Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

BACKGROUND INFORMATION: Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. RESULTS: Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. CONCLUSIONS AND SIGNIFICANCE: Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression.

In this study, the effects of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.

Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells.

In this study, the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.

Manejo de la pancreatitis enfisematosa / Management of emphysematous necrotizing pancreatitis

La pancreatitis enfisematosa es una complicación grave y rara de la pancreatitis aguda grave, cuyo diagnóstico se hace mediante tomografía computadorizada, ante el hallazgo de gas en la celda pancreática. Su manejo depende en gran medida del estado general del paciente, de forma que se comienza con un manejo conservador inicial, planteándose opciones más agresivas ante el empeoramiento del cuadro clínico. Hoy en día, han tomado mayor auge los tratamientos mínimamente invasivos, aunque el desbridamiento quirúrgico sigue siendo el método de referencia en estos pacientes. Se presenta una revisión amplia de su manejo quirúrgico a raiz de un caso fatal en nuestro servicio.

Emphysematous pancreatitis is a rare and serious complication of severe acute pancreatitis. The diagnosis is made by computed tomography in the presence of gas in the pancreatic cell. Its management will depend largely on the condition of the patient, with a conservative a management at the start, and considering more aggressive options when worsening of symptoms. There has been a recent boom with minimally invasive procedures, but surgical debridement remains the gold standard. We present a subject review after a fatal case of emphysematous pancreatitis that we had in our service.

Hemangiomatosis neonatal difusa / Diffuse neonatal hemangiomatosis

Introducción. La hemangiomatosis neonatal difusa es una entidad poco frecuente. Es un trastorno grave en el que aparecen múltiples hemangiomas cutáneos y viscerales de distribución amplia y, en ocasiones, puede ser fatal. Las complicaciones incluyen falla cardiaca, hemorragias, falla hepática, entre otras. El tratamiento está encaminado a evitar los efectos adversos y las complicaciones. Caso clínico. Se trata de un paciente masculino de 53 días de vida que acudió al hospital por presentar lesiones cutáneas y sangrado de tubo digestivo alto. Durante su internamiento se encontraron hemangiomas viscerales en pulmón, bazo y mucosa gástrica. Recibió tratamiento con interferón alfa-2b y corticosteroides. Los hemangiomas de mucosa gástrica fueron coagulados con argón plasma. Conclusiones. La hemangiomatosis neonatal diseminada es una entidad rara que puede ser mortal si no recibe tratamiento. El paciente evolucionó satisfactoriamente y actualmente se encuentra asintomático.

Introduction. Diffuse neonatal hemangiomatosis is a rare entity and is a serious disorder in which multiple cutaneous and visceral hemangiomas are widely distributed and sometimes may be fatal. Complications include heart failure, bleeding, and liver failure, among others. Treatment is aimed at avoiding adverse effects and complications. Case report. We report the case of a 53-day-old male patient who presented to the hospital for skin lesions and upper gastrointestinal bleeding. During his hospital stay, visceral hemangiomas were found in the lung, spleen and gastric mucosa. He was treated with interferon alpha-2b corticosteroids and hemangiomas of gastric mucosa were treated with argon plasma coagulation. Conclusions. Disseminated neonatal hemangiomatosis is a rare entity and can be fatal if untreated. Our patient experienced a satisfactory evolution and is currently asymptomatic.

Interaction of CYP2C8 and CYP2C9 genotypes modifies the risk for nonsteroidal anti-inflammatory drugs-related acute gastrointestinal bleeding.

OBJECTIVES: To analyze whether gene variants leading to impaired drug metabolism are related with acute gastrointestinal bleeding after nonsteroidal anti-inflammatory drugs (NSAID) use. METHODS: Common CYP2C8 and CYP2C9 polymorphisms were studied in a cross-sectional study, involving 134 NSAID-related bleeding patients and in 177 patients receiving NSAID with no adverse effects. RESULTS: Among patients receiving NSAID that are CYP2C8/9 substrates the frequencies for carriers of variant alleles versus control patients were CYP2C8*3: 0.50 vs. 0.23 [odds ratio (OR); 95% confidence interval (CI)=3.4; 1.5-7.5; P=0.002], CYP2C9*2: 0.48 vs. 0.26 (OR; 95% CI=2.7; 1.2-5.8; P=0.013) and CYP2C9*3: 0.24 vs. 0.20 (OR; 95% CI=1.3; 0.5-3.1; P=0.578). The frequencies for carriers of the CYP2C8*3+CYP2C9*2 genotype were 0.40 vs. 0.15 (OR; 95% CI=3.7; 1.6-8.9; P=0.003). These findings were not influenced by sex, age, smoking or drinking habits. Among bleeding patients receiving NSAID that are not extensively metabolized by CYP2C8/9, no differences in genotypes or allele frequencies were observed as compared with control patients. CONCLUSION: The combined presence of CYP2C8*3 and CYP2C9*2 (CYP2C8*3+CYP2C9*2 genotype), is a relevant determinant in the risk to develop gastrointestinal bleeding in patients receiving NSAID that are CYP2C8/9 substrates.

Synthesis of new self-assembled PdII and PtII rectangular metallomacrocycles: a comparative study of their inclusion complexes.

New palladium and platinum metallocycles have been synthesized by reacting 4,4'-bipyridinium-based ligands with PdII and PtII complexes. Strict thermodynamic self-assembly of 1 and [M(en)(NO3)2] (M=Pd, Pt) 6 a,b afforded metallocycles 7 a,b. However, the synthesis of 8 a,b and 9 a,b required a self-assembly process that used sodium p-phenylenediacetate (12) as a template. Finally, metallocycles 10 a,b were synthesized under high dilution conditions from ligand 4. The formation of inclusion complexes between metallocycles 7-10 and substrates 13 and 14 were studied by low-temperature 1H NMR, and the association constants were determined in nitromethane and water by following the characteristic charge-transfer band that these metallomacrocycles show in their UV-visible absorption spectra. A clear correlation between the affinity for a substrate and the dimensions of the metallocycle was observed. Metallocycles 8 b and 9 b exhibited the highest binding constants in water and nitromethane. This observation is in agreement with the DFT (B3LYP)-optimized geometries obtained for the different metallomacrocycles, which indicate that only macrocycles 8 and 9 possess a cavity with a width larger than 3.5 A. The insertion of hydroquinone or diol 13 into the cavity of metallocycle 11 a was confirmed by single-crystal X-ray crystallography.

The effect of the cytochrome P450 CYP2C8 polymorphism on the disposition of (R)-ibuprofen enantiomer in healthy subjects.

AIMS: To study the effect of CYP2C8*3, the most common CYP2C8 variant allele on the dis-position of (R)-ibuprofen and the association of CYP2C8*3 with variant CYP2C9 alleles. METHODS: Three hundred and fifty-five randomly selected Spanish Caucasians were screened for the common CYP2C8 and CYP2C9 mutations. The pharmacokinetics of (R)-ibuprofen were studied in 25 individuals grouped into different CYP2C8 genotypes. RESULTS: The allele frequency of CYP2C8*3 (0.17) was found to be higher than that reported for other Caucasian populations (P = 0.0001). The frequencies of CYP2C9*2 and CYP2C9*3 were 0.19 (0.16-0.21) and 0.10 (0.08-0.12), respectively. An association between CYP2C8*3 and CYP2C9*2 alleles was observed, occurring together at a frequency 2.4-fold higher than expected for a random association of alleles (P = 0.0001). The presence of the CYP2C8*3 allele was found to influence the pharmacokinetics of (R)-ibuprofen in a gene-dose effect manner. Thus, after administration of 400 mg ibuprofen, the plasma half-life (95% confidence intervals) for individuals with genotypes CYP2C8*1/*1, CYP2C8*1/*3 and CYP2C8*3/*3, was 2.0 h (1.8-2.2), 4.2 h (1.9-6.5; P < 0.05) and 9.0 h (7.8-10.2; P < 0.002), respectively. A statistically significant trend with respect to the number of variant CYP2C8*3 alleles was also observed for the area under the concentration-time curve (P < 0.025), and drug clearance (P < 0.03). CONCLUSION: Polymorphism of the CYP2C8 gene was found to be common, with nearly 30% of the population studied carrying the variant CYP2C8*3 allele. The presence of the latter caused a significant effect on the disposition of (R)-ibuprofen. This suggests that a substantial proportion of Caucasian subjects may show alterations in the disposition of drugs that are CYP2C8 substrates.

Sustitución esofágica en un hospital de tercer nivel de atención pediátrica / Esophageal replacement in a third level pediatric center

Introducción. La ingeste de sosa cáustica en los niños es un problema frecuente en nuestro medio, y en ocasiones las secuelas son tan importantes que nos obligan a realizar una sustitución esofágica. Otras causas menos frecuentes que requieren este procedimiento, son: estenosis por reflujo gastroesofágico, estenosis congénitas y la atresia de esófago. Una buena opción para sustituir el esófago es la utilización de colon, con lo que se ha obtenido buenos resultados en diferentes reportes en la literatura. El motivo del presente reporte es mostrar nuestra experiencia en la sustitución de esófago con colon. Material y métodos. Se hizo la revisión de los pacientes sometidos a sustitución esofágica en los últimos 30 años, se analizan: edad, sexo, etiología quirúrgica, complicaciones y evolución. La técnica quirúrgica utilizada fue empleando colon derecho íleon terminal, isoperistáltico, retroesternal, con anastomosi íleo-esofágica en 2 planos y cologástrica en 2 planos. Resultados. Se han efectuado 25 procedimientos de sustitución esofágica. Los diagnósticos fueron: estenosis por cáusticos en 20 pacientes, atresia de esófago 2, estenosis congénita 1, estenosi por reflujo 1, ruptura de aneurisma aórtico 1 caso. Como complicaciones un paciente presentó necrosis del injerto, resuelto en forma satisfactoria con un segundo procedimiento.. Sólo se presentaron 8 por ciento de fístulas proximales y 12 por ciento de estenosis; 24 pacientes se encuentran en seguimiento actual con buena ingesta de alimentos y ganancia ponderal, un paciente falleció 2 años después de la cirugía por causas no atribuibles a la misma. Conclusiones. La sustitución esofágica con colon es una alternativa de manejo, efectiva en casos graves, en los que la función esofágica no es adecuada y permite una calidad de vida mejor para estos pacientes. La técnica empleada con anastomosis íleo-esofágica en 2 planos ofrece como ventaja una disminusión en la incidencia de fístulas y estenosis ya que se logra una mejor anastomosis, por la similitud de calibres entre el esófago y el íleon. Las complicaciones tardías son escasas y en caso de presentarse, como por ejemplo las estenosis, su resolución es favorable