Britain (NAP5) published in the British Journal of Anaesthesia and in Anaesthesia. The NAP5 evaluated patient reports of accidental awareness during general anesthesia (AAGA) in a multicenter manner in more than 2.7 million cases and indicated incidences of awareness of only 1:19,600, a frequency 20 times less than previously reported. These results gave rise to some controversy. It seems likely that, due to the absence of structured interviews, the NAP5 data only demonstrated the tip of the iceberg and may have vastly underestimated the real incidence of intraoperative awareness. The present overview summarizes the current knowledge about awareness. Furthermore, it addresses the question whether the awareness problem has been overestimated by evidence-based criteria or underestimated by the results of the NAP5. The responsibility for sufficient anesthesia in the clinical routine requires knowledge of awareness risks and potential sequelae. A formal recommendation from the German Society of Anesthesia and Intensive Care Medicine (DGAI) concerning awareness is not yet available; however, the recognition of evidence-based strategies in the management of anesthesia may minimize the occurrence of awareness and its sequelae.

Large-area 2D periodic crystalline silicon nanodome arrays on nanoimprinted glass exhibiting photonic band structure effects.

Two-dimensional silicon nanodome arrays are prepared on large areas up to 50 cm² exhibiting photonic band structure effects in the near-infrared and visible wavelength region by downscaling a recently developed fabrication method based on nanoimprint-patterned glass, high-rate electron-beam evaporation of silicon, self-organized solid phase crystallization and wet-chemical etching. The silicon nanodomes, arranged in square lattice geometry with 300 nm lattice constant, are optically characterized by angular resolved reflection measurements, allowing the partial determination of the photonic band structure. This experimentally determined band structure agrees well with the outcome of three-dimensional optical finite-element simulations. A 16% photonic bandgap is predicted for an optimized geometry of the silicon nanodome arrays. By variation of the duration of the selective etching step, the geometry as well as the optical properties of the periodic silicon nanodome arrays can be controlled systematically.

Testing of the WHO Infection Prevention and Control Assessment Framework at acute healthcare facility level.

BACKGROUND: Monitoring and evaluation are an essential part of infection prevention and control (IPC) implementation. The authors developed an IPC assessment framework (IPCAF) to support implementation of the World Health Organization (WHO) guidelines on core components of IPC programmes in acute healthcare facilities. AIM: To evaluate the usability and reliability of the IPCAF tool for global use. METHODS: The IPCAF is a questionnaire with a scoring system to measure the level of IPC implementation according to the eight WHO core components. The tool was pre-tested qualitatively, revised and translated selectively. A convenience sample of hospitals was invited to participate in the final testing. At least two IPC professionals from each hospital independently completed the IPCAF and a usability questionnaire online. The tool's internal consistency and interobserver reliability or intraclass correlation coefficient (ICC) were assessed, and usability questions were summarized descriptively. FINDINGS: In total, 46 countries, 181 hospitals and 324 individuals participated; 52 (16%) and 55 (17%) individual respondents came from low- and lower-middle income countries, respectively. Fifty-two percent of respondents took less than 1 h to complete the IPCAF. Overall, there was adequate internal consistency and a high ICC (0.92, 95% confidence interval 0.89-0.94). Ten individual questions had poor reliability (ICC <0.4); these were considered for revision according to usability feedback and expert opinion. CONCLUSIONS: The WHO IPCAF was tested using a robust global study and revised as necessary. It is now an effective tool for IPC improvement in healthcare facilities.

Anesthetics drug pharmacodynamics.

Anesthesia cannot be defined in an unambiguous manner. The essential components of general anesthesia are absence of consciousness and pain. This translates into two particular qualities: (1) sedation and hypnosis, i.e., mental blockade and (2) analgesia/antinociception, i.e., sensory blockade. Anesthetic actions on these two subcomponents are difficult to separate. On the one hand, very few anesthetics act exclusively on one of these components. On the other hand, these components are closely related to each other. Unconsciousness prevents (conscious) perception of pain, and nociception may serve as an arousal stimulus and change the level of sedation and hypnosis. The art of anesthesia lies in adequate dosing of drugs to reach both mental and sensory blockade. Drug administration can be based on pharmacokinetic considerations. Pharmacokinetic models allow an estimation of what happens to the administered drug in the body. Models with an effect site compartment may facilitate a tailored administration of anesthetic drugs. Finally, the quantification of pharmacodynamic effects allows a precise titration of drugs. Clinical assessment of mental blockade is often dichotomous, and therefore not very helpful to guide drug administration. Several scoring systems exist, but once consciousness is lost they become less reliable, in particular because reaction to stimuli is assessed, which mixes assessment of mental blockade with assessment of sensory blockade. Clinical assessment of analgesia requires a conscious patient, so antinociception is difficult to measure. Several methods of objective quantification on the basis of electrical brain activity are discussed including EEG and evoked potentials. Despite numerous indexes of the hypnotic component of anesthesia, there is no parameter that unambiguously quantifies the level of mental or sensory blockade.

The significance of shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection.

The plasma membrane remodeling, including the early transverse redistribution of phosphatidylserine, is a general feature occurring in cells in which a death program has been induced. In most cases, studies of this kind have focused mainly on cells. In this study, we report a clear correlation between the degree of apoptosis induced by a variety of agents in several types of cultured cells and the amount of shed membrane microparticles captured in the corresponding supernatants by insolubilized annexin V, a protein showing a strong affinity for phosphatidylserine. Such particles carry membrane antigens specific of the cells they stem from, and through which capture is also feasible. Homologous circulating microparticles were captured in peripheral blood from individuals with HIV-1 infection. A substantial proportion bore CD4 antigen. In some cases, CD4+ particles could be detected even in the absence of circulating CD4+ T cells, testifying to the presence of such resident cells in lymphoid tissues. These results suggest that shed membrane particles are one of the hallmarks of programmed cell death, of particular interest when the corresponding cells are hardly accessible.

No evidence for mutations in the alpha- and beta-catenin genes in human gastric and breast carcinomas.

Disturbed function of E-cadherin and/or of one of its anchoring proteins, the catenins, is thought to destabilize E-cadherin-mediated cell-cell adhesion, which may enhance the invasiveness of epithelial cells and thus favor carcinoma progression. Reduced expression of E-cadherin and alpha-catenin, as well as mutations in the E-cadherin gene, have been found in various carcinomas, whereas mutations in the alpha- and beta-catenin genes have been described only in carcinoma cell lines. Using reverse transcription-PCR, followed by agarose gel electrophoresis and single-strand conformational polymorphism, we examined 16 diffuse- and 5 intestinal-type gastric carcinomas, as well as 9 lobular and 2 ductal breast carcinomas, for mutations of alpha- and beta-catenin cDNA. All of the investigated tumors were analyzed previously for E-cadherin mutations. Comparing tumorous and nontumorous samples, we detected neither deletions nor aberrant single-strand conformational polymorphism patterns. At nucleotide 2220 of the alpha-catenin gene, we identified one frequent polymorphism. Our findings suggest that, in contrast to E-cadherin, mutations of alpha- and beta-catenin do not contribute to the pathogenesis or the diffuse growth patterns of gastric or breast carcinomas.

At-wavelength metrology facility for soft X-ray reflection optics.

A new Optics Beamline coupled to a versatile UHV reflectometer is successfully operating at BESSY-II. It is used to carry out at-wavelength characterization and calibration of in-house produced gratings and novel nano-optical devices as well as mirrors and multilayer systems in the UV and XUV spectral region. This paper presents most recent commissioning data of the beamline and shows their correlation with initial beamline design calculations. Special attention is paid to beamline key parameters which determine the quality of the measurements such as high-order suppression and stray light behavior. The facility is open to user operation.

Induction, by adriamycin and mitomycin C, of modifications in lipid composition, size distribution, membrane fluidity and permeability of cultured RDM4 lymphoma cells.

Adriamycin and mitomycin C were previously found to modulate the sensitivity of lymphoma cells to lysis by certain effectors of immunity and this modulation was dependent on drug concentration. In the present studies, RDM4 lymphoma cells were treated with different concentrations of the two drugs for 24 h in culture. These treatments resulted in changes in the lipid composition, membrane fluidity, cell size distribution, and permeability to 51CrO4, Trypan blue, Acridine orange and trimethylaminodiphenylhexatriene (TMA-DPH) of the cells. Changes in some of these parameters, as a function of drug concentration, resulted in dose-response curves which were bell-like shaped, hence paradoxical similarities between non-drug-treated cells and cells treated with higher drug concentrations were observed.

Safety and efficacy of a new transpulmonary echo contrast agent in echocardiographic studies in patients.

OBJECTIVES: This study was designed to investigate in patients the effect of a new transpulmonary echo contrast agent, made from 5% human serum albumin (Albunex), on systemic and pulmonary hemodynamics and the influence of the contrast doses on left ventricular opacification. BACKGROUND: New intravenous transpulmonary echo contrast agents are promising, allowing contrast stress echocardiography and myocardial contrast echocardiography. Nevertheless, some shortcomings still remain. Thus, the pulmonary hypertension observed in pigs after Albunex injection should be investigated in humans, and the optimal dose of contrast agent remains to be determined because previous experiments indicated that the left ventricular opacification and attenuation are dose dependent. METHODS: Albunex in doses of 0.08 and 0.22 ml/kg was successively injected intravenously in 20 catheterized patients; in 11 of them, anti-inflammatory drugs were withdrawn to avoid the blocking of an eventual thromboxane-mediated pulmonary artery hypertension. Systemic blood pressure and pulmonary artery, capillary wedge and right atrial pressures were continuously monitored. Cardiac output, left ventricular fractional shortening and blood gases were determined 5 min before and 5 and 10 min after each injection. The left ventricular opacification was qualitatively assessed by three independent observers using a grading scale from 0 to 3, with 0 indicating an absence of contrast effect and 3 indicating full opacification. RESULTS: No clinical, hemodynamic or respiratory adverse reactions were observed in any patient. Irrespective of doses, a left ventricular opacification grade > or = 2 was observed in 74% of the 35 injections that could be evaluated. This percentage increased to 94% when the higher dose group was considered alone. CONCLUSIONS: This first report of the effect of Albunex injected intravenously on pulmonary artery pressures in humans demonstrates that this contrast agent appears to be safe. The significant left ventricular opacification obtained in a majority of patients without an important increase in attenuation supports the use of the higher dose of the contrast agent.

Knowledge-based reconstruction of random porous media.

An evolutionary optimization technique is used to reconstruct digitized material models of 300(3) nm3 size for mesoporous two-phase systems. The models are adapted to the two-point probability (TPP) and to a volume-based pore-size distribution (PSD) which were derived from SANS and adsorption experiments and which carry statistical information about morphology and topology of the pore system. To avoid extreme update-costs, the bulk of mutations are assessed by means of a suitable approximation of the PSD; it is demonstrated that a sporadic insertion of the PSD suffices to drive the algorithm towards satisfactory models in acceptable time. Our approach is knowledge-based in the sense that (i) the mutations are restricted to expedient exchanges of phase-voxels by a heuristic rule, and (ii) the sporadic calculation of the PSD from the current state of the model, in essence, provides an efficient self-control for the evolutionary process. We applied the method to reconstruct periodic models of the xerogel Gelsil 200. Such reconstructs of real mesoporous solids could be utilized, for instance, to verify theories of adsorption and capillary condensation.

Apoptosis at the interface of immunosuppressive and anticancer activities: the examples of two classes of chemical inducers, oxysterols and alkylating agents.

Recent progresses in the understanding of molecular and biochemical pathways involved in apoptotic cell death offer novel perspectives for therapeutic interventions, in particular in immunosuppressive and anti-cancer therapies. In this review, we examine some chemical, biological, and mechanistic aspects of two classes of apoptosis chemical inducers: oxysterols and alkylating agents. Oxysterols represent a vast family of oxygenated derivatives of sterols. Found in both animal and vegetal kingdoms, they can be considered as ultimate products of an oxidative stress, and are chemically inert. Some of them (7beta-hydroxycholesterol, 25-hydroxycholesterol and 7, 25-dihydroxycholesterol) are cytotoxic at micromolar concentrations towards normal and tumor cells in culture, particularly lymphocytes, and reduce the growth of murine transplanted tumors. Thus, possible applications of oxysterols in medicine as immunosuppressants or as anticancer agents may be considered. Alkylating agents, on the other hand, have been widely used in cancer chemotherapy for decades. There toxicity results from their high chemical reactivity, causing lesions to macromolecules through covalent linkage. Some representative members of this class, mainly bifunctional derivatives which possess dichloroethyl groups, such as Chlormethine, Cyclophosphamide and Chlorabucil, express a pronounced cytotoxicity against lymphoid cells, and have therefore potent immunosuppressive properties. Because they triggers apoptosis via both common and distinct mechanisms, oxysterols and alkylating agents provide unique tools for exploring the initiation of this phenomenon in lymphoid cells, and may help design novel pharmacological approaches based on apoptotic modulation of these cells.

Membrane-related oxysterol function: preliminary results on the modification of protein kinase C activity and substrate phosphorylation by 7 beta,25-dihydroxycholesterol.

Oxysterols exhibit a wide variety of biological activities, including potent immunosuppressive effects. 7 beta,25-Dihydroxycholesterol (7,25-OHC), a synthetic oxysterol, has been shown to strongly inhibit the lymphocyte response to different stimuli. This compound has been chosen as a model compound to investigate the mechanisms underlying the immunosuppressive effects of oxysterols. As protein kinase C (PKC) constitutes a key enzyme in the pathways leading to cell activation, we have studied the effect of 7,25-OHC on PKC activity in the cytosolic and particulate fractions of spleen cells. Lymphocytes treated with 7,25-OHC showed a decrease of the relative PKC activity in the particulate fractions compared to control cells. These results are confirmed by the observation that 7,25-OHC also reduces the phosphorylation of the endogenous PKC substrates. Thus oxysterols interfere with two membrane related phenomena, ie the modification of membrane PKC activity and the inhibition of the phosphorylation of the substrates of PKC located in the membrane. Previous results obtained by fluorescence polarisation revealed a modification of the membrane fluidity after oxysterol treatment. Furthermore, it has been demonstrated that oxysterols are incorporated into cell membranes. The alteration of the cell membrane could impair the signal transduction and may explain the immunosuppressive activity of oxysterol. Thus, along with other biological effects previously reported, oxysterols decrease membrane associated PKC activity in immune cells.

Monophosphoric acid diesters of 7 beta-hydroxycholesterol and of pyrimidine nucleosides as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity.

7 beta-Hydroxycholesterol, which has been shown to be selectively cytotoxic toward tumor cells cultured in vitro, was converted into the corresponding water-soluble phosphoric acid ester and linked to a pyrimidine nucleoside such as 5-fluoro-2'-deoxyuridine or 2'-deoxyuridine. 2-Chlorophenyl phosphorodichloridate (3), without activation, was used directly to phosphorylate the protected oxygenated sterol. The intermediate phosphorylated the 5'-OH group of nucleoside selectively, leading to compounds 1a and 1b after deprotection. These compounds were screened for their antiproliferative activity toward EL-4 murine leukemia cells in vitro and for their antitumor activity against the mice bearing Krebs II ascitic carcinoma in vivo.

Estimation of extracellular fluid volume from plasma clearance on technetium-99m DTPA by a single-injection, two-sample method.

A simple method is presented for estimating extracellular fluid volume from the plasma clearance of [99mTc])DTPA or [169Yb]DTPA. Two plasma samples are required, at 1 and 3 hr, following a single intravenous injection. (The same plasma samples can be used for measurement of glomerular filtration rate.) Using the complete plasma clearance curve as a reference (eight samples at 10 to 240 min), the error of the two-sample method in 40 patients was 1.5 I for [99mTc]DTPA, 2.1 I for [169Yb]DTPA (residual standard deviation).

Quality control of Tc-99m DTPA for measurement of glomerular filtration: concise communication.

When technetium-99m DTPA is used to measure glomerular filtration rate (GFR), the accuracy depends on the supplier of the radiopharmaceutical. The error in GFR is due to protein binding, as we have shown by direct measurement. In 19 patients, GFR measured with Tc-DTPA and corrected for protein binding agreed with that measured simultaneously using Yb-169 DTPA (correlation coefficient 0.991). Without correction, Tc-DTPA gave falsely low values in patients having good renal function, in whom unbound activity cleared rapidly while bound activity remained in the circulation. When Tc-DTPA is used to measure GFR, the in vivo protein binding should be measured and used to correct the data.

Measurement of glomerular filtration rate: single injection plasma clearance method without urine collection.

Glomerular filtration rate (GFR) can be calculated from the plasma clearance of any of several radiopharmaceuticals that are excreted by glomerular filtration. Simplified methods have been proposed that require only one or two plasma samples in lieu of a more complete clearance curve. We examined the error introduced by this simplification. Forty patients were studied using a dual-isotope technique employing [99mTc]DTPA and [169Yb]DTPA, obtaining eight plasma samples for each clearance curve at intervals from 10 to 240 min after injection. Data were fit to several empirical or semiempirical formulae and also to a two-compartment computer model that permitted GFR estimation from only one or two data points. The computer model gave good fit, but so did several simpler methods. The error that results from replacing the complete clearance curve by a single 3-hr sample was about 8 ml/min (residual s.d.). By using two samples (at 1 and 3 hr), the error could be reduced to 4 ml/min. Recommended one- and two-sample methods are presented.