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.

BedMachine v3: Complete Bed Topography and Ocean Bathymetry Mapping of Greenland From Multibeam Echo Sounding Combined With Mass Conservation.

Greenland's bed topography is a primary control on ice flow, grounding line migration, calving dynamics, and subglacial drainage. Moreover, fjord bathymetry regulates the penetration of warm Atlantic water (AW) that rapidly melts and undercuts Greenland's marine-terminating glaciers. Here we present a new compilation of Greenland bed topography that assimilates seafloor bathymetry and ice thickness data through a mass conservation approach. A new 150 m horizontal resolution bed topography/bathymetric map of Greenland is constructed with seamless transitions at the ice/ocean interface, yielding major improvements over previous data sets, particularly in the marine-terminating sectors of northwest and southeast Greenland. Our map reveals that the total sea level potential of the Greenland ice sheet is 7.42 ± 0.05 m, which is 7 cm greater than previous estimates. Furthermore, it explains recent calving front response of numerous outlet glaciers and reveals new pathways by which AW can access glaciers with marine-based basins, thereby highlighting sectors of Greenland that are most vulnerable to future oceanic forcing.

MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib/IIa randomised, double-blind, placebo-controlled, dose-escalation trial.

OBJECTIVES: To determine the safety, tolerability and signs of efficacy of MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), in patients with rheumatoid arthritis (RA). METHODS: Patients with active, moderate RA were enrolled in a randomised, multicentre, double-blind, placebo-controlled, dose-escalation trial of intravenous MOR103 (0.3, 1.0 or 1.5 mg/kg) once a week for 4 weeks, with follow-up to 16 weeks. The primary outcome was safety. RESULTS: Of the 96 randomised and treated subjects, 85 completed the trial (n=27, 24, 22 and 23 for pooled placebo and MOR103 0.3, 1.0 and 1.5 mg/kg, respectively). Treatment emergent adverse events (AEs) in the MOR103 groups were mild or moderate in intensity and generally reported at frequencies similar to those in the placebo group. The most common AE was nasopharyngitis. In two cases, AEs were classified as serious because of hospitalisation: paronychia in a placebo subject and pleurisy in a MOR103 0.3 mg/kg subject. Both patients recovered fully. In exploratory efficacy analyses, subjects in the MOR103 1.0 and 1.5 mg/kg groups showed significant improvements in Disease Activity Score-28 scores and joint counts and significantly higher European League Against Rheumatism response rates than subjects receiving placebo. MOR103 1.0 mg/kg was associated with the largest reductions in disease activity parameters. CONCLUSIONS: MOR103 was well tolerated and showed preliminary evidence of efficacy in patients with active RA. The data support further investigation of this monoclonal antibody to GM-CSF in RA patients and potentially in those with other immune-mediated inflammatory diseases. TRIAL REGISTRATION NUMBER: NCT01023256.

In-depth characterization of polyolefin plastomers/elastomers (ethylene/1-octene copolymers) through hyphenated chromatographic techniques.

The chemical composition distribution (CCD) of three single site made ethylene/1-octene copolymers was investigated through offline-hyphenation of solvent gradient interaction chromatography (SGIC) with 1H NMR. Thus, a clear, non-linear correlation between SGIC elution time and chemical composition was found under the specific measurement conditions applied here. The application of 1H NMR as detection allowed to determine the CCD with unprecedented accuracy. 2D-LC of the copolymers revealed the correlation between CCD and molar mass distribution (MMD) in a quantitative manner. Furthermore, this approach allowed a comparison between the response behavior of an evaporative light scattering detector (ELSD, semi-quantitative, commonly applied in SGIC) and that of an infrared (IR) detector (quantitative, commonly applied in SEC). As a result, it could be shown that ELSD results are close to IR results for the system investigated here, in other words, the often-criticized semi-quantitative response behavior of the ELSD is affecting results in an acceptable manner.

Shock metamorphism in lunar samples.

Indications of shock metamorphism produced by pressures up to the megabar region have been observed in the fine material and the breccias, but very rarely in the coarser fragments of crystalline rocks. These indications are deformation structures in plagioclase and pyroxene, diaplectic plagioclase glasses, and glasses formed by shock-induced melting of lunar rocks. Two sources of shock waves have been distinguished: primary impact of meteorites and secondary impact of crater ejecta. There are two major chemical types of shock-induced melts. The differences in chemistry may be related to impact sites in mare and highland areas.

Serum serotonin concentrations in dogs with degenerative mitral valve disease.

BACKGROUND: Increased serotonin (5HT) signaling has been implicated in valvular disease of humans and animals, including canine degenerative mitral valve disease (DMVD). High circulating 5HT concentration is a potential source of increased signaling, and serum 5HT concentrations have not been previously reported in dogs with DMVD. HYPOTHESIS: Dogs with DMVD and small breed dogs predisposed to DMVD have higher serum 5HT concentrations than large breed controls. ANIMALS: Fifty dogs affected with DMVD, 34 dogs predisposed to DMVD but without cardiac murmur or echocardiographic evidence of DMVD, and 36 healthy large breed control dogs. METHODS: Prospective analysis. Serum 5HT concentration was measured by an ELISA test. RESULTS: Median serum 5HT concentration was significantly higher in dogs with DMVD and in dogs predisposed to DMVD as compared with controls (DMVD, 765.5 ng/mL [interquartile range, 561.3-944.4]; predisposed, 774.9 ng/mL [528.3-1,026]; control, 509.8 ng/mL [320.8-708.8]; P= .0001). Subgroup analysis of predisposed dogs indicated significantly higher serum 5HT concentrations in Cavalier King Charles Spaniel (CKCS) dogs than in other breeds (CKCS, 855.0 ng/mL [635.8-1,088]; non-CKCS, 554.2 ng/mL [380.6-648.4]; P= .0023). Age, platelet count, and platelet morphology were not correlated with 5HT concentration in any group. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with DMVD had significantly higher serum 5HT concentrations when compared with large breed control dogs. Healthy CKCS dogs had significantly higher serum 5HT concentrations than other healthy dogs predisposed to DMVD. Additional investigation into a possible role of 5HT in the pathogenesis of DMVD is warranted.

High performance liquid chromatography of polyolefin plastomers/elastomers (ethylene/1-octene copolymers) - Comparison of different solvent systems.

A series of ethylene/1-octene copolymers with different chemical composition was separated in six binary mobile phases using solvent gradients and a column packed with porous graphite Hypercarb™. It was found that the elution volumes of the samples were to a larger extent influenced by the choice of desorption promoting solvent (desorli: 1,2-dichlorobenzene vs. 1,2,4-trichlorobenzene) than by the choice of adsorption promoting solvent (2-ethyl-1-hexanol, 1-decanol, n-decane). Elution volumes increased with decreasing number of chlorine atoms in the desorlis as well as with increasing polarity of the adsorlis. The resolution of HPLC systems depended pronouncedly on the choice of solvent pair: While in the majority of the tested HPLC systems, the chromatograms of the polymer samples indicate a shoulder, in n-decane→TCB the samples eluted without indication of a shoulder. In addition to the influence of different solvents on the samples elution behavior, the response of the employed detector, an evaporative light scattering detector (ELSD), was investigated. Its response was found to depend pronouncedly on the nature of the used solvents. Overall, the solvent pair 1-decanol→TCB appears to be the optimal compromise between the considered parameters and thus the best choice for HPLC of ethylene/1-octene copolymers.

Cytokines, NF-kappaB, microenvironment, intestinal inflammation and cancer.

Inflammation and cancer have been viewed as closely linked for many years. This link is not merely a loose association but causative. In colorectal cancer (CRC), chronic inflammation as observed in inflammatory bowel (IBD) disease is a key predisposing factor and IBD-associated CRC comprises five percent of all CRCs. Although the molecular mechanisms linking IBD with CRC are not well understood, recent results obtained in preclinical models point to the transcription factor NF-kappaB as a central player. On the one hand, NF-kappaB regulates the expression of various cytokines and modulates the inflammatory processes in IBD. On the other, NF-kappaB stimulates the proliferation of tumor cells and enhances their survival through the regulation of anti-apoptotic genes. Furthermore, it has been clearly established that most carcinogens and tumor promoters activate NF-kappaB, while chemopreventive agents generally suppress this transcription factor. Actually, several lines of evidence suggest that activation of NF-kappaB may cause cancer. These include the finding that NF-kappaB genes can be oncogenes, and that this transcription factor controls apoptosis, cell-cycle progression and proliferation, and possibly also cell differentiation.

Scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDX) and aerosol time-of-flight mass spectrometry (ATOFMS) single particle analysis of metallurgy plant emissions.

The chemical composition of single particles deposited on industrial filters located in three different chimneys of an iron-manganese (Fe-Mn) alloy manufacturing plant have been compared using aerosol time-of-flight mass spectrometry (ATOFMS) and scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDX). Very similar types of particles were observed using both analytical techniques. Calcium-containing particles dominated in the firing area of the sintering unit, Mn and/or Al-bearing particles were observed at the cooling area of the sintering unit, while Mn-containing particles were dominant at the smelting unit. SEM-EDX analysis of particles collected downstream of the industrial filters showed that the composition of the particles emitted from the chimneys is very similar to those collected on the filters. ATOFMS analysis of ore samples was also performed to identify particulate emissions that could be generated by wind erosion and manual activities. Specific particle types have been identified for each emission source (chimneys and ore piles) and can be used as tracers for source apportionment of ambient PM measured in the vicinity of the industrial site.

Adenosine does not evoke pain from venous and paravascular nociceptors in the human.

OBJECTIVE: The pain evoking and pain modulating properties of adenosine were studied at venous and paravascular nociceptors in humans. METHODS: In six volunteers, adenosine (3 to 15.10(-3) M) was perfused continuously through vascularly isolated segments of dorsal hand veins or injected into occluded finger veins. The effects of adenosine on pain evoked by intravenous electrostimulation of hand veins were also studied. The subjects rated the pain intensities on a visual analogue scale. RESULTS: Adenosine neither evoked pain nor altered the intensities of electrically evoked pain. CONCLUSIONS: Adenosine does not mediate or modulate pain via venous or paravascular nociceptors. Therefore these nociceptors are unlikely to be involved in adenosine related ischaemia pain.

Reversible blockade of cardiac efferents with procaine instilled into the pericardium of cats.

The blocking of afferent signals from the heart by the instillation of local anaesthetics into the pericardium has frequently been used for studying cardiac reflexes. Yet no attempts have been made to define what effects this has on efferent cardiac drive. Therefore, in nine chloralosed cats with open chests and catheters sewn into the pericardium the procaine concentrations were determined which would block the heart rate responses to electrostimulation of the vagi nerves or the stellate ganglia. The procaine effects on arterial pressure and left ventricular peak positive dP/dt were also evaluated and in 11 cats the vagolytic effects of atropine (0.25 mg . kg-1, intravenous) and intrapericardial procaine (0.1%) were compared. As little as 0.05% procaine attenuated the bradycardia associated with stimulation of the stellate ganglia by only about 30%. Virtual elimination of both sympathetic and vagal drive was achieved with 0.5% procaine having only mild effects on blood pressure and left ventricular dP/dt. Atropine (0.25 mg . kg-1, intravenously) and procaine (0.1% intrapericardially) affected heart rate equally. With procaine in the pericardium it is possible to reversibly and selectively block cardiac efferents and to separate vagal and sympathetic drive by choosing the appropriate concentration of the local anaesthetic.

Reversible blockade of myelinated and non-myelinated cardiac afferents in cats by instillation of procaine into the pericardium.

Pericardial local anaesthesia was instituted in 33 chloralosed cats through catheters implanted into the pericardium. The activity of 1531 single fibres teased from either vagus in the neck was identified and classified according to source. Random testing with 2% procaine instilled into the pericardium revealed that only cardiac afferents could be blocked. Arterial and other receptors were unaffected. Blocking concentrations were determined for 38 non-myelinated cardiac afferents (mean conduction velocity 1.3 m.s-1) and for 43 myelinated fibres (mean conduction velocity 19 m.s-1). Blocking concentrations varied widely but equally for both fibre categories between 0.05% and 2.0% procaine. Non-myelinated fibres required a mean concentration of 0.26% procaine for block, whereas myelinated fibres needed a mean concentration of 0.45% (P less than 0.01). The large overlap in the frequency distributions of the blocking concentrations for both sets of fibres, would not, however, allow the blocking of one fibre category without affecting the other. Additionally two cardiac sympathetic afferents were blocked at 0.25 and 0.5% procaine. Mean times for block onset and recovery for non-myelinated fibres were 3.05/1.98 min and for myelinated fibres 1.4/0.59 min, respectively. Pericardial local anaesthesia is selective for cardiac afferents but does not allow for differential block of a given fibre category. A 1% solution of procaine will eliminate the heart as a reflexogenic area.

Cerebral blood flow during migraine attacks without aura and effect of sumatriptan.

OBJECTIVE: To study regional cerebral blood flow (rCBF) during migraine attacks without aura and after treatment with sumatriptan. DESIGN AND INTERVENTION: We performed three technetium Tc99m hexemethyl-propyleneamineoxime single photon emission computed tomography scanning procedures in patients with migraine who participated in a double-blind, placebo-controlled, randomized clinical trial (1) outside an attack, (2) during an attack, and (3) after treatment of the attack with 6 mg of subcutaneous sumatriptan. SETTING: University hospital. PATIENTS: We studied 20 patients with migraine without aura, 15 of whom were evaluated under all three conditions and five of whom were evaluated under only two conditions. OUTCOME MEASURES: The single photon emission computed tomographic images were evaluated semiquantitatively with regard to (1) the degree of asymmetry of the rCBF between the headache side and the nonheadache side and (2) the ratio of the rCBF in regions of interest to the rCBF in two reference areas (cerebellum or frontal cortex). RESULTS: We found no significant rCBF asymmetries outside or during the attack or after treatment with sumatriptan, and there were no significant changes of the rCBF ratios during the attack (compared with outside the attack) or after treatment of the attack (compared with during the attack). CONCLUSION: Migraine attacks without aura and treatment of the attacks with 6 mg of subcutaneous sumatriptan are not associated with detectable focal changes of the rCBF.

Nitric oxide as a chemical link in the generation of pain from veins in humans.

In humans, both nitric oxide (NO) and bradykinin, a naturally occurring algetic and a potent NO liberator, evoke pain from hand veins. The afferent innervation of these veins consists solely of polymodal nociceptors which are located close to the endothelium, a well-known source of NO, thus suggesting NO as a chemical link in nociception. Consistent with this hypothesis, our observations show that neither bradykinin, nor hyperosmolar solutions (a noxious physicochemical stimulus) evoke pain from hand vein segments that have been exposed to the NO-synthase (NOS) inhibitor NG-mono-methyl-L-arginine. An intact NOS pathway is therefore a prerequisite for pain to be evoked by bradykinin and hyperosmolar solutions from veins, indicating for the first time in humans that vascular pain is mediated by NO. Thus, new directions for research on analgesics may be opened.

The role of pain from veins for the formation of perivenous edema in humans.

To test the hypothesis that nociception from veins plays a role in the formation of perivenous edema, we looked at edema along hand veins of humans during painful noxious stimulation in the presence and absence of nerve conduction block. Pain from vascularly isolated hand vein segments was evoked by perfusion with hyperosmolar saline and rated with the help of an electronically controlled visual analogue scale. Perivenous edema measured as changes in skin altitude was continuously recorded by means of infrared reflection. To alternately block the innervation of skin and vein segment we used a perivenous block (vein but not skin numbed), a distal ulnar nerve block (skin but not vein numbed), and a proximal ulnar nerve block (both vein and skin numbed). Without nerve block, hyperosmolar saline always evoked both pain and a continuous increase in perivenous edema to a maximum of 2.0-3.2 mm after 30 min. On painless control perfusions with isoosmolar saline, edema increased slightly (0.2-0.8 mm) to a plateau which was maintained until the end of perfusion. When the vein was denervated by perivenous or proximal ulnar nerve block, hyperosmolar saline evoked a slight increase in edema which resembled that of control perfusions in both extent and time course. On distal ulnar nerve block, which numbed the skin but not the vein, both pain and substantial edema were evoked. These observations show that nociception from veins is a prerequisite for perivenous edema to occur.

Nalbuphine does not act analgetically in electrical painful tooth pulp stimulation in man.

Nalbuphine is a mixed opioid agonist/antagonist, the analgesic properties of which are still open to debate. In a randomized and placebo-controlled protocol, we compared its effects (0.2 mg/kg) on man's perception of multimodal stimuli (i.e., nociceptive, acoustic and visual) to those of aspirin (acetylsalicylic acid, 10 mg/kg) and meperidine (0.5 mg/kg). Amplitudes and latencies of the evoked potentials (EP), tolerance maxima to painful tooth pulp stimuli, and subjective intensity ratings were measured as indicators of drug induced perception differences. After nalbuphine, the EP amplitudes markedly decreased while the stimuli of each modality were rated by the subjects to be of higher intensity. Also, the tolerance maxima of painful tooth pulp stimulation were reduced by nalbuphine, and naloxone did not have additional effects. In contrast, after aspirin and meperidine, the subjective pain ratings corresponded to the reduction of nociceptive EP amplitudes. Tolerance maxima to painful stimulation were also increased by both drugs. While aspirin did not influence acoustic and visual perception, meperidine caused a slight increase in EP amplitudes as well as in the intensity ratings of these stimuli relative to placebo. Thus, at the dose studied, nalbuphine did not act analgetically. The amplitude reduction of nociceptive EP suggested that nalbuphine had analgetic properties. These were, however, not confirmed by subjective pain ratings nor by changes in tolerance maxima.

Novel 3-oxa lipoxin A4 analogues with enhanced chemical and metabolic stability have anti-inflammatory activity in vivo.

Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

Prospective comparative study of technetium-99m-WBCs and indium-111-granulocytes for the examination of patients with inflammatory bowel disease.

In a prospective comparative study of 14 patients with inflammatory bowel disease (IBD), the abilities of 99mTc-HMPAO labeled white blood cells (WBCs) and 111In-granulocytes to assess the presence and location of active disease were compared. The two examinations were carried out within 2 wk of each other. Scintigraphically concordant positive or discordant segments were evaluated by radiologic or endoscopic examination performed within 14 days. When bowel segments were compared, concordance was found for 102/111 (91.8%) segments between 99mTc-WBC images obtained at 1 hr after injection and 3-hr 111In-granulocyte images. For five of five 99mTc-WBCs positive/111In-granulocyte negative segments, it could be proven that the 99mTc-WBC result was caused by active disease. For patients, 99mTc-WBC scintigraphy detected four more patients with active disease than 111In-granulocytes (11 and 7 patients, respectively). Technetium-WBCs was superior in the assessment of active disease, especially for small bowel segments. We conclude that early imaging 1 hr after the injection of 99mTc-WBCs can reliably replace 111In-granulocyte scintigraphy in IBD patients because the radiopharmaceutical is available on a daily basis. Thus, there is less radiation burden to the patient and cell separation is simpler and less time-consuming.

Evidence for the induction of protease activity on cultured tumour cells as a consequence of implantation into nude mice.

We have studied the enzymic status of a tumour associated protease on human colonic tumour cells cultured in vitro and grown in vivo in nude mice. The cultured tumour cells lack this protease. The cells of the tumour colonies in the mice possess active enzyme although the tissue contains an inhibitor capable of inactivating this tumour cell protease. The evidence indicates that the induction of this protease on the tumour cells is a consequence of implantation of the cultured cells into the nude mouse.

Three-dimensional dose mapping from gamma knife treatment using a dosimeter gel and MR-imaging.

A new method has been investigated for the mapping of dose distributions in three dimensions delivered by the Leksell gamma knife. The irradiation unit is used to selectively treat small volumes in the brain with single high doses of ionising radiation--a treatment procedure known as radiosurgery. The dosimetry method investigated utilises a dosimeter gel consisting of ferrous sulphate solution and agarose which is, prior to irradiation, loaded into a cavity in a spherical phantom. Chemical changes induced in the gel by the radiation are measured by means of an MR-scanner. This imaging method permits rapid evaluation of the dose distribution in an irradiated volume. It thus offers a potential verification of individual radiation intracranial target treatment regimes as well as quality assurance measurements, assuming that the precision and accuracy of the dose mapping are adequate. The dose and its distribution registered by the gel dosimeter, in this initial experiment, are in good agreement with corresponding computed data obtained with the KULA treatment planning system of the gamma knife. The gel has thus the potential of being an attractive alternative dose mapping method to those used at present in radiosurgery, i.e. radiographic film and small ionisation chambers. The precision of the dosimeter gel is, however, not yet sufficient high to be used as a basic dosimetry system for the gamma knife.