[Antirreflux ureteral catheters.] / Catéteres ureterales antirreflujo.

Double-J ureteral stents disposal is associated with the appearance of side effects in up to 90% of the patients. The main causes of these symptoms are related to stent's design and the materials they are manufactured from. Vesicoureteral reflux and bladder trigone irritation are the etiopathogenic causes of ureteral stents associated morbidity. Due to this, and in order to improve patients' quality of life, stents that avoid reflux have been developed. Among anti-reflux designs, the first was a double-J stent the bladder tip of which is provided with a polymeric membrane that prevents retrograde flow of urine through its internal drainage channel. This design showed satisfactory vesicaresults, although not statistically significant. Their use in renal transplantation has also been assessed not only to decrease morbidity and ascending infection but also to improve graft survival. Other designs try to thin the distal end and even change it to a surgical suture thread, with the aim of eliminating the internal drainage channel in order to cause the minimum interference with the UVJ. Recently, two prototypes were evaluated in animal models and have achieved reduction of VUR. The first consists of a valve attached to the distal end of a traditional double-J stent, acting as a backflow prevention device. The second design is an intra-ureteral stent that acts like a double-J stent, but without crossing the UVJ and therefore preventing reflux completely. Nowadays, the use of these devices is not implemented in hospitals due to the absence of scientific evidence supporting the superiority of these designs over conventional stents.

[Biodegradable catheters and urinary stents. When?] / Catéteres y stents urinarios biodegradables. ¿Para cuándo?

One of the main wishes in the field of urinary catheters and stents is to arm them with biodegradable characteristics because we consider a failure of these devices the need for retrieval, the forgotten catheter syndrome as well as the adverse effects permanent devices cause after fulfilling their aim. The efforts focused in new designs, coatings and biomaterials aim to increase the biocompatibility of theses internal devices. Lately, there have been correct advances to answer the main challenges regarding biodegradable ureteral devices. Thus, modulation of the rate of degradation has been achieved thanks to new biomaterials and the use of copolymers that enable to choose the time of permanence as it is programmed with conventional double J catheters. Biocompatibility has improved with the use of new polymers that adapt better to the urine. Finally, one of the main problems is elimination of degraded fragments and experimentally it has be demonstrated that new designs elicit controlled degradation, from distal to proximal; using stranding and combination of copolymers degradation may be caused by dilution, reducing fragmentation to the last stages of life of the prosthesis. Moreover, it has been demonstrated that biodegradable catheters potentially may cause less urinary tract infection, less encrustation and predictably they will diminish catheter morbidity, since their degradation process reduces adverse effects. Regarding the development of biodegradable urethral stents, it is necessary to find biomaterials that enable maintaining their biomechanical properties in the long term, keeping open the urethral lumen both in patients with BPH and urethral stenosis. Modulation of the time of degradation of the prosthesis has been achieved, but the appearance of urothelial hyperplasia is still a constant in the initial phases after implantation. The development of drug eluting stents, anti-proliferative or anti-inflammatory, as well as biodegradable stents biocoated is a field from which it is expected the arrival of the solution of theses adverse effects. Therefore, many features need to be improved to obtain biodegradable stents, but over the last years some turning points have been accomplished thanks to the advances in Bioengineering, allowing to foresee safe and effective solutions in the nearest future.

[Metallic ureteral stents. Present and future.] / Stents ureterales metálicos. Presente y futuro.

The management of ureteral obstruction of malignant origin or complicated benign obstruction continues to be a challenge for the urological community. In this sense, the use of metallic stents could be considered a useful alternative to the conventional drainage techniques, because it accomplishes the resolution of obstruction in a single procedure, without external diversions and without the adverse effects of current diversions. Another important advantage they offer is that they do not need replacement as frequently as double J catheters or nephrostomy tubes require. From their first applications in the upper urinary tract until now the design of metallic stents has experienced a notable evolution. The main obstacle at the beginning was the use of stents intended for other organic territories, which caused a high rate of failures, since they did not take into consideration in their designs the hostile environment represented by urine for this type of devices, neither the existence of ureteral peristaltism. Thanks to subsequent metallic designs (Memokath, Uventa, Allium Medical URS-stent, Resonance), the current generation of ureteral metallic stents has improved the success rate in comparison to classical designs, accommodating to ureteral dynamics and improving the coating and alloys. Despite these advances, today, their application is limited to very selected patients due to the onset of undesirable effects still associated with theses stents, such as obstructive urothelial hyperplasia, encrustation or migration. The precise knowledge of the physiopathological mechanisms responsible for the cited adverse effects, together with the application of Bioengineering enabling the development of drug eluting metallic stents, biocoated stents, or new biodegradable metallic materials that mitigate or diminish their effects, may be the key to allow the development of the ideal metallic stent.

Validity of insertion depth measurement in double-balloon endoscopy.

BACKGROUND AND STUDY AIMS: In double-balloon enteroscopy (DBE) the use of a reliable and practical method to calculate the insertion depth of the endoscope could help to improve diagnosis accuracy and optimize the effort and cost of the technique. The objectives of this work were to compare and evaluate two methods of estimating the insertion depth and to obtain a descriptive model capable of representing the exploration dynamics and efficiency in terms of advanced distance and time. METHODS: Oral DBE was performed in 25 pigs. Insertion depth was calculated during the procedure by: 1) estimation of time and distance for each push and pull cycle during progression; and 2) estimation of distance during withdrawal. At the maximum insertion depth a tattoo was placed, and the observed measures for the two methods were compared with the distance between the pylorus and the mark after euthanasia and necropsy of the animals 1 week after DBE. RESULTS: The average insertion depth during progression, withdrawal, and after necropsy was 324.92 cm, 317.23 cm, and 342.05 cm, respectively (P Anova = 0.72). The Pearson correlation coefficient (r > 0.85; P < 0.001) and paired Brand - Altman plots demonstrated high agreement between progression and necropsy (0.03 % difference) and between withdrawal and necropsy (6.9 % difference). The exploration dynamics and efficiency in terms of advanced distance per cycle and time fitted to potential and logarithmic regression models, respectively. CONCLUSIONS: Measurement of insertion depth in vivo was validated in the porcine model during progression and withdrawal. Estimation during progression was more accurate and allowed exploration dynamics and efficiency to be plotted, which might be used as approximate reference values for humans.

Estudio experimental con Ecografía Doppler en uropatía obstructiva parcial crónica / Experimental study with Doppler Ultrasound in partial chronic obstructive uropathy

Objetivo: La valoración de los parámetros hemodinámicos índice de resistencia renal (IR), velocidad pico sistólica (PSV), velocidad mínima diastólica (EDV) y flujo de la arteria renal (FR) mediante ecografía Doppler para el diagnóstico y monitorización posquirúrgica de la uropatía obstructiva parcial crónica. Material y métodos: Se emplean 50 animales de la especie porcina. El estudio se divide en 3 fases. La fase I consta de la valoración ecográfica dúplex-Doppler de ambos riñones, determinando los parámetros objeto de estudio. La ratio de cada índice se calcula como la diferencia entre el valor del riñón en estudio y su contralateral. El examen termina con la realización de cistografía de compresión, urografía excretora y ureteropielografía retrógrada. Seguidamente, se procede a la creación de un modelo de obstrucción parcial del uréter derecho. Transcurridas 6 semanas, comienza la fase II mediante la realización de las pruebas descritas anteriormente y posterior resolución endourológica de la obstrucción. La fase III consiste en un seguimiento a los 6 meses del tratamiento mediante las pruebas realizadas en las fases previas. Resultados: De los parámetros estudiados la EDV y su ratio son los que mayor sensibilidad y especificidad muestran como marcador diagnóstico de uropatía obstructiva. En el seguimiento posquirúrgico se observa como el IR y principalmente la EDV retornan a los valores basales. Conclusiones: La ΔEDV es el parámetro que mayor eficacia muestra para el diagnóstico de uropatía obstructiva parcial crónica; pero esta es insuficiente para relegar las técnicas de diagnóstico convencionales. Todos los parámetros, principalmente la EDV, han mostrado ser útiles como pruebas complementarias de monitorización y pronóstico tras la resolución endourológica de la uropatía obstructiva (AU)

Introduction: This study has aimed to assess the hemodynamic parameters, Renal Resistive Index (RI), Peak Systolic Velocity (PSV), End-Diastolic Velocity (EDV) and Blood Flow of the Renal Artery (FR) by Doppler Ultrasound for diagnosis and monitoring postsurgical partial chronic obstructive uropathy. Material and methods: Fifty pigs were used. The experiment was divided into three phases. Phase I consisted of a duplex-Doppler evaluation of the both kidneys to determine the parameters under study. The ratio of each index is calculated as the difference between the value of study kidney and the contralateral. After, a fluoroscopic examination was performed by compressive cystography, excretory urography and retrograde ureteropyelography. Finally, a model of partial right ureteral obstruction was created. After six weeks of the obstructive model, Phase II was begun with the diagnosis of the uropathy, by means of the aforementioned diagnostic methods and the endourological treatment was completed. Phase III is a follow-up performed at 6 months of treatment using the same methods as in the previous phases. Results: Of the parameters studied, the EDV and its ratio showed greater sensitivity and specificity as a diagnostic marker of obstructive uropathy. In the postoperative monitoring, it was observed that the RI and the EDV returned to baseline levels, with the baseline values. Conclusions: The ΔEDV and its ratio is the parameter that shows the greater efficacy for the diagnosis of chronic partial obstructive uropathy, however, it is insufficient to avoid conventional diagnostic techniques. All the parameters, mainly the EDV, have proven useful as complementary tests for monitoring after endourologic resolution of obstructive uropathy (AU)

[Experimental study with Doppler ultrasound in partial chronic obstructive uropathy]. / Estudio experimental con Ecografía Doppler en uropatía obstructiva parcial crónica.

INTRODUCTION: This study has aimed to assess the hemodynamic parameters, Renal Resistive Index (RI), Peak Systolic Velocity (PSV), End-Diastolic Velocity (EDV) and Blood Flow of the Renal Artery (FR) by Doppler Ultrasound for diagnosis and monitoring postsurgical partial chronic obstructive uropathy. MATERIAL AND METHODS: Fifty pigs were used. The experiment was divided into three phases. Phase I consisted of a duplex-Doppler evaluation of the both kidneys to determine the parameters under study. The ratio of each index is calculated as the difference between the value of study kidney and the contralateral. After, a fluoroscopic examination was performed by compressive cystography, excretory urography and retrograde ureteropyelography. Finally, a model of partial right ureteral obstruction was created. After six weeks of the obstructive model, Phase II was begun with the diagnosis of the uropathy, by means of the aforementioned diagnostic methods and the endourological treatment was completed. Phase III is a follow-up performed at 6 months of treatment using the same methods as in the previous phases. RESULTS: Of the parameters studied, the EDV and its ratio showed greater sensitivity and specificity as a diagnostic marker of obstructive uropathy. In the postoperative monitoring, it was observed that the RI and the EDV returned to baseline levels, with the baseline values. CONCLUSIONS: The ΔEDV and its ratio is the parameter that shows the greater efficacy for the diagnosis of chronic partial obstructive uropathy, however, it is insufficient to avoid conventional diagnostic techniques. All the parameters, mainly the EDV, have proven useful as complementary tests for monitoring after endourologic resolution of obstructive uropathy.

Bosentan inhibits cigarette smoke-induced endothelin receptor expression in pulmonary arteries.

The endothelin (ET) system contributes to lung vascular tension and remodelling in smokers and chronic obstructive pulmonary disease (COPD) patients. This study examined the effect of cigarette smoke (CS) on ET receptor A (ET(A)) and B (ET(B)) expression in human pulmonary artery smooth muscle cells (HPASMCs) and human small intrapulmonary arteries, as well as their functional consequences. CS extract (CSE) increased ET(A) and ET(B) expression in HPASMCs and small intrapulmonary arteries, which was attenuated by bosentan, the ET(A) antagonist BQ123 and the ET(B) antagonist BQ788, and by blocking ET-1 with a monoclonal antibody against ET-1, suggesting a feed-forward mechanism mediated by ET-1 release. ET receptor (ETR) antagonism attenuated the CSE-induced HPASMC proliferation. Furthermore, CSE exposure increased the acute ET-1-induced small intrapulmonary artery contraction, which was attenuated by bosentan, BQ123 and BQ788. Pulmonary arteries from smokers and COPD patients showed a higher expression of ET(A) and ET(B) than those of nonsmoker patients. These results show a novel mechanism by which ETR blockade attenuates CS-induced ETR overexpression and, subsequently, small intrapulmonary artery tension. These data may be of potential value to explain therapeutic effects of bosentan in some forms of disproportionate pulmonary hypertension in COPD patients.

Oxidative stress-induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages.

BACKGROUND: Oxidative stress is present in airway diseases such as severe asthma or Chronic Obstructive Pulmonary Disease and contributes to the low response to glucocorticoids through the down-regulation of histone deacetylase (HDAC) activity. OBJECTIVE: To study the effects of the phosphodiesterase (PDE)-3 and 4 inhibitors and their combination vs. glucocorticoids in a model of lipopolysaccharide (LPS)-induced cytokine release in alveolar macrophages under oxidative stress conditions. METHODS: Differentiated U937 or human alveolar macrophages were stimulated with H(2) O(2) (10-1000 µM) or cigarette smoke extract (CSE, 0-15%) for 4 h before LPS (0.5 µg/mL, 24 h) addition. In other experiments, cells were pre-treated with dexamethasone or budesonide (10(-9) -10(-6) M), with the PDE4 inhibitor rolipram (10(-9) -10(-5) M), PDE3 inhibitor motapizone (10 µM), 3',5'-cyclic monophosphate enhancer PGE(2) (10 nM), or with the combination of rolipram (10(-6) M)+PGE(2) (10 nM)+motapizone (10 µM) 15 min before oxidants. IL-8 and TNF-α were measured by ELISA and HDAC activity by a colorimetric assay. RESULTS: Budesonide and dexamethasone produced a concentration-dependent inhibition of the LPS-induced IL-8 and TNF-α secretion with an E(max) about 90% of inhibition, which was reduced by approximately 30% in the presence of H(2)O(2) or CSE. Pre-treatment with rolipram, motapizone or PGE2 only reached about 20% of inhibition but was not affected by oxidative stress. In contrast, PDE4/PDE3 combination in presence of PGE2 effectively inhibited the LPS-induced cytokine secretion by about 90% and was not affected by oxidative stress. Combined PDE4 and PDE3 inhibition reversed glucocorticoid resistance under oxidative stress conditions. HDAC activity was reduced in the presence of oxidative stress, and in contrast to glucocorticoids, pre-treatment with PDE4/PDE3 combination was able to prevent HDAC inactivity. CONCLUSIONS & CLINICAL RELEVANCE: This study shows that the combination of the PDE3/PDE4 inhibitors prevents alveolar macrophage activation in those situations of glucocorticoid resistance, which may be of potential interest to develop new effective anti-inflammatory drugs in airway diseases.

Increased lung neutrophil apoptosis and inflammation resolution in nonresponding pneumonia.

Neutrophil activation state and its relationship with an inflammatory environment in community-acquired pneumonia (CAP) remain insufficiently elucidated. We aimed to evaluate the neutrophil apoptosis and cytokine pattern in CAP patients after 72 h of treatment, and their impact on infection resolution. Apoptosis of blood and bronchoalveolar lavage (BAL) neutrophils was measured in nonresponding CAP (NCAP), in responding CAP (blood only) and in patients without infection (control). Pro-inflammatory (interleukin (IL)-6, IL-8) and anti-inflammatory (IL-10) cytokines were measured. Main outcomes were clinical stability and days of hospitalisation. Basal neutrophil apoptosis was higher in the BAL and blood of NCAP, whereas spontaneous apoptosis (after 24 h culture) was lower. Cytokines in NCAP were higher than in responding CAP and control: IL-6 was increased in BAL and blood, IL-8 in BAL and IL-10 in blood. An increased basal apoptosis (≥20%) in BAL of NCAP was associated with lower systemic IL-10 (p<0.01), earlier clinical stability (p=0.05) and shorter hospital stay (p=0.02). A significant correlation was found for systemic IL-6 and IL-10 with days to reach stability and length of stay. After 72 h of treatment, an increased basal alveolar neutrophil apoptosis might contribute to downregulation of inflammation and to faster clinical stability.

Haemodynamic effects of long-term administration of sildenafil in normotensive pregnant and non-pregnant rats.

OBJECTIVE: To determine the effects of chronic administration of sildenafil citrate on healthy pregnant rats. DESIGN: In vivo animal experimental study. SETTING: Fundación IVI-Instituto Universitario IVI, Valencia, Spain. SAMPLE: Pregnant and non-pregnant Wistar rats exposed to chronic administration of sildenafil. METHODS: Placental cross-barrier and feto-maternal relationship levels, maternal blood pressure, and haemodymamic effects on uterine arteries were evaluated. The effect of growth on weight and fetal tissues, and on perinatal outcome, was investigated. MAIN OUTCOME MEASURES: Maternal blood pressure, blood viscosity, vascular indices of uterine arteries and fetal ductus venosus, plasmatic levels of sildenafil, embryo/fetal and litter weights, perinatal/postnatal survival rates. RESULTS: Sildenafil citrate crossed the placenta. The maternal and fetal levels of sildenafil, and its metabolite desmethyl-sildenafil, demonstrated a positive linear correlation in treated pregnant animals versus controls; a selective maternal hypotensive effect without changes in uterine vascular resistance was noted on days E8 and E11 (embryonic day). The lower pulsatility index of the ductus venosus on day E18 suggests fetal overflow at the end of the pregnancy. Effects on offspring were placental and liver enlargement, and increased fetal weight gain in the second half of pregnancy (irrespective of liver enlargement) and at birth. Perinatal and postnatal survival rates in the sildenafil group remained unaltered. No haemodynamic effects were evident in non-pregnant animals. CONCLUSIONS: In normotensive rats, sildenafil appears to have a selective effect at the onset of pregnancy, implying increased fetal blood supply, and increased fetal weight, and placental and liver enlargement, but no increased perinatal mortality.

Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways.

Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 µM) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC5AC mRNA and protein expression with potency (half maximal inhibitory concentration) ~1 nM in human bronchus and cultured airway epithelial cells. AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, inhibited carbachol-induced MUC5AC responses, indicating EGFR transactivation. Aclidinium inhibited carbachol-induced phospho-EGFR and phospho-p44/42 MAPK expression. In cultured airway epithelial cells transfected with small interfering (si)RNA against muscarinic receptor subtypes, siRNA-M3 but not siRNA-M2 blocked carbachol-induced MUC5AC expression. Cigarette smoke-induced MUC5AC upregulation in cultured airway epithelial cells was suppressed by aclidinium. In conclusion, aclidinium decreases carbachol and tobacco smoke-induced MUC5AC overexpression in human airway epithelial cells. This effect may contribute to the clinical efficacy of aclidinium in mucus hypersecretory diseases including COPD.

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences.

BACKGROUND AND PURPOSE: Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET(B) ) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction. EXPERIMENTAL APPROACH: ET(B) receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca(2+) , F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A(2) and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively. KEY RESULTS: Cigarette smoke extract dose-dependently increased ET(B) receptor expression in HPAECs after 24h incubation. CSE-induced ET(B) expression was attenuated by bosentan, the ET(B) receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET(B) receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET(B) receptor expression, mimicking the effect of CSE. CSE-induced ET(B) receptor overexpression caused greater cell contraction; increased intracellular Ca(2+) ; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA(2) and ROS production; and decreased NO after acute ET-1 (10nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine. CONCLUSIONS AND IMPLICATION: Cigarette smoke extract induced ET(B) receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET(B) receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells.

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca(2+)-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (-logEC(50)=4.67+/-0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20muM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca(2+)-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G(q)-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, alpha(1)(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca(2+) mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.

Taurine chloramine inhibits functional responses of human eosinophils in vitro.

BACKGROUND: Eosinophils are prominent effectors of allergic inflammation. Taurine-chloramine (TauCl), a derivative of the amino acid taurine, shows antioxidant properties in different cell systems but its effects on eosinophils have not been reported. OBJECTIVE: To study the effects of TauCl and taurine on functional responses of isolated human eosinophils activated by different stimuli. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of TauCl and taurine (0.1-1 mM) were investigated on the generation of superoxide anion (ferricytochrome-c reduction microassay), calcium signal (fluorimetry), p47phox-p67phox translocation (Western blot), leukotriene C4 (LTC4) production (enzymeimmunoassay), eosinophil peroxidase (EPO) release (spectrophotometry), eosinophil cationic protein (ECP) release (radioimmunoassay), apoptosis (flow cytometry with annexin V-propidium iodide), and nuclear factor-kappaB (NF-kappaB) activation (Western blot). RESULTS: TauCl inhibited superoxide anion generation triggered by N-formyl-Met-Leu-Phe (fMLP; 30 nM), phorbol myristate acetate (1 nM) and serum opsonized zymosan (0.5 mg/mL) with similar potency (IC50 approximately 200 microM) for the three stimuli, while taurine (0.1-1 mM) was scarcely effective. TauCl but not taurine inhibited p47phox-p67phox translocation. TauCl (200 microM) and taurine (1 mM) did not modify the [Ca2+]i responses to fMLP. TauCl inhibited the release of EPO (IC50 approximately 200 microM) and reduced ECP and LTC4 production from fMLP-activated eosinophils while taurine was without significant effects. TauCl (1 mM) did not change constitutive apoptosis but significantly attenuated the ability of granulocyte-monocyte colony-stimulating factor (GM-CSF) and IL-5 to prevent apoptosis. The activation of eosinophil NF-kappaB induced by GM-CSF and IL-5 was suppressed by TauCl. CONCLUSION: Taurine is without significant in vitro effects on human eosinophil functions but its derivative TauCl inhibits oxidative burst and generation of inflammatory mediators, and reverses the survival effect produced by inflammatory cytokines. Therefore, endogenous TauCl may help to suppress excessive inflammatory response in eosinophils at inflammatory sites.

Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury.

BACKGROUND AND PURPOSE: The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in 'preventive' and 'therapeutic' protocols and compared with glucocorticoids. EXPERIMENTAL APPROACH: Roflumilast (1 and 5 mg.kg(-1).d(-1), p.o.) or dexamethasone (2.5 mg.kg(-1).d(-1), p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U.kg(-1)). In Wistar rats, roflumilast (1 mg.kg(-1).d(-1), p.o.) was compared with methylprednisolone (10 mg.kg(-1).d(-1), p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillation of bleomycin (7.5 U.kg(-1)). Analyses were performed at the end of the treatment periods. KEY RESULTS: Preventive. Roflumilast reduced bleomycin-induced lung hydroxyproline, lung fibrosis and right ventricular hypertrophy; muscularization of intraacinar pulmonary vessels was also attenuated. The PDE4 inhibitor diminished bleomycin-induced transcripts for tumour necrosis factor (TNFalpha), transforming growth factor (TGFbeta), connective tissue growth factor, alphaI(I)collagen, endothelin-1 and the mucin, Muc5ac, in lung, and reduced bronchoalveolar lavage fluid levels of TNFalpha, interleukin-13, TGFbeta, Muc5ac, lipid hydroperoxides and inflammatory cell counts. Therapeutic. In mice, roflumilast but not dexamethasone reduced bleomycin-induced lung alphaI(I)collagen transcripts, fibrosis and right ventricular hypertrophy. Similar results were found in the rat. CONCLUSIONS AND IMPLICATIONS: Roflumilast prevented the development of bleomycin-induced lung injury, and alleviated the lung fibrotic and vascular remodeling response to bleomycin in a therapeutic protocol, the latter being resistant to glucocorticoids.

[Quantification of liver iron concentration using 1-Tesla MRI]. / Cuantificación de la concentración de hierro en hígado mediante RM de 1 Tesla.

OBJECTIVE: To evaluate the quantification of liver iron concentration using 1-Tesla magnetic resonance imaging (MRI) and its ability to diagnose or rule out hemochromatosis. To evaluate the role of 1.5-Tesla MRI in inconclusive cases. MATERIAL AND METHOD: Between 2002 and 2006, we used 1-Tesla MRI (Gandon method) and liver biopsy to quantify the liver iron concentration in 31 patients. Moreover, we used 1.5-Tesla MRI (according to Alústiza's model) and liver biopsy to determine the liver iron concentration in 10 additional patients and to check the results of 10 patients in whom 1-Tesla MRI detected iron overload. RESULTS: In the first group of 31 patients, liver biopsy classified the liver iron concentration as normal (<36 micromol.Fe/g) in 11 patients, as hemosiderosis (36-80 micromol.Fe/g) in 15, and as hemochromatosis (>80 micromol.Fe/g) in 5. The correlation with the values calculated at MRI was 100% in the 5 cases with hemochromatosis; in the 15 patients with hemosiderosis, 5 were correctly classified and the liver iron concentration was overestimated in 10; of the 11 patients with normal liver iron concentration, 6 were correctly classified and 5 were overestimated. Quantification >80 at MRI has a sensitivity and negative predictive value of 100% and specificity of 50% for the diagnosis of hemochromatosis. Quantification <36 at MRI has a positive predictive value and specificity of 100% to identify the absence of iron overload. In the 10 patients with liver biopsy that underwent 1.5-Tesla MRI, there was a high correlation between the two techniques. CONCLUSION: The reliability of the evaluation of liver iron concentration using 1-Tesla MRI is useful for ruling out hemochromatosis and identifying patients without iron overload. We observed a tendency to overestimate liver iron concentration in both patients with overload and in those without, and this limits the reliability of the technique. 1.5-Tesla MRI is a good alternative for quantifying liver iron concentration more precisely.

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability.

BACKGROUND AND PURPOSE: The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. EXPERIMENTAL APPROACH: In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor alpha (TNFalpha)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced endothelial permeability was evaluated. Flow cytometry was used to determine the effect of roflumilast on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced CD11b upregulation on human neutrophils. KEY RESULTS: In vivo, roflumilast, given 1 h before lipopolysaccharide (LPS), dose-dependently reduced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venules. It also diminished histamine-induced microvascular permeability. Immunohistochemical analyses revealed that roflumilast prevented LPS-induced endothelial P- and E-selectin expression. In vitro, roflumilast N-oxide concentration-dependently suppressed neutrophil adhesion to TNFalpha-activated HUVEC and CD11b expression on fMLP-stimulated neutrophils. It also reduced TNFalpha-induced E-selectin expression on HUVEC, when PDE3 activity was blocked. HUVEC permeability elicited by thrombin was concentration-dependently suppressed by roflumilast N-oxide. While roflumilast N-oxide was as potent as roflumilast at inhibiting stimulated endothelial cell and neutrophil functions, both compounds were significantly more potent than the structurally unrelated PDE4 inhibitors, rolipram or cilomilast. CONCLUSIONS AND IMPLICATIONS: These findings further support earlier observations on the inhibition of inflammatory cell influx and protein extravasation by roflumilast in vivo.

Modulatory effects of N-acetyl-L-cysteine on human eosinophil apoptosis.

Eosinophils are oxidant-sensitive cells considered relevant in allergic inflammation. The present study aimed to examine the effects of the antioxidant N-acetyl-L-cysteine (NAC) on constitutive and cytokine-delayed apoptosis in human isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic separation system. Apoptosis and cellular glutathione were assessed by cytofluorometric analysis and nuclear factor (NF)-kappaB binding activity assessed by electrophoresis mobility shift assay. The rate of spontaneous apoptosis of human eosinophils after 24 h culture, as assessed by annexin-V-positive staining, was mean+/-sem 48.2+/-1.4%, n = 5. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng.mL(-1)) decreased apoptosis to 19.4+/-1.8%, n = 5. NAC (5 mM) inhibited spontaneous apoptosis (33.6+/-2.7%, n = 5) but augmented apoptosis in the presence of GM-CSF (30.9+/-1.5%, n = 5). NAC (5 mM) also increased the rate of apoptosis in the presence of tumour necrosis factor (TNF)-alpha (10 ng.mL(-1)) and interleukin-5 (5 ng.mL(-1)). NAC (5 mM) increased eosinophil glutathione content. The increase in eosinophil NF-kappaB binding activity induced by GM-CSF and TNF-alpha was suppressed by NAC. In conclusion, N-acetylcysteine modulates eosinophil apoptosis by inhibiting constitutive apoptosis but reversing the survival effect produced by inflammatory cytokines in human eosinophils.