Publications by authors named "O Bulbena"

89 Publications

Macrophage activation in exacerbated COPD with and without community-acquired pneumonia.

Eur Respir J 2010 Aug 23;36(2):285-91. Epub 2009 Dec 23.

Dept Experimental Pathology, Institut d'Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas IIBBCSIC, Insitut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

In large series of nonresponding community-acquired pneumonia (CAP) patients, chronic obstructive pulmonary disease (COPD) was observed to be a protective factor for nonresponse to initial antibiotics. This intriguing fact may be linked to changes in the phenotype of inflammatory cells and, in particular, to the induction of classical-M1 or alternative-M2 activation of macrophages, which result in different inflammatory profiles. We evaluated the effect of sputum obtained from patients with acute exacerbation of COPD (AECOPD), CAP and COPD+CAP on the phenotypic changes in macrophages. Human THP1 cells differentiated to macrophages were incubated with sputum from patients with AECOPD, CAP or COPD+CAP, and expression of tumour necrosis factor-alpha, interleukin-6, mannose receptor and arginase was measured to evaluate the phenotype acquired by macrophages. We found that sputum from CAP patients induced the M1 phenotype and that from AECOPD patients induced an M2-like phenotype. Sputum from CAP+COPD patients did not present a clear M1 or M2 phenotype. These results indicate that the microenvironment in the lung modulates the activation of macrophages, resulting in different phenotypes in AECOPD, CAP and COPD+CAP patients. This different type of activation induces different inflammatory responses and may be involved in the different outcome observed when COPD and CAP present simultaneously.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1183/09031936.00118909DOI Listing
August 2010

Biological consequences of oxygen desaturation and respiratory effort in an acute animal model of obstructive sleep apnea (OSA).

Sleep Med 2009 Sep 1;10(8):892-7. Epub 2009 Apr 1.

Laboratori de la Son. Servei de Pneumologia, Sleep Lab, Hospital Clinic Provincial-IDIBAPS, C/Casanoves 170, Barcelona, Spain.

Background: An animal model mimicking all the factors involved in obstructive sleep apnea (OSA) is useful for investigating mechanisms because the associated comorbidity usually present in such patients is an important limitation.

Aim: To test the hypothesis that hypoxia/normoxia and respiratory effort have different effects on the induction of inflammatory response and endothelial dysfunction in an acute rat model of OSA.

Methods: Four groups of anesthetized rats were studied (n=8): (1) sham; (2) apnea: obstructions (15s each, 60/h, for 3h); (3) apnea+O(2): obstructions and breathing oxygen-enriched air to avoid hypoxia and (4) intermittent hypoxia/normoxia. Inflammatory and endothelial mediators were measured as outcomes along with NF-kappaB in the lung and diaphragm.

Results: TNF-alpha and IL-1beta significantly increased in all groups compared with sham. NF-kappaB in the lung was increased in apnea and hypoxia/normoxia groups, but not in apnea+O(2) group. In diaphragm tissue, NF-kappaB was only significant in apnea compared to sham. Significant differences were found in the ratio thromboxane-B2/6-keto-Prostaglandin-F1alpha between apnea and hypoxia/normoxia compared to sham but not in apnea+O(2).

Conclusions: Oxygen desaturations and respiratory efforts play a role in the induction of systemic inflammation but only hypoxia/normoxia induces endothelial dysfunction. These data suggest a potential role for oxygen therapy in patients with OSA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sleep.2008.09.014DOI Listing
September 2009

Oxidised lipids present in ascitic fluid interfere with the regulation of the macrophages during acute pancreatitis, promoting an exacerbation of the inflammatory response.

Gut 2008 May 18;57(5):642-8. Epub 2008 Jan 18.

Department of Ischemia and Inflammation, Institut d'Investigacions Biomèdiques de Barcelona, Barcelona, Spain.

Background: Pancreatitis-associated ascitic fluid (PAAF) plays a critical role in the pathogenesis of acute pancreatitis. Taking into consideration that damaged pancreas exudes high concentrations of lipolytic enzymes in the peritoneal cavity, large amounts of lipid metabolism derived products could occur in PAAF. In this study, we have examined the involvement of the lipid fraction of PAAF generated in the early stages of experimental acute pancreatitis.

Methods: Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. After 3 h, PAAF was collected and its lipid fraction was obtained. Lipid composition and levels of lipid peroxidation were measured. Toxicity was evaluated by measuring the effects of the PAAF lipid fraction on cell viability of hepatic and macrophage cell lines. In vivo effects on the liver were also evaluated. Effects on the inflammatory response were determined by measuring the levels of nuclear factor kappa B (NF kappa B) activation, the effect on the inhibitory activity of 15-deoxy-PGJ(2) and the possible interference on PPAR gamma activation.

Results: High concentrations of oxidised free fatty acids were detected in PAAF. Besides the direct cell toxicity, the PAAF-derived lipid extract interfered with the anti-inflammatory pathway mediated by PPAR gamma. Addition of this lipid extract to macrophage cell cultures had no direct effect on the activation of NF kappa B, but abolished the inhibitory activity of endogenous PPAR gamma agonists such as 15-deoxy-PGJ(2).

Conclusions: Oxidised free fatty acids present in PAAF interfere with the endogenous regulatory mechanism of the inflammatory response, thus promoting an exacerbation of macrophage activation in acute pancreatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gut.2007.127472DOI Listing
May 2008

Intratracheal transplantation of alveolar type II cells reverses bleomycin-induced lung fibrosis.

Am J Respir Crit Care Med 2007 Dec 19;176(12):1261-8. Epub 2007 Jul 19.

Department of Experimental Pathology, IIBB-CSIC-IDIBAPS, C/Rosselló, 161, 7, 08036 Barcelona, Spain.

Rationale: Transplantation of stem cells has been proposed as a strategy for repair of lung fibrosis. Nevertheless, many studies have yielded controversial results that currently limit the potential use of these cells as an efficient treatment. Alveolar type II cells are the progenitor cells of the pulmonary epithelium and usually proliferate after epithelial cell injury. During lung fibrosis, however, the altered regeneration process leads to uncontrolled fibroblast proliferation.

Objectives: To investigate whether intratracheal transplantation of isolated alveolar type II cells can halt and reverse the fibrotic process in an experimental model of bleomycin-induced lung fibrosis in rats.

Methods: Lung fibrosis was induced in syngeneic female Lewis rats by a single intratracheal instillation of bleomycin (2.5 U/kg). Animals were transplanted with alveolar type II cells from male animals at a dose of 2.5 x 10(6) cells per animal 3, 7, and 15 days after endotracheal bleomycin instillation. Animals were killed 21 days after the induction of lung fibrosis.

Measurements And Main Results: Lung fibrosis was assessed by histologic study and determination of hydroxyproline content. Engraftment of transplanted cells was measured by real-time polymerase chain reaction for the Y chromosome and by fluorescence in situ hybridization for the Y chromosome. Transplantation of alveolar type II cells into damaged lung 3, 7, or 15 days after bleomycin instillation led to reduced collagen deposition, and reduction in the severity of pulmonary fibrosis.

Conclusions: This study demonstrates the potential role of alveolar type II cell transplantation in designing future therapies for lung fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1164/rccm.200610-1491OCDOI Listing
December 2007

Losartan attenuates bleomycin induced lung fibrosis by increasing prostaglandin E2 synthesis.

Thorax 2006 Jul 6;61(7):604-10. Epub 2006 Apr 6.

Servei de Pneumologia, Institut Clínic del Tórax, Hospital Clínic, c/Villarroel 170, Barcelona 08036, Spain.

Background: The angiotensin system has a role in the pathogenesis of pulmonary fibrosis. This study examines the antifibrotic effect of losartan, an angiotensin II type 1 receptor antagonist, in bleomycin induced lung fibrosis and its possible implication in the regulation of prostaglandin E(2) (PGE(2)) synthesis and cyclooxygenase-2 (COX-2) expression.

Methods: Rats were given a single intratracheal instillation of bleomycin (2.5 U/kg). Losartan (50 mg/kg/day) was administrated orally starting one day before induction of lung fibrosis and continuing to the conclusion of each experiment.

Results: Losartan reduced the inflammation induced by bleomycin, as indicated by lower myeloperoxidase activity and protein content in the bronchoalveolar lavage fluid. Collagen deposition induced by bleomycin was inhibited by losartan, as shown by a reduction in the hydroxyproline content and the amelioration of morphological changes. PGE(2) levels were lower in fibrotic lungs than in normal lungs. Losartan significantly increased PGE(2) levels at both 3 and 15 days. A reduction in COX-2 expression by bleomycin was seen at 3 days which was relieved by losartan.

Conclusions: The antifibrotic effect of losartan appears to be mediated by its ability to stimulate the production of PGE(2). Losartan, which is already widely used clinically, could be assessed as a new treatment in lung fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/thx.2005.051946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2104675PMC
July 2006

Influence of portal blood on the development of systemic inflammation associated with experimental acute pancreatitis.

Surgery 2005 Feb;137(2):186-91

Department of Surgery, Hospital Clinic, University of Barcelona, c/Rosselló 161, 7o 08036 Barcelona, Spain.

Background: The liver is a source of systemic proinflammatory mediators in acute pancreatitis. We have investigated the effects of blood from the pancreas and intestine in liver activation and lung inflammation during early stages of experimental acute pancreatitis in a rat model.

Methods: A portosystemic shunt and a mesosystemic shunt were created to prevent the passage of blood coming from the pancreas and the intestine, respectively, to the liver. Pancreatitis was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. After 3 hours, the inflammatory process in the lung and intestine, plasma levels of tumor necrosis factor (TNF)-alpha and their soluble receptor, and mRNA expression of inflammatory mediators in the lung were evaluated.

Results: Portocaval shunting of blood prevented the inflammatory process in the lung, an increase in plasma TNF-alpha concentration, and the expression of TNF-alpha, interleukin (IL)-1beta, and heat-shock protein (HSP)-72 in the lung, but had no effect on plasma levels of soluble TNF-alpha receptor or on expression of inducible nitric oxide synthase (iNOS) and macrophage inflammatory protein (MIP)-2 in the lung. In contrast, mesocaval shunting of blood did not modify any of the parameters evaluated.

Conclusions: Pancreatic blood, but not intestinal blood, plays a key role in liver activation during experimental acute pancreatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.surg.2004.06.039DOI Listing
February 2005

Induction of c-fos messenger RNA by 3-(N-phenylamino)-1,2-propanediol esters, compounds related to Toxic Oil Syndrome.

Chem Biol Interact 2004 Oct;149(2-3):117-23

Deparment of Experimental Pathology, IIBB-CSIC, IDIBAPS, c/ Rosselló 161, 7 08036 Barcelona, Spain.

The Toxic Oil Syndrome (TOS) was a toxic epidemic disease, related to the consumption of rapeseed oil denatured with aniline that affected more than 20,000 people in Spain and resulted in more than 330 deaths after its sudden appearance in 1981. It has been reported that the fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have shown a strong association with TOS. These PAP-esters could be absorbed and metabolized in a similar way than phospholipids. This is of interest because some products of phospholipid metabolism are important mediators in downstream pathways involved in the regulation of different nuclear factors. In particular, phospholipase D activity is involved in the activation of c-fos. Thus, we have investigated the effect of different PAP-esters in the induction of c-fos in lung fibroblasts. Results indicate that PAP-esters rapidly induced the expression of c-fos in a dose-dependent manner. In addition, both butanol and propranolol prevent this induction pointing to the involvement of phospholipase D in this activation. These results suggest that deregulation of some nuclear factors such as AP-1 could be involved in the pathogenesis of TOS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbi.2004.07.003DOI Listing
October 2004

Oxygen in the alveolar air space mediates lung inflammation in acute pancreatitis.

Free Radic Biol Med 2004 Nov;37(10):1640-7

Department of Experimental Pathology, Institut d'Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain.

During the early stages of acute pancreatitis, acute respiratory distress syndrome often occurs. This is associated with the release of proinflammatory mediators into the blood, but it remains unclear why these mediators induce inflammation especially in the lung. One of the first events occurring during the progression of acute pancreatitis is the induction of P-selectin expression in the endothelial cells of the lung. This expression has been associated with the generation of superoxide radicals by circulating xanthine oxidase. Because this enzyme needs molecular oxygen to perform the reaction, we have hypothesized that oxygen present in the alveolar space favors the generation of free radicals by xanthine oxidase and explains why P-selectin is expressed only in the lung. For this purpose, we evaluated the progression of the inflammatory process in rats with induced acute pancreatitis and one lung breathing nitrogen while the other lung continued breathing air. Acute pancreatitis was induced by intraductal administration of taurocholate and myeloperoxidase; P-selectin expression was measured 3 h after induction. Results indicated that, in the absence of oxygen in the alveolar space, the xanthine oxidase-dependent P-selectin expression did not occur and lung inflammation was significantly reduced.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2004.07.036DOI Listing
November 2004

Mobilization of xanthine oxidase from the gastrointestinal tract in acute pancreatitis.

BMC Gastroenterol 2004 Jan 19;4. Epub 2004 Jan 19.

Dept, of Experimental Pathology, Institut d'Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas (IIBB-CSIC), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Background: Xanthine oxidoreductase has been proposed to play a role in the development of local and systemic effects of acute pancreatitis. Under physiologic conditions, the enzyme exists mainly as xanthine dehydrogenase (XDH) but can be converted by proteolytic cleavage to its superoxide-generating form xanthine oxidase (XOD). In addition to its intracellular location XDH/XOD is also associated to the polysaccharide chains of proteoglycans on the external endothelial cell membrane. In the early stages of acute pancreatitis, this enzyme seems to be arising from its mobilization from the gastrointestinal endothelial cell surface. Taking into account the ability of alpha-amylase to hydrolyze the internal alpha-1,4 linkages of polysaccharides, we wanted to elucidate the involvement of alpha-amylase in XDH/XOD mobilization from the gastrointestinal endothelial cell surface and the relevance of the ascitic fluid (AF) as the source of alpha-amylase in experimental acute pancreatitis.

Methods: Acute pancreatitis was induced in male Wistar rats by intraductal administration of 5% sodium taurocholate. In another experimental group 3000 U/Kg alpha-amylase was i.v. administered. The concentrations of XDH, XOD and alpha-amylase in plasma and AF and myeloperoxidase (MPO) in lung have been evaluated. In additional experiments, the effect of peritoneal lavage and the absorption of alpha-amylase present in the AF by an isolated intestine have been determined.

Results: Similar increase in XDH+XOD activity in plasma was observed after induction of acute pancreatitis and after i.v. administration of alpha-amylase. Nevertheless, the conversion from XDH to XOD was only observed in the pancreatitis group. Lung inflammation measured as MPO activity was observed only in the pancreatitis group. In addition peritoneal lavage prevented the increase in alpha-amylase and XDH+XOD in plasma after induction of pancreatitis. Finally, it was observed that alpha-amylase is absorbed from the AF by the intestine.

Conclusions: During the early stages of acute pancreatitis, alpha-amylase absorbed from AF through the gastrointestinal tract could interfere with the binding of XDH/XOD attached to glycoproteins of the endothelial cells. Proteolytic enzymes convert XDH into its oxidase form promoting an increase in circulating XOD that has been reported to be one of the mechanisms involved in the triggering of the systemic inflammatory process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-230X-4-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331409PMC
January 2004

In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats.

Br J Pharmacol 2003 Mar;138(6):1037-48

Department of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona (IIBB-IDIBAPS), CSIC, Barcelona, Spain.

1. This study examines the activity of the antioxidant N-acetylcysteine on bleomycin-induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. 2. Rats receiving N-acetylcysteine (300 mg kg(-1) day(-1), intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. 3. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N-acetylcysteine. Alveolar macrophages from bleomycin-exposed rats released augmented amounts of superoxide anion and nitric oxide. N-Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. 4. N-Acetylcysteine suppressed the bleomycin-induced increased activation of lung NF-kappaB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor-alpha, interleukin-beta, interleukin-6 and macrophage inflammatory protein-2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. 5. At 15 days postbleomycin, N-acetylcysteine decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content: 6351+/-669 and 4626+/-288 micro g per lung in drug vehicle- and N-acetylcysteine-treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N-acetylcysteine-treated rats compared to those receiving bleomycin alone. 6. These results indicate that N-acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.bjp.0705138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573750PMC
March 2003

Nuclear factor-kappaB activity is down-regulated in nasal polyps from aspirin-sensitive asthmatics.

Allergy 2003 Feb;58(2):122-6

Servei de Pneumologia i Allèrgia Respiratòria, Institut Clínic de Pneumologia i Cirurgia Toràcica, Universitat de Barcelona, Barcelona, Spain.

Background: We examined whether a decreased activity of nuclear factor(NF)-kappaB), a transcriptional regulator of cyclooxygenase-2 (COX-2), could account for down-regulation of COX-2 in nasal polyps of aspirin-sensitive asthmatics.

Methods: Nasal polyps were obtained from 17 aspirin-intolerant asthma/rhinitis patients (AIAR; 7 men, mean age 48 +/- 12 years) and 23 aspirin-tolerant asthma/rhinitis patients (ATAR; 12 men, mean age 65 +/- 11 years). COX-2 mRNA expression was measured using semiquantitative reverse transcriptase competitive polymerase chain reaction (RT-PCR), and the results were expressed as mean +/- standard error of 106 molecules of mRNA/ micro g of total RNA. NF-kappaB binding was measured with 32P-labeled oligonucleotides and electrophoretic mobility shift assay (EMSA), and the results were expressed as a percentage with respect to the mean EMSA obtained in 19 healthy nasal mucosa.

Results: The mean levels of COX-2 mRNA expression (0.25 +/- 0.06) and NF-kappaB activity (89 +/- 13) in nasal polyps from AIAR were significantly lower than in polyps from ATAR (COX-2 = 1.58 +/- 0.50, and NF-kappaB = 143 +/- 12, P < 0.01 and P < 0.05, respectively). Levels of COX-2 mRNA and NF-kappaB activity in polyps from patients on corticosteroid therapy did not differ statistically from those who were not on this therapy before polypectomy.

Conclusion: This study shows that the low expression of COX-2 mRNA in nasal polyps from aspirin-sensitive patients is associated with a down-regulation of NF-kappaB activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1034/j.1398-9995.2003.23792.xDOI Listing
February 2003

Heparin mobilizes xanthine oxidase and induces lung inflammation in acute pancreatitis.

Crit Care Med 2003 Feb;31(2):525-30

Department of Experimental Pathology, Institut d' Investigacions Biomèdiques de Barcelona-Consejo Superior de Investigaciones Científicas, Spain.

Objective: To evaluate the effect of low molecular weight heparin on plasma xanthine oxidase concentrations and lung inflammatory response during acute pancreatitis.

Design: Randomized, controlled trial.

Setting: Experimental laboratory.

Subjects: Male Wistar rats.

Interventions: Acute pancreatitis was induced by intraductal administration of 5% sodium taurocholate. Low molecular weight heparin (0, 30, 90, or 300 units/kg) was administered immediately after induction of pancreatitis.

Measurements And Main Results: Lipase and xanthine oxidase plasma concentrations were measured 3 hrs after pancreatitis induction. Expression of P-selectin messenger RNA and myeloperoxidase activity as a marker of neutrophil infiltration were determined in the lung. An increase in xanthine oxidase plasma concentrations was observed during pancreatitis. Administration of heparin also increased plasma xanthine oxidase activity in both control and pancreatitis animals. Measures of xanthine oxidase present in the endothelial surface indicate that during pancreatitis, the enzyme is released from the gastrointestinal endothelium. By contrast, heparin mobilizes xanthine oxidase from almost all organs evaluated. Neutrophil infiltration was increased in the lung during pancreatitis. Heparin administration further increased, in a dose-dependent manner, myeloperoxidase activity and P-selectin expression in the lung in animals with pancreatitis. By contrast, in control animals, heparin had no effect on myeloperoxidase activity and did not induce P-selectin up-regulation.

Conclusion: During acute pancreatitis, heparin administration might mobilize xanthine oxidase attached to endothelial cells, originating a free radical-generating system in the circulation that would trigger an inflammatory response in the lung.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.CCM.0000049948.64660.06DOI Listing
February 2003

P-selectin upregulation in bleomycin induced lung injury in rats: effect of N-acetyl-L-cysteine.

Thorax 2002 Jul;57(7):629-34

Department of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona (IIBB-IDIBAPS), CSIC, Barcelona, Spain.

Background: A number of adhesion molecules are involved in the process of neutrophil infiltration into the lung. P-selectin is one of these neutrophil-endothelial cell adhesion molecules. A study was undertaken to examine the involvement of P-selectin in the development of bleomycin induced inflammation and the ability of N-acetyl-L-cysteine to reduce the potential expression of this selectin in rats.

Methods: N-acetyl-L-cysteine (3 mmol/kg po) was administered daily for seven days prior to bleomycin administration (2.5 U/kg). The kinetics of P-selectin expression and the effect of N-acetyl-L-cysteine after bleomycin treatment were measured using radiolabelled antibodies. P-selectin localisation was evaluated by immunohistochemistry and neutrophil infiltration was assessed by myeloperoxidase activity.

Results: Bleomycin administration resulted in an upregulation of P-selectin at 1 hour, returning to baseline at 3 hours. Myeloperoxidase activity showed a significant increase at 6 hours after bleomycin administration that lasted for 3 days. N-acetyl-L-cysteine treatment completely prevented these increases.

Conclusion: Upregulation of P-selectin in the lung is associated with neutrophil recruitment in response to bleomycin. The beneficial effect of N-acetyl-L-cysteine on bleomycin induced lung injury may be explained in part by the prevention of neutrophil recruitment in the inflammatory stage of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1746384PMC
http://dx.doi.org/10.1136/thorax.57.7.629DOI Listing
July 2002

Ischemic preconditioning: a defense mechanism against the reactive oxygen species generated after hepatic ischemia reperfusion.

Transplantation 2002 Apr;73(8):1203-11

Department of Clinical and Biological Sciences, Section Genral Pathology, University of Turin, Italy.

Background: Preconditioning protects against both liver and lung damage after hepatic ischemia-reperfusion (I/R). Xanthine and xanthine oxidase (XOD) may contribute to the development of hepatic I/R.

Objective: To evaluate whether preconditioning could modulate the injurious effects of xanthine/XOD on the liver and lung after hepatic I/R.

Methods: Hepatic I/R or preconditioning previous to I/R was induced in rats. Xanthine and xanthine dehydrogenase/xanthine oxidase (XDH/XOD) in liver and plasma were measured. Hepatic injury and inflammatory response in the lung was evaluated.

Results: Preconditioning reduced xanthine accumulation and conversion of XDH to XOD in liver during sustained ischemia. This could reduce the generation of reactive oxygen species (ROS) from XOD, and therefore, attenuate hepatic I/R injury. Inhibition of XOD prevented postischemic ROS generation and hepatic injury. Administration of xanthine and XOD to preconditioned rats led to hepatic MDA and transaminase levels similar to those found after hepatic I/R. Preconditioning, resulting in low circulating levels of xanthine and XOD activity, reduced neutrophil accumulation, oxidative stress, and microvascular disorders seen in lung after hepatic I/R. Inhibition of XOD attenuated the inflammatory damage in lung after hepatic I/R. Administration of xanthine and XOD abolished the benefits of preconditioning on lung damage.

Conclusions: Preconditioning, by blocking the xanthine/XOD pathway for ROS generation, would confer protection against the liver and lung injuries induced by hepatic I/R.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/00007890-200204270-00004DOI Listing
April 2002

Strategies to modulate the deleterious effects of endothelin in hepatic ischemia-reperfusion.

Transplantation 2000 Dec;70(12):1761-70

Departamento de Bioanalítica Médica, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Spain.

Background: This study evaluates whether bosentan (endothelin [ET] receptor antagonist) or preconditioning (mechanism that inhibits the postischemic ET release) could reduce the microvascular disorders and the injurious effects of tumor necrosis factor (TNF) associated with hepatic ischemia-reperfusion (I/R).

Methods: Hepatic I/R was induced in rats and the effects of bosentan or preconditioning on the deleterious effects of ET in hepatic I/R were evaluated. Transaminase and TNF levels in plasma; edema, vascular permeability, lactate, ET, and TNF levels in liver; and edema and myeloperoxidase activity levels in lung were measured after hepatic reperfusion.

Results: The administration of bosentan or the induction of preconditioning previous to I/R attenuated the increase in vascular permeability, edema and lactate levels observed in liver after I/R. However, the addition of ET before preconditioning abolished its benefits. Preconditioning prevented both the increase in hepatic TNF and its release from the liver into the systemic circulation. This resulted in an attenuation of liver and lung damage. Addition of ET or TNF to the preconditioned group abolished the benefits of preconditioning, whereas the previous inhibition of TNF release with GdCl3 in the preconditioned group pretreated with ET did not modify the effects of preconditioning. The inhibition of ET with bosentan prevented the increase of both hepatic and plasma TNF, thus attenuating the liver and lung injury, whereas TNF addition abolished the benefits of bosentan.

Conclusions: These findings suggest that both bosentan and preconditioning, by inhibition of ET could attenuate the microvascular disorders and the deleterious effect of TNF on the liver and lung elicited by hepatic I/R.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/00007890-200012270-00016DOI Listing
December 2000

Constitutive nuclear factor-kappaB activity in human upper airway tissues and nasal epithelial cells.

Eur Respir J 2000 Mar;15(3):582-9

Dept of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona, CSIC-IDIBAPS, Spain.

Respiratory epithelial cells are actively involved in the host defence and inflammatory reactions of the airways. Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in many cellular responses to environmental changes. The inducible nitric oxide synthase (iNOS) isoform has been implicated in airway inflammation as well as in normal airway function. In this study, the hypothesis that NF-kappaB may be associated with iNOS expression in airway epithelium, not only in inflammatory processes but also under physiological conditions was examined. NF-kappaB deoxyribonucleic acid-binding activity was assayed by means of electrophoretic mobility shift assay (EMSA) and iNOS expression examined using immunohistochemical techniques in healthy nasal mucosa and chronically inflamed nasal polyps. Further NF-kappaB activity was assayed; by means of EMSA, in nasal epithelial cells isolated from both tissues. NF-kappaB was activated in nasal polyps, but also to the same extent in healthy nasal mucosa. Uniform iNOS expression was localized within the airway epithelium in both inflamed and noninflamed tissues. Along with iNOS expression, concomitant NF-kappaB activation was found in nasal epithelial cells obtained from both tissues and no differences were observed when nasal mucosa and nasal polyp were compared. These results suggest that constitutive nuclear factor-kappaB and concurrent inducible nitric oxide synthase expression in epithelial cells may play a physiological role in airway function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1034/j.1399-3003.2000.15.26.xDOI Listing
March 2000

[Nitric oxide synthase activity in nasal mucosa].

Acta Otorrinolaringol Esp 1998 Oct;49(7):503-7

Departamento de ORL, Hospital General Universitario Valle de Hebrón, Barcelona.

Nitric oxide (NO) plays an important regulatory role in airway function and seems to be implicated in the pathophysiology of several airway diseases. We studied the presence of NO synthase activity in human nasal mucosa and nasal polyp tissues obtained from patients undergoing septoplasty or polypectomy, respectively. NO synthase activity was quantified in tissue homogenates using citrulline release assay and was located in tissue sections using NADPH-diaphorase histochemistry. The results indicated that nasal polyps contain higher levels of total NO synthase activity than nasal mucosa tissue. In addition, nasal polyps contained mainly inducible NO synthase activity whereas all NO synthase activity detected in the nasal mucosa was in constitutive form. In both cases, NO synthase activity was localized in epithelial cells. In view of these results, we conclude that NO may be an important inflammatory mediator in the respiratory system and that the epithelium may be a source of NO production.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 1998

Effect of subcutaneous administration of octreotide on endogenous vasoactive systems and renal function in cirrhotic patients with ascites.

Dig Dis Sci 1998 Oct;43(10):2184-9

Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Splanchnic and systemic arteriolar vasodilation plays an important role in ascites formation in cirrhosis. Octreotide produces splanchnic vasoconstriction, but the effects on systemic hemodynamics and renal function are controversial. This study evaluated the effect of subcutaneous octreotide administration on systemic hemodynamics, endogenous vasoactive systems, and renal function in cirrhotic patients with ascites. Twenty patients were included: 10 received octreotide 250 microg/12 hr subcutaneously (for five days), and 10 did not. No statistically significant changes were found in mean arterial pressure and cardiac rate. Octreotide induced a statistically significant decrease in plasma renin activity (P < 0.01), plasma aldosterone (P = 0.01) and plasma glucagon (P < 0.05). No significant variations were observed in other systemic vasoactive substances (nitric oxide and prostacyclin). Renal function was not modified in either group. In conclusion, in cirrhotic patients with ascites, subcutaneous octreotide administration decreases plasma glucagon, renin activity, and aldosterone without changing in systemic hemodynamics or renal function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1023/a:1026698001921DOI Listing
October 1998

Cytoprotective activity in the gastric mucosa of rats exposed to carbon tetrachloride-induced liver injury.

Inflammation 1997 Oct;21(5):475-88

Department of Pharmacology, Laboratorios Viñas, S.A., Barcelona, Spain.

The present study was undertaken to evaluate the cytoprotective activity in the gastric mucosa of rats subjected to CCl4-induced liver injury. Response of gastric mucosa to absolute ethanol insult or acid (pylorus ligation) after CCl4 challenge was analyzed. Intraperitoneal administration of CCl4 increased plasma AST and ALT, but liver protein and glycogen levels were decreased; in addition, gastric acid secretion was significantly increased with respect to control animals (1541 +/- 266 vs. 629 +/- 25 mu eq H+; p < 0.001). Microscopical gastric erosions were observed in 3/10 animals after CCl4 challenge. Pylorus-ligated as well as CCl4-challenged rats developed increased susceptibility to gastric lesions, compared to control (lesion indices: 4.6 +/- 0.20 vs 2.8 +/- 0.13; p < 0.05), while showing increased resistance to absolute ethanol-induced gastric damage (30.4 +/- 11.2 vs 89.7 +/- 9.7 mm, p < 0.01). PGE2 levels in the gastric mucosa were not influenced by exposure to CCl4. Ultrastructural studies revealed the presence of continuous ethanol-resistant and apparently more adherent layer of mucus in CCl4-challenged animals. Morphological evaluation revealed an increase in Alcian blue-stained mucus. A dual condition of enhanced sensitivity to HCl with increased tolerance to ethanol was observed in gastric mucosa of CCl4-treated animals. These observations could be explained by the amount and/or composition of protective mucus layer in the gastric mucosa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1023/a:1027355528929DOI Listing
October 1997

Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine.

Hepatology 1997 Apr;25(4):934-7

Department of Medical Bioanalysis, Institut d'Investigacions Biomèdiques de Barcelona, CSIC, Spain.

The effects of ischemic preconditioning on rat liver integrity, as well as the implication of nitric oxide (NO) and adenosine in this process, has been evaluated. Preconditioning before ischemia-reperfusion prevented the increases in alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) levels, but did not modify blood flow. Adenosine or NO administration previous to hepatic ischemia-reperfusion simulated the effect of preconditioning, whereas inhibition of adenosine or NO synthesis abolished the protective effect of hepatic preconditioning. Nevertheless, inhibition of adenosine and simultaneous administration of NO in preconditioned animals offered similar results to those found in the preconditioned group, indicating that, in the absence of adenosine, NO is able to maintain the preconditioning benefits. It is suggested that, in preconditioning, adenosine stimulates NO production to protect against the injury associated with ischemia-reperfusion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.510250424DOI Listing
April 1997

Calcium channel blockers in experimental acute pancreatitis: effect on tissue prostanoids and oxygen free radicals.

Pancreas 1996 Mar;12(2):178-82

Department of Medical Bioanalysis, Centro de Investigacion y Desarrollo, CSIC, Barcelona, Spain.

The effect of verapamil administration on the changes of prostanoid synthesis, and on free radical production associated with acute pancreatitis, has been evaluated. A necrohemorrhagic model of pancreatitis was induced in Wistar rats by intraductal administration of sodium taurocholate (3.5%). This model is associated with initial increases in prostanoid synthesis and peroxidative damage. Verapamil, administered before pancreatitis induction, prevented initial increases in 6-keto prostaglandin F1alpha (PGF1alpha) and thromboxane B2 (TXB2) but had no effect on PGF2alpha or PGE2 or on lipoperoxidative damage. These results indicate that verapamil administration prevents the increases in pancreatic vasoactive prostanoids (TXB2 and 6-keto PGF1alpha) without affecting the increased levels of PGE2 and PGF2alpha and has no effect on oxygen free radical production in the initial stages of experimental acute pancreatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/00006676-199603000-00012DOI Listing
March 1996

Differential activity of nitric oxide synthase in human nasal mucosa and polyps.

Eur Respir J 1996 Feb;9(2):202-6

Dept of Medical Bionalysis, CSIC, Barcelona, Spain.

Nitric oxide (NO) plays an important regulatory role in airway function and seems to be implicated in the pathophysiology of several airway diseases. To better understand the involvement of NO in the upper airways, we examined the presence of nitric oxide synthase (NOS) activity in human nasal mucosa and nasal polyp tissues. Nasal mucosa was obtained from seven patients undergoing septoplasty, and nasal polyps came from nine patients following polypectomy. NOS activity was quantified in tissue homogenates using the citrulline release assay and localized in tissue sections using reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. The results showed that nasal polyps (n = 9) contained higher levels of total NOS activity (mean +/- SD 5.94 +/- 5.71, range 1.29-18.0 pmol.min-1.mg protein) than nasal mucosa tissues (n = 7) (0.28 +/- 0.22, range 0.01-0.57 pmol.min-1.mg protein). In addition, nasal polyps mainly contained inducible NOS activity (4.67 +/- 4.57, range 1.23-15.5 pmol.min-1.mg protein) whereas in nasal mucosa all NOS activity detected was in constitutive form. In both cases, NOS activity was localized in the epithelial cells. Since NO synthase is induced in inflamed upper airways, we conclude that NO may be an important inflammatory mediator in the respiratory system and that the epithelium may be a source of NO production in the human upper airways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1183/09031936.96.09020202DOI Listing
February 1996

Transmucosal potential difference in experimental colitis in rats.

Inflammation 1995 Aug;19(4):445-55

Department of Surgery, Sagunto Hospital, Valencia, Spain.

Colon transmucosal potential difference (TPD), macro- and microscopic lesions, myeloperoxidase activity, and leukotriene levels were studied after the induction of experimental colitis in the rat. Forty-three male Wistar rats were subjected to the instillation of 200 mg/ml 2,4,6-trinitrobenzenesulfonic acid (TNB) solution through a rectal cannula. TPD measurements were made at different distances from the anus before and 24 h and one, two, three, and four weeks after lesion induction. Leukotriene B4 levels were assayed by intracolonic dialysis 24 h and one, two, three and four weeks after lesion induction. Macro- and microscopic evaluations were made of the bowel lesions, and myeloperoxidase activity was assayed. The mean basal TPD was -46.06 mV at 1 cm from the anus, and +10.86 mV in the proximal colon. Twenty-four hours after lesion induction the values proved markedly positive. This was correlated with an abrupt increase in LTB4 levels and myeloperoxidase activity. After one week the TPD values exhibited a greater electronegativity, returning to basal values by the fourth week after lesion induction. This coincided with an improved macroscopic lesion index, LTB4 levels, and myeloperoxidase activity. In conclusion, TPD is a useful indicator of acute colonic lesions and correlates well with LTB4 and myeloperoxidase assays. Moreover, the parameter is able to delimit lesion evolution, reflecting possible ad integrum restoration of the bowel mucosa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/BF01534578DOI Listing
August 1995

[Scanning electron microscopy in gastric pathology].

Authors:
O Bulbena M L Bravo

Gastroenterol Hepatol 1995 Aug-Sep;18(7):396-403

Departamento de Farmacología, Laboratorios Viñas, S. A. Barcelona.

View Article and Find Full Text PDF

Download full-text PDF

Source
November 1995

Cytoprotective and antisecretory activity of a ranitidine-zinc complex.

Prostaglandins Leukot Essent Fatty Acids 1995 Jun;52(6):393-7

Pharmacology Department, Laboratorios Viñas, S.A., Barcelona, Spain.

The effects of a ranitidine-zinc complex and ranitidine alone were compared in three different experimental models (pyloric ligation, ethanol and indomethacin) of gastric ulceration in the rat. In the pyloric ligation model, the ranitidine-zinc complex (50, 100 and 150 mg/kg p.o.) showed antiulcerogenic activity similar to that observed with equimolar doses of ranitidine (35, 70 and 105 mg/kg p.o.). Both the ranitidine-zinc complex and ranitidine significantly reduced (p < 0.05) gastric acid secretion in a dose-dependent manner. The protective effect of the ranitidine-zinc complex (100 and 150 mg/kg p.o.) against gastric damage developing after p.o. administration of absolute ethanol or indomethacin was enhanced (p < 0.05) with respect to that obtained with equimolar doses of ranitidine (70 and 105 mg/kg p.o.). The presence of zinc in the ranitidine-zinc complex does not interfere with the antisecretory effects of ranitidine on the gastric mucosa, while it confers an additional cytoprotective action to the final compound.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/0952-3278(95)90067-5DOI Listing
June 1995

Effects of omega-toxins on noradrenergic neurotransmission in beating guinea pig atria.

Eur J Pharmacol 1995 Apr;276(3):231-8

Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.

The effects of four omega-toxins, known to block various subtypes of neuronal voltage-activated Ca2+ channels, on the beating guinea pig left atrium have been analyzed. Atria were suspended in oxygenated Krebs-bicarbonate solution at 32 degrees C and driven with electrical pulses delivered by a stimulator at 1 Hz, 1 ms, 4 V. A 10-fold increase of voltage caused a potent and rapid enhancement of the size of contractions (about 3- to 4-fold above basal), which reflects the release of endogenous noradrenaline from sympathetic nerve terminals. omega-Conotoxin MVIIC, omega-conotoxin MVIIA and omega-conotoxin GVIA inhibited the inotropic responses to 10 x V stimulation with IC50 values of 191, 44 and 20.4 nM, respectively. omega-Agatoxin IVA did not affect the contractile responses. The inotropic responses to exogenous noradrenaline were unaffected by the toxins. The potent blocking effects of omega-conotoxin GVIA were present even in conditions in which the release of noradrenaline was strongly facilitated by presynaptic alpha 2-adrenoceptor blockade by phenoxybenzamine. These effects were not reversed upon repeated washing of the tissue with toxin-free medium. In contrast, the blockade induced by omega-conotoxin MVIIC and omega-conotoxin MVIIA were fully reversed, with t1/2 of 13.5 and 31.2 min, respectively. omega-Conotoxin MVIIC (1 microM) protected against the irreversibility of the blockade induced by omega-conotoxin GVIA (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/0014-2999(95)00032-gDOI Listing
April 1995

Nitric oxide and arachidonate metabolism in ischemia-reperfusion associated with pancreas transplantation.

Transplantation 1995 Feb;59(3):417-21

Molecular Pathology Unit, Centro de Investigación y Desarrollo, CSIC, Barcelona, Spain.

The role of eicosanoid metabolism and its relationship with nitric oxide production in the ischemia-reperfusion associated with pancreas transplantation in the rat is explored in this study. Twenty-six male Sprague-Dawley rats were randomized into 3 groups, as follows: group 1, control animals not surgically manipulated; group 2, pancreas transplantation, after 12 hr of organ preservation in University of Wisconsin solution; group 3, same as group 2 but with administration of NG-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor) (10 mg/kg) before organ revascularization. The results show posttransplantation increases in edema and in 6-keto-prostaglandin F1 alpha (x1.9), thromboxane B2 (x4), and prostaglandin E2 (x5) levels in pancreatic tissue. Nitric oxide synthase inhibition reversed the increases in edema and eicosanoid production, which suggests that eicosanoid generation in the recipient rat would be mediated, in part, through a nitric oxide-dependent mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
February 1995

Mass spectrometric identification of N-phenyllinoleamide metabolites in mouse peritoneal macrophages.

Rapid Commun Mass Spectrom 1995 ;9(9):753-60

Departamento de Bioanalítica Médica, CID-CSIC, Barcelona, Spain.

N-Phenyllinoleamide (NPLA), the anilide of linoleic acid, has been regarded as a marker of the case oils associated with toxic oil syndrome, but the mechanisms of toxic injury remain enigmatic. Experimental data have related an increased systemic toxic effect of heated linoleic anilides to chemical structural modifications that might also be possible by in vivo metabolism; however, little is known about their metabolism. Taking into account that NPLA is a derivative of linoleic acid, a fatty acid that can be metabolized by lipoxygenase activity to a vast array of derivatives possessing biological activity, the objective has been to elucidate the oxidative metabolism of NPLA by mouse peritoneal macrophages, a cellular model with high lipoxygenase activity. Cells were incubated with 0.1 mM NPLA spiked with N-phenyl[1-14C]linoleamide. The metabolites were separated by high-performance liquid chromatography and individually collected prior to GC/MS analysis. Identification of trihydroxy-, monohydroxy- and epoxy-derivatives of NPLA, suggests that this xenobiotic can be metabolized via the same oxidative processes as for linoleic acid. Furthermore, identification of the non-amidated monohydroxylated and trihydroxylated derivatives of linoleic acid arising from NPLA suggests an amidase-like activity with release of aniline and the free fatty acid. These results provide information about possible biological structures arising from NPLA, and open the way to evaluate the biological significance of these metabolites in the inflammatory reactions associated with toxic oil syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.1290090907DOI Listing
October 1995

Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion.

Pflugers Arch 1994 Dec;429(2):231-9

Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.

Zn2+ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca2+ independent; in fact, in the absence of extracellular Ca2+, the secretory effects of Zn2+ were enhanced. At low concentrations (3-10 microM), Zn2+ enhanced the secretory responses to 10-s pulses of 100 microM 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K+. In the presence of DMPP, secretion was increased 47% above controls and in high-K+ solutions, secretion increased 54% above control. These low concentrations of Zn2+ did not facilitate the whole-cell Ca2+ (ICa) or Ba2+ (IBa) currents in patch-clamped chromaffin cells. Higher Zn2+ concentrations inhibited the currents (IC50 values, 346 microM for ICa and 91 microM for IBa) and blocked DMPP- and K(+)-evoked secretion (IC50 values, 141 and 250 microM, respectively). Zn2+ permeated the Ca2+ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba2+, Ca2+, Sr2+ and Zn2+ were 991, 734, 330 and 7.4 pA, respectively. Zn2+ entry was also evidenced using the fluorescent Ca2+ probe fura-2. This was possible because Zn2+ causes an increase in fura-2 fluorescence at the isosbestic wave-length for Ca2+, i.e. 360 nm. There was a slow resting entry of Zn2+ which was accelerated by stimulation with DMPP or high-K+ solution. The entry of Zn2+ was concentration dependent, slightly antagonized by 1 mM Ca2+ and completely blocked by 5 mM Ni2+. The entry of Ca2+ evoked by depolarization with high-K+ solution was antagonized by Zn2+.(ABSTRACT TRUNCATED AT 250 WORDS)
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/BF00374317DOI Listing
December 1994