Measurement of the α-Particle Monopole Transition Form Factor Challenges Theory: A Low-Energy Puzzle for Nuclear Forces?

We perform a systematic study of the α-particle excitation from its ground state 0_{1}^{+} to the 0_{2}^{+} resonance. The so-called monopole transition form factor is investigated via an electron scattering experiment in a broad Q^{2} range (from 0.5 to 5.0 fm^{-2}). The precision of the new data dramatically supersedes that of older sets of data, each covering only a portion of the Q^{2} range. The new data allow the determination of two coefficients in a low-momentum expansion, leading to a new puzzle. By confronting experiment to state-of-the-art theoretical calculations, we observe that modern nuclear forces, including those derived within chiral effective field theory that are well tested on a variety of observables, fail to reproduce the excitation of the α particle.

First principles description of the giant dipole resonance in 16O.

We present a calculation of the giant dipole resonance in (16)O based on a nucleon-nucleon (NN) interaction from chiral effective field theory that reproduces NN scattering data with high accuracy. By merging the Lorentz integral transform and the coupled-cluster methods, we extend the previous theoretical limits for breakup observables in light nuclei with mass numbers (A ≤ 7) and address the collective giant dipole resonance of (16)O. We successfully benchmark the new approach against virtually exact results from the hyperspherical harmonics method in (4)He. Our results for (16)O reproduce the position and the total strength (bremsstrahlung sum rule) of the dipole response very well. When compared to the cross section from photoabsorption experiments, the theoretical curve exhibits a smeared form of the peak. The tail region between 40 and 100 MeV is reproduced within uncertainties.

Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase.

Analysis of living cells grown on different titanium surfaces by time-lapse confocal microscopy.

In this study we have combined fluorescence- and reflection-confocal laser scanning microscopy for the simultaneous visualization of living cells and surface topography beneath them. To this purpose we have designed a specific flow chamber and we have tested it with osteoblasts grown on an opaque, thick support, made of smooth or sandblasted titanium. Cells were loaded with Calcein-AM or tetramethylrhodamine methyl ester (TMRM), two probes employed as indicators of cell viability/morphology and mitochondrial membrane potential, respectively. Besides the acquisition of stacks of confocal sections, the system allowed also vertical views and faithful three-dimensional reconstruction of the samples. Confocal microscope implemented with our flow chamber proved to be a promising tool for time-lapse investigation of cell-biomaterial interactions.

Cytoskeletal reorganization in skeletal muscle differentiation: from cell morphology to gene expression.

Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.

Ethanol increases the paracellular permeability of monolayers of CAPAN-1 pancreatic duct cells.

When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.

EuCliD--a medical registry.

OBJECTIVES: The European Clinical Database EuCliD small star, filled has been developed as a tool for supervising selected quality indicators of about 200 European dialysis centers. Major efforts had to be made to comply with local and European laws regarding data security. METHOD: EuCliD is a Lotus Notes based flat-file database currently containing medical data of more than 14,000 dialysis patients from 10 European countries. Another 15,000 patients from 150 centers in 4 South-American countries will be added soon. Data are entered either manually or by means of interfaces to existing local data managing systems. This information is transferred to a central Lotus Notes Server. Data evaluation was performed with statistical tools like SPSS. RESULTS: EuCliD is used as a part of the CQI (Continuous Quality Improvement) management system of Fresenius Medical Care (FMC) dialysis units. Each participating dialysis center receives (currently every half year) benchmarking reports at a regular interval. The benchmark for all quality parameters is the weighted mean of the corresponding data of all centers. CONCLUSIONS: An obvious impact of data sampling and data evaluation on the quality of the treatments could be observed within the first one and a half years of working with EuCliD. This also concerns important outcome predictors like Kt/V and hemoglobin concentration as the outcome itself expressed in hospitalization days and survival rates. With the help of EuCliD the user is able to sample clinical data, identify problems, search for solutions with the aim of improving the dialysis treatment quality and guarantee a high-class treatment quality for all patients.

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts.

The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.

Normal human IgG prevents endothelial cell activation induced by TNFalpha and oxidized low-density lipoprotein atherogenic stimuli.

Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-alpha (TNFalpha) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFalpha induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)'2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies.

Results from EuCliD (European Clinical Dialysis Database): impact of shifting treatment modality.

BACKGROUND: The use of biocompatible high-flux membranes is more efficient than low-flux membranes in controlling a number of hemodialysis-related diseases. The aim of this cooperative study was to evaluate the 6-month effect of a switch from low- to high-flux dialysers on patients treated in 39 Spanish dialysis centres. METHODS: The clinical data used in this analysis were prospectively collected by the EuCliD database, developed to monitor the quality of treatment delivered in a large network of European Dialysis Centres. Inclusion criteria for the study were the condition of end-stage renal disease (ESRD) on chronic hemodialysis and low-flux dialysis for at least six months before the switch to high-flux dialysis. Of 1,543 patients enrolled in the study between 2000 and 2001, 1,046 patients were considered for the analysis. 497 patients were excluded because they did not complete the follow-up. Outcome measures were the reduction of pre-dialysis beta-2 microglobulin, the improvement of anemia or reduction in rHu-EPO dose required to maintain best correction of anemia, reduction of inflammatory parameters (CRP), improvement in lipid profile (Total and HDL cholesterol, tryglycerides), maintenance of nutritional status. Albumin and "dry" (post-hemodialysis) body weight were both evaluated as nutritional indexes. RESULTS: During the six months of high-flux hemodialysis, there was a significant increase in hemoglobin (from 11.55 +/- 1.41 to 11.88 +/- 1.43 g/L; p < 0.001). Considering the temporarily untreated patients on a 0 U/week dose, erythropoietin remained stable (from 5,670 +/- 4,199 to 5,657 +/- 4,411 U/week). During the second part of the follow-up, the lipid profile significantly improved (Fig. 3). Total cholesterol and triglycerides decreased significantly (p < 0.001), while HDL cholesterol increased (p = 0.006). Calculated levels of LDL cholesterol also significantly decreased (p = 0.001). Dry body weight remained stable (64.7 +/- 11.9 vs. 64.7 +/- 12.0 kg) as well as in albumin levels (3.93 +/- 0.43 vs. 3.94 +/- 0.43 g/dL) between the two modalities of treatment. The level of beta2-microglobulin significantly decreased during high-flux dialysis (33.5 +/- 14.4 vs. 26.3 +/- 8.6 mg/dL, p < 0.001). CONCLUSION: All above mentioned results may have as a common denominator an improved blood purification from uremic toxins and a reduced level of chronic sub-clinical inflammation. All together, these results seem to confirm the superiority of high-flux dialysis in terms of clinical and physiological outcomes.

Control de la anemia en hemodiálisis. Base de datos EuCliD (European Clinical Database) en España / Anaemia management in haemodialysis. EuCliD® database in Spain

Nos planteamos evaluar el manejo de la anemia en los centros Fresenius Medical Care de España. Presentamos datos de 4.426 pacientes en hemodiálisis prevalentes en el año 2001 recogidos en la base de datos European Clinical Database (EuCliD®). Evaluamos mediante el índice de respuesta a la Eritropoyetina (IRE): cociente entre dosis de Eritropoyetina (UI/kg de peso/semana) y Hemoglobina (g/dl), la respuesta a la eritropoyetina en función de diferentes variables. Comparamos nuestros resultados con los aparecidos en el estudio ESAM2.El 70 por ciento de la población alcanza una hemoglobina superior a 11 g/dl utilizando dosis de eritropoyetina de 111,9 UI/kg de peso/semana (n = 3.700; SD 74,9).Resultado similar al medido al inicio del ESAM2 donde es del 65 por ciento. La dosis empleada de eritropoyetina es ligeramente superior a la utilizada en el ESAM2 (111,9 UI/kg de peso/semana y 105,5 UI/kg de peso/semana respectivamente), quizá relacionado con la menor proporción de pacientes en tratamiento por vía subcutánea (70 por ciento frente a 79 por ciento) y con la inclusión de pacientes en diálisis peritoneal dentro del estudio ESAM2. Efectivamente según nuestros datos, el IRE es mayor en los pacientes que reciben tratamiento por vía intravenosa en comparación con la vía subcutánea (11,66 y 9,6 respectivamente p 20 por ciento (135 UI/kg de peso/semana frente a 110,52 UI/kg de peso/semana respectivamente, p < 0,005).Observamos también que variables relacionados de forma directa o indirecta con la inflamación como la elevación de proteína C reactiva, la hipoalbuminemia o la elevación de la ferritina, presentan peor respuesta a la eritropoyetina (AU)

Ultrastructural basis for the efficiency of an ileal orthotopic neobladder 27 years after surgery.

The morphological and functional basis of the excellent clinical outcome of ileal orthotopic neobladders are largely unknown. Only long-term follow-up studies will provide an adequate answer to this unsettled question. We have studied a patient who underwent this type of surgery over 27 years ago. Besides an important secretive adaptation we have found, at the ultrastructural level, that the monolayered epithelium does not show signs of true metaplasia and that changes had occurred in the intercellular junctions, namely that desmosomes are significantly increased. Although limited to a single case, these features, if confirmed by further observations, suggest a working hypothesis for the understanding of the definitive phenotypic adaptation of the ileal epithelium to the new aggressive environment.

Employment of confocal microscopy for the dynamic visualization of domes in intact epithelial cell cultures.

Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.

[Anemia management in haemodialysis. EuCliD database in Spain]. / Control de la anemia en hemodiálisis. Base de datos EuCliD (European Clinical Database) en España.

We present the results on Anaemia Management in Fresenius Medical Care Spain dialysis centres as reported by EuCliD (European Clinical Database), evaluating a population of 4,426 patients treated in Spain during the year 2001. To analyse the erythropoietin dose and the haemoglobin levels we divided the population in two groups according to the time with dialysis treatment: patients treated less than six months and patients between six months, and four years on therapy. We compared our results with the evidence based recommendations Guidelines: the European Best Practice Guidelines (EBPG) and the US National Kidney Foundation (NKF-K/DOQI). We also compared our results with those presented by the ESAM2 on 2,618 patients on dialysis in Spain carried out in the second half of the year 2000. We observed that 70% of the population reaches an haemoglobin value higher that 11 g/dl, with a mean erythropoietin (rHu-EPO) dose of 111.9 Ul/kg weight/week (n = 3,700; SD 74.9). However, for those patients on treatment for less than six months, the mean Haemoglobin only reaches 10.65 g/dl (n = 222; SD 1.4). The rHu-EPO was administrated subcutaneously in 70.2% of the patients. About the iron therapy, 86% of the patients received iron treatment and the administration route was intravenous in 93% of the population. The ferritin levels were below 100 micrograms/dl in 10% of the patients and 26.4% showed a transferrin saturation index (TSAT) below 20%. The erythropoieting resistance index (ERI), as rHu-EPO/haemoglobin, has been used to evaluate the response to rHu-Epo, according to different variables. It was observed that the following factors lead to a higher rHu-EPO resistance: intravenous rHu-EPO as administration route, the presence of hypoalbuminemia, increase of protein C reactive, Transferrin saturation below 20% and starting dialysis during the last six months.

Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.

OBJECTIVE: To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. RESULTS: Unstimulated skin fibroblasts from SSc patients released more O2- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of alpha1(I) and alpha2(I) collagen messenger RNA. CONCLUSION: The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2-, H2O2, IL-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

Risk factors for acquiring Helicobacter pylori infection in a group of Tuscan teenagers.

Risk factors for acquiring Helicobacter pylori infection include hygienic, social, and environmental conditions. Some of these conditions usually change over time. We therefore investigated the existence of risk factors in a group of teenagers living in a place with the same environmental characteristics, in which hygienic and crowding conditions have not changed significantly in the last 20 years. A group of 164 students, mostly borne in 1977, attending four different schools, were examined serologically for H. pylori infection and CagA status. The importance of the risk factors for the transmission of the infection were evaluated by the chi2 test. P values <0.05 were considered significant. Twenty-two students (13.4%) were H. pylori seropositive. Students attending teachers' college and high school of arts were infected significantly more often than those attending high school (P = 0.011 and P = 0.012, respectively). Students who smoked and students whose parents had a manual job had an increased risk of acquiring the infection (P = 0.002, and P = 0.036, respectively). Crowding conditions and the presence of domestic animals were close to being statistically significant. Other factors, such as gender, number of bathrooms and bedrooms, sharing the bed with adults as a child, presence of a sexual partner, and a family history of peptic ulcer and gastric cancer, did not increase the risk of infection. The prevalence of seropositivity for CagA was similar in the various risk groups. Manual job of parents and smoking were the most important factors for acquiring H. pylori infection.

Accuracy and safety of online clearance monitoring based on conductivity variation.

BACKGROUND: Haemodialysis dose has been shown to have a distinct impact upon the morbidity and mortality rate in patients on regular dialysis therapy. Accordingly, the adequacy of dialysis treatment should be guaranteed. METHODS: In 200 dialysis sessions two or three +/-10% dialysate conductivity variations were applied to test patient compliance and the accuracy of an electrolyte based online clearance measurement (OCM) reflecting the total clearance of urea. RESULTS: Using a step profile the electrolytic clearance showed highly significant correlation with the reference data in the blood side (n=118, r=0.867, P<0.001) and dialysate side (n=118, r=0.820, P<0.001) if only reference values were taken into account for which the error in mass balance did not exceed 5%. Kt/V according to the single pool model (n=35, r=0.940, P<0.001), the equilibrated single pool variable volume kinetic model (n=36, r=0.982, P<0.001), Daugirdas formula (n=34, r=0.951, P<0.001) and direct quantification of dialysance via spent dialysate (n=26, r=0.900, P<0.001) showed outstanding correlations with electrolyte-based Kt/V at mass balance error below 5%. No adverse clinical effect of OCM was reported. Serum sodium, body weight, heart rate and breathing rate at rest, arterial pO(2) and pCO(2) and blood pressure before haemodialysis remained unaffected in OCM measurements in comparison with baseline parameters. A small influx of sodium (1.53+/-7.62 mmol) into the patient was seen following the impulse, but no signs associated with fluid overload were observed during the study period of 10 consecutive dialysis sessions. CONCLUSIONS: The OCM option of the haemodialysis machine provides a safe and accurate tool for continuous online monitoring of total urea clearance.

EuCliD (European Clinical Database): a database comparing different realities.

Quality and variability of dialysis practice are generally gaining more and more importance. Fresenius Medical Care (FMC), as provider of dialysis, has the duty to continuously monitor and guarantee the quality of care delivered to patients treated in its European dialysis units. Accordingly, a new clinical database called EuCliD has been developed. It is a multilingual and fully codified database, using as far as possible international standard coding tables. EuCliD collects and handles sensitive medical patient data, fully assuring confidentiality. The Infrastructure: a Domino server is installed in each country connected to EuCliD. All the centres belonging to a country are connected via modem to the country server. All the Domino Servers are connected via Wide Area Network to the Head Quarter Server in Bad Homburg (Germany). Inside each country server only anonymous data related to that particular country are available. The only place where all the anonymous data are available is the Head Quarter Server. The data collection is strongly supported in each country by "key-persons" with solid relationships to their respective national dialysis units. The quality of the data in EuCliD is ensured at different levels. At the end of January 2001, more than 11,000 patients treated in 135 centres located in 7 countries are already included in the system. FMC has put the patient care at the centre of its activities for many years and now is able to provide transparency to the community (Authorities, Nephrologists, Patients.....) thus demonstrating the quality of the service.