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INTRODUCTION: Thiamine is a key cofactor for aerobic metabolism, previously shown to improve mortality and neurological outcomes in a mouse model of cardiac arrest. We hypothesized that thiamine would decrease lactate and improve outcomes in post-arrest patients. METHODS: Single center, randomized, blinded, placebo-controlled, Phase II trial of thiamine in adults within 4.5 hours of return of spontaneous circulation after out-of-hospital cardiac arrest (OHCA), with coma and lactate ≥ 3 mmol/L. Participants received 500 mg IV thiamine or placebo twice daily for 2 days. Randomization was stratified by lactate > 5 or ≤ 5 mmol/L. The primary outcome of lactate was checked at baseline, 6, 12, and 24 hours, and compared using a linear mixed model to account for repeated measures. Secondary outcomes included SOFA score, pyruvate dehydrogenase, renal injury, neurological outcome, and mortality. RESULTS: Of 93 randomized patients, 76 were enrolled and included in the analysis. There was no difference in lactate over 24 hours (mean difference 0.34 mmol/L (95% CI: -1.82, 2.50), p = 0.43). There was a significant interaction between randomization lactate subgroup and the effect of the intervention on mortality (p = 0.01) such that mortality was higher with thiamine in the lactate > 5 mmol/L group and lower with thiamine in the < 5 mmol/L group. This subgroup difference prompted the Data and Safety Monitoring Board to recommend the study be terminated early. PDH activity increased over 72 hours in the thiamine group. There were no differences in other secondary outcomes. CONCLUSION: In this single-center randomized trial, thiamine did not affect lactate over 24 hours after OHCA.

Asunto(s)

Ácido Láctico , Paro Cardíaco Extrahospitalario , Tiamina , Paro Cardíaco Extrahospitalario/mortalidad , Paro Cardíaco Extrahospitalario/terapia , Humanos , Tiamina/uso terapéutico , Tiamina/administración & dosificación , Masculino , Femenino , Persona de Mediana Edad , Anciano , Ácido Láctico/sangre , Reanimación Cardiopulmonar/métodos , Complejo Vitamínico B/uso terapéutico , Complejo Vitamínico B/administración & dosificación , Método Doble Ciego

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Objective: To determine if psychophysiologic symptom relief therapy (PSRT) will reduce symptom burden in patients suffering from post-acute sequelae of COVID-19 (PASC) who had mild/moderate acute COVID-19 disease without objective evidence of organ injury. Patients and Methods: Twenty-three adults under the age of 60 with PASC for at least 12 weeks following COVID-19 infection were enrolled in an interventional cohort study conducted via virtual platform between May 18, 2021 and August 7, 2022. Participants received PSRT during a 13 week (approximately 44 hour) course. Participants were administered validated questionnaires at baseline and at 4, 8, and 13 weeks. The primary outcome was change in somatic symptoms from baseline, measured using the Somatic Symptom Scale-8 (SSS-8), at 13 weeks. Results: The median duration of symptoms prior to joining the study was 267 days (IQR: 144, 460). The mean SSS-8 score of the cohort decreased from baseline by 8.5 (95% CI: 5.7-11.4), 9.4 (95% CI: 6.9-11.9), and 10.9 (95% CI: 8.3-13.5) at 4, 8, and 13 weeks respectively (all p<.001). Participants also experienced statistically significant improvements across other secondary outcomes including changes in dyspnea, fatigue, and pain (all p<.001). Conclusion: PSRT may effectively decrease symptom burden in patients suffering from PASC without evidence of organ injury. The study was registered on clinicaltrials.gov (NCT04854772).

RESUMEN

BACKGROUND: Widespread reports suggest the characteristics and disease course of coronavirus disease 2019 (COVID-19) and influenza differ, yet detailed comparisons of their clinical manifestations are lacking. OBJECTIVE: Comparison of the epidemiology and clinical characteristics of COVID-19 patients during the pandemic with those of influenza patients in previous influenza seasons at the same hospital DESIGN: Admission rates, clinical measurements, and clinical outcomes from confirmed COVID-19 cases between March 1 and April 30, 2020, were compared with those from confirmed influenza cases in the previous five influenza seasons (8 months each) beginning September 1, 2014. SETTING: Large tertiary care teaching hospital in Boston, MA PARTICIPANTS: Laboratory-confirmed COVID-19 and influenza inpatients MEASUREMENTS: Patient demographics and medical history, mortality, incidence and duration of mechanical ventilation, incidences of vasopressor support and renal replacement therapy, and hospital and intensive care admissions. RESULTS: Data was abstracted from medical records of 1052 influenza patients and 582 COVID-19 patients. An average of 210 hospital admissions for influenza occurred per 8-month season compared to 582 COVID-19 admissions over 2 months. The median weekly number of COVID-19 patients requiring mechanical ventilation was 17 (IQR: 4, 34) compared to a weekly median of 1 (IQR: 0, 2) influenza patient (p=0.001). COVID-19 patients were significantly more likely to require mechanical ventilation (31% vs 8%) and had significantly higher mortality (20% vs. 3%; p<0.001 for all). Relatively more COVID-19 patients on mechanical ventilation lacked pre-existing conditions compared with mechanically ventilated influenza patients (25% vs 4%, p<0.001). Pneumonia/ARDS secondary to the virus was the predominant cause of mechanical ventilation in COVID-19 patients (94%) as opposed to influenza (56%). LIMITATION: This is a single-center study which could limit generalization. CONCLUSION: COVID-19 resulted in more weekly hospitalizations, higher morbidity, and higher mortality than influenza at the same hospital.

Asunto(s)

COVID-19 , Gripe Humana , Hospitalización , Humanos , Gripe Humana/epidemiología , Gripe Humana/terapia , Pandemias , SARS-CoV-2 , Centros de Atención Terciaria

RESUMEN

INTRODUCTION: Vitamin C deficiency has been described in patients with sepsis. The post-cardiac arrest syndrome shares similarities to sepsis, however vitamin C levels in post-arrest patients have been incompletely characterized. We assessed vitamin C levels in a post-arrest population. METHODS: This was a retrospective observational study at a tertiary care center. A convenience sample of post-arrest, sepsis, and healthy control patients was selected from prior studies. Vitamin C levels were measured from samples obtained within 6-h of emergency department admission. A subset of cardiac arrest patients had vitamin C levels additionally measured 24-h later. RESULTS: A total of 84 patients (34 healthy controls, 25 post-arrest, and 25 septic patients) were included. The median baseline vitamin C level in cardiac arrest patients was 0.33 mg/dL (0.05-0.83), as compared to 0.91 mg/dL (0.69-1.48) in the healthy control group (p < 0.01) and 0.28 mg/dL (0.11-0.59) in the septic group (p = 0.36). Vitamin C levels for cardiac arrest patients fell between the two time points, but the change was not statistically significant (median decrease 0.26 mg/dL, p = 0.08). CONCLUSIONS: Serum vitamin C levels were lower in post-arrest patients compared to controls and were similar to patients with sepsis. Future studies of vitamin C levels and supplementation following cardiac arrest may be warranted.

Asunto(s)

Paro Cardíaco Extrahospitalario , Sepsis , Ácido Ascórbico , Humanos , Estudios Retrospectivos , Sobrevivientes

RESUMEN

(1) Background: Thiamine is an important cofactor for multiple metabolic processes. Its role in cancer has been debated for years. Our aim is to determine if thiamine can convert the cellular metabolic state of breast cancer cells from anaerobic to aerobic, thus reducing their growth. (2) Methods: Breast cancer (MCF7) and non-tumorigenic (MCF10A) cell lines were treated with various doses of thiamine and assessed for changes in cell growth. The mechanism of this relationship was identified through the measurement of enzymatic activity and metabolic changes. (3) Results: A high dose of thiamine reduced cell proliferation in MCF7 (63% decrease, p < 0.0001), but didn't affect apoptosis and the cell-cycle profile. Thiamine had a number of effects in MCF7; it (1) reduced extracellular lactate levels in growth media, (2) increased cellular pyruvate dehydrogenase (PDH) activities and the baseline and maximum cellular oxygen consumption rates, and (3) decreased non-glycolytic acidification, glycolysis, and glycolytic capacity. MCF10A cells preferred mitochondrial respiration instead of glycolysis. In contrast, MCF7 cells were more resistant to mitochondrial respiration, which may explain the inhibitory effect of thiamine on their proliferation. (4) Conclusions: The treatment of MCF7 breast cancer cells with 1 µg/mL and 2 µg/mL of thiamine for 24 h significantly reduced their proliferation. This reduction is associated with a reduction in glycolysis and activation of the PDH complex in breast cancer cells.

Asunto(s)

Glucólisis/efectos de los fármacos , Ácido Láctico/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Tiamina/farmacología , Anaerobiosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7

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Tissue-engineered cardiovascular patches, cardiac valves, and great vessels are emerging solutions for the surgical treatment of congenital cardiovascular abnormalities due to their potential for adapting with the growing child. The ideal pediatric cardiovascular patch/graft is non-thrombogenic, phenotypically compatible, and matches the compliance and mechanical strength of the native tissue, both initially and throughout growth. Bottom-up tissue engineering approaches, in which three-dimensional tissue is built layer-by-layer from scaffold-less cell sheets in vitro, offer an exciting potential solution. Cell source variability, sheet patterning, and scaffold-less fabrication are promising advantages offered by this approach. Here we review the latest developments and next steps in bottom-up tissue engineering targeted at meeting the necessary design criteria for successful pediatric cardiac tissue-engineered grafts.

Asunto(s)

Enfermedades Cardiovasculares/terapia , Células Madre Fetales/citología , Válvulas Cardíacas/citología , Ingeniería de Tejidos/métodos , Prótesis Valvulares Cardíacas , Humanos

RESUMEN

BACKGROUND: Intra-abdominal adhesions are the most frequent postoperative complication after abdominopelvic surgery. Our laboratory has previously shown that an intraoperative peritoneal lavage containing either the histone deacetylase inhibitor valproic acid (VPA) or a neurokinin-1 receptor antagonist (NK-1RA) reduced adhesions by approximately 50% in a rat model. The objective of this study was to determine whether the combination of these 2 drugs was more effective in reducing adhesions than either alone. METHODS: Rats underwent laparotomy with creation of peritoneal ischemic buttons to induce adhesions. A single dose of VPA (25 mg/kg), NK-1RA (50 mg/kg), a combination of both, or 0.9% saline was lavaged intraperitoneally just before wound closure. On postoperative day 7, adhesions were quantified. To investigate early mechanisms of adhesiogenesis, adhesions were created as described and adhesive button tissue was harvested at 30 minutes and 3 hours postoperatively and fibrinogen and vascular endothelial growth factor (VEGF) protein levels, both indices of peritoneal extravasations, were determined by Western blot analysis. Peritoneal fluid was collected in similar experiments at 30 minutes, and 3 and 6 hours to measure fibrinolytic activity, an index of the ability of the peritoneum to degrade fibrinous adhesions. RESULTS: The coadministration of VPA plus NK-1RA reduces adhesions by 72.6% relative to saline (P < .001); this reduction was greater than either compound alone (P < .001). Peritoneal fibrinolytic activity was significantly increased at 3 and 6 hours postoperatively in animals administered the combination therapy versus saline (P = .01). VPA plus NK-1RA significantly decreased fibrinogen and VEGF protein levels at 3 and 6 hours compared with saline controls. CONCLUSION: These results suggest that a combined pharmacologic approach targeting multiple adhesiogenic pathways provides optimal adhesion prevention.

Asunto(s)

Inhibidores de Histona Desacetilasas/administración & dosificación , Antagonistas del Receptor de Neuroquinina-1/administración & dosificación , Adherencias Tisulares/prevención & control , Abdomen , Animales , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Fibrinógeno/análisis , Cuidados Intraoperatorios , Masculino , Ratas , Ratas Wistar , Ácido Valproico/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/análisis , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

BACKGROUND: Intra-abdominal adhesions are a common source of postoperative morbidity. Previous studies in our laboratory have shown that a neurokinin 1 receptor antagonist (NK-1RA) reduces abdominal adhesion formation and increases peritoneal fibrinolytic activity. However, the cellular pathway by which the antagonist exerts its effects is unclear, as cultured peritoneal mesothelial cells exposed to the NK-1RA show increases in fibrinolytic activity despite having very low expression of neurokinin 1 receptor (NK-1R) messenger RNA and protein. Our aim was to determine whether the NK-1R plays an essential role in the adhesion-reducing effects of the NK-1RA, or if the NK-1RA is acting independently of the receptor. METHODS: Homozygous NK-1R knockout mice and age matched wild-type mice underwent laparotomy with cecal cautery to induce adhesions. At the time of surgery, mice received a single intraperitoneal dose of either NK-1RA (25 mg/kg) or saline alone. Adhesion severity at the site of cecal cautery was assessed on postoperative day 7. In a separate experiment, peritoneal fluid was collected from wild type and NK-1R knockout mice 24 h after laparotomy with cecal cautery and administration of either NK-1RA or saline. Tissue plasminogen activator levels, representative of total fibrinolytic activity, were then measured in peritoneal fluid. RESULTS: In wild-type mice, NK-1RA administration significantly decreased adhesion formation compared with saline controls. Among the NK-1R knockout mice, there was no significant reduction in adhesion formation by the NK-1RA. Fibrinolytic activity increased 244% in wild-type mice administered NK-1RA compared with saline controls; however, the NK-1RA did not raise fibrinolytic activity above saline controls in NK-1R knockout mice. CONCLUSIONS: These data indicate that the NK-1R mediates the adhesion-reducing effects of the NK-1RA, in part, by the upregulation of peritoneal fibrinolysis, and suggest that the NK-1R is a promising therapeutic target for adhesion prevention.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Peritoneo/metabolismo , Receptores de Neuroquinina-1/metabolismo , Adherencias Tisulares/metabolismo , Adherencias Tisulares/prevención & control , Animales , Líquido Ascítico/metabolismo , Líquido Ascítico/patología , Ciego/lesiones , Ciego/cirugía , Femenino , Fibrosis/metabolismo , Fibrosis/patología , Fibrosis/prevención & control , Laparotomía/efectos adversos , Masculino , Ratones Noqueados , Antagonistas del Receptor de Neuroquinina-1/farmacología , Peritoneo/patología , Receptores de Neuroquinina-1/genética , Sustancia P/metabolismo , Adherencias Tisulares/patología , Activador de Tejido Plasminógeno/metabolismo

RESUMEN

BACKGROUND: We previously demonstrated that postoperative peritoneal injury and inflammation contribute to adhesiogenesis. Recent evidence suggests that in addition to their role of interfering with the acetylation status of nuclear histone proteins, histone deacetylase inhibitors (HDACIs) including valproic acid (VPA) can target nonhistone proteins to resolve inflammation and modulate immune cells. We hypothesized that HDACIs could reduce adhesions. METHODS: Seventy-two rats underwent laparotomy with creation of 6 peritoneal ischemic buttons to induce adhesions. A single intraperitoneal (IP) dose of 50 mg/kg VPA was administered intraoperatively, whereas controls received vehicle. To evaluate the timing, 25 rats underwent ischemic button creation with either an intraoperative or a delayed IP dose of VPA at 1, 3, or 6 hours postoperatively. On postoperative day 7, adhesions were quantified. To investigate mechanisms, ischemic buttons were created in 24 rats and either VPA or saline was administered in 1 intraoperative dose. At 3 or 24 hours later, peritoneal fluid was collected and fibrinolytic activity measured. Alternatively, button tissue was collected 30 minutes postoperatively to measure tissue factor, fibrinogen, and vascular endothelial growth factor (VEGF) by real-time polymerase chain reaction or Western blot. RESULTS: A single intraoperative dose of VPA reduced adhesions by 50% relative to controls (P < .001). Delayed dosing did not reduce adhesions. In operated animals, peritoneal fibrinolytic activity was not different between groups. Tissue factor mRNA was downregulated by 50% (P = .02) and protein by 34% (P < .01) in animals administered VPA versus saline. VPA decreased fibrinogen protein by 56% and VEGF protein by 25% compared with saline (P = .03). CONCLUSION: These findings suggest that VPA rapidly reduces the extravasation of key adhesiogenic substrates into the peritoneum. A single, intraoperative intervention provides an ideal dosing strategy and indicates an exciting new role for HDACIs in adhesion prevention.

Asunto(s)

Fibrinógeno/metabolismo , Inhibidores de Histona Desacetilasas/administración & dosificación , Peritoneo/metabolismo , Adherencias Tisulares/prevención & control , Ácido Valproico/administración & dosificación , Animales , Benzamidas/administración & dosificación , Fibrina/metabolismo , Ácidos Hidroxámicos/administración & dosificación , Intestinos/cirugía , Cuidados Intraoperatorios , Masculino , Peritoneo/efectos de los fármacos , Piridinas/administración & dosificación , Ratas , Ratas Wistar , Tromboplastina/metabolismo , Adherencias Tisulares/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Vorinostat , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

BACKGROUND: The substance P (SP) or neurokinin-1 receptor pathway has been implicated in intra-abdominal adhesion formation, in large part through its effects on peritoneal fibrinolysis. This study investigates the role of SP as an early mediator of the messenger RNA (mRNA) expression of key components of the peritoneal fibrinolytic system and other fundamental adhesiogenic pathways. MATERIALS AND METHODS: Intra-abdominal adhesions were surgically induced in 28 rats using the ischemic button model. mRNA levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), hypoxia-inducible factors (HIFs) 1α and 2α, and vascular endothelial growth factor A (VEGF-A) were measured in adhesive button tissue taken at time 0 and 1, 3, 6, 12, and 24h after surgery in rats receiving an intraoperative peritoneal bolus (25mg/kg) of a neurokinin-1 receptor antagonist (NK-1RA) or saline. Peritoneal fluid fibrinolytic activity was measured in peritoneal lavages taken at the same time points. RESULTS: SP levels increased (P≤0.05) within 1h postoperatively followed by an increase (P≤0.05) in tPA mRNA expression from 3 to 6h after surgery along with a striking increase (P≤0.05) in PAI-1 mRNA expression from 3 to 12h. NK-1RA administration further increased (P≤0.05) tPA mRNA expression and significantly blunted the increase in PAI-1 mRNA levels. The NK-1RA increased (P≤0.05) fitbrinolytic activity in peritoneal fluid at 3, 12, and 24h after surgery. HIF-1α and VEGF-A mRNA expressions increased from 3 to 12h (P≤0.05) and from 1 to 3h (P≤0.05) after surgery, respectively, whereas HIF-2α mRNA expression steadily decreased. NK-1RA delayed the rise in HIF-1α mRNA and ablated the changes in HIF-2α and VEGF-A mRNAs. CONCLUSIONS: SP is a pleiotropic early regulator of mRNA levels of key adhesiogenic mediators after surgery, suggesting that it may be a viable therapeutic target.

Asunto(s)

Fibrinólisis , Sustancia P/fisiología , Adherencias Tisulares/etiología , Animales , Líquido Ascítico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Masculino , Antagonistas del Receptor de Neuroquinina-1 , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/análisis , Ratas , Ratas Wistar , Activador de Tejido Plasminógeno/genética , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

BACKGROUND: Intraperitoneal adhesions occur in more than 94% of patients after abdominal surgery. Mechanisms that decrease oxidative stress and upregulate peritoneal fibrinolysis reduce adhesions. N-acetyl-l-cysteine (NAC) is a clinically relevant antioxidant whose effect on peritoneal fibrinolysis and ability to decrease adhesions has not been established. The aims of this study were to determine if NAC reduces adhesions and to characterize its potential mechanism(s) of action. METHODS: Male Wistar rats (n = 92) received 0.9% saline (OP Control), intraperitoneal NAC (150 mg/kg, OP + NAC), or oral NAC (1200 mg/kg) twice daily on preoperative day 1, day of operation, and postoperative day 1. Adhesions were induced on the day of operation using our previously described ischemic button model. Animals were killed on postoperative day 7 for adhesion scoring. Peritoneal tissue and fluid from the intraperitoneal NAC group were measured at 24 hours for fibrinolytic activity, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), total glutathione, and 8-isoprostane (8-IP). The effect of NAC on tPA and PAI-1 production was tested in vitro in human mesothelial cells. The effect of NAC on intestinal wound healing was measured using colonic anastomotic burst pressures. RESULTS: Intraperitoneal NAC reduced adhesions by 53% (P < .001) compared to OP Controls without affecting anastomotic wound healing. NAC increased the tPA/PAI-1 protein ratio and peritoneal fibrinolytic activity by 69% and 127%, respectively, compared to OP Controls (P < .05). NAC did not restore total glutathione levels in peritoneal adhesion tissue but decreased 8-IP by 46% and 65% (P < .05) in peritoneal tissue and fluid, respectively, compared to OP Controls. Human mesothelial cells incubated with NAC exhibited a concentration-dependent increase in the tPA/PAI-1 ratio, which supported in vivo observations (P < .05). Oral NAC did not decrease adhesions. CONCLUSION: NAC administered intraperitoneally decreased adhesion formation while upregulating peritoneal fibrinolytic activity and antioxidant defenses without affecting normal anastomotic wound healing. These data suggest a potential new therapeutic use for NAC in adhesion prevention.

Asunto(s)

Abdomen/cirugía , Acetilcisteína/uso terapéutico , Fibrinólisis/fisiología , Peritoneo/metabolismo , Adherencias Tisulares/metabolismo , Adherencias Tisulares/prevención & control , Regulación hacia Arriba/fisiología , Acetilcisteína/administración & dosificación , Acetilcisteína/farmacología , Animales , Antioxidantes/administración & dosificación , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Células Cultivadas , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glutatión/metabolismo , Humanos , Inyecciones Intraperitoneales , Masculino , Modelos Animales , Estrés Oxidativo/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas , Ratas Wistar , Activador de Tejido Plasminógeno/metabolismo , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

BACKGROUND: Mounting evidence indicates that postoperative oxidative stress may be linked to decreased fibrinolytic activity and, subsequently, the development of intraabdominal adhesions. The goal of this study was to determine if methylene blue, a highly redox active dye that has been shown to inhibit adhesion formation (1) acts as an antioxidant in the postoperative peritoneum, and (2) subsequently affects fibrinolytic activity. MATERIALS AND METHODS: Intraabdominal adhesions were surgically induced in rats receiving methylene blue (30 mg/kg) or vehicle (sterile water) intraperitoneally at surgery. At 24 h and 7 d following surgery, adhesion formation, oxidative stress, and peritoneal fibrinolytic activity were assessed. RESULTS: Methylene blue did not affect adhesion formation at 24 h, but did induce a >50% regression in adhesions after 7 d (P < 0.05). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO) activities, and 8-isoprostane and thiobarbituric acid-reactive substances were all significantly increased in peritoneal tissue samples (P < 0.05) by 24 h following surgery. Methylene blue inhibited NADPH oxidase by 98% and MPO activity by 78% in the 24 h tissue samples, and blunted the corresponding surgery-induced increases in tissue lipid and protein oxidation. Furthermore, methylene blue significantly increased (P < 0.05) fibrinolytic activity in peritoneal fluid at 24 h. CONCLUSIONS: Methylene blue acts as an antioxidant in this experimental system and may reduce intraabdominal adhesion formation by enhancing peritoneal fibrinolytic activity following surgery.

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Abdomen/cirugía , Inhibidores Enzimáticos/farmacología , Fibrinólisis/efectos de los fármacos , Azul de Metileno/farmacología , Estrés Oxidativo/efectos de los fármacos , Adherencias Tisulares/prevención & control , Animales , Antioxidantes/metabolismo , Líquido Ascítico/metabolismo , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Inyecciones Intraperitoneales , Masculino , NADPH Oxidasas/metabolismo , Peroxidasa/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Adherencias Tisulares/metabolismo

RESUMEN

Oxidative stress has been implicated in intra-abdominal adhesion formation. Substance P, a neurokinin-1 receptor (NK-1R) ligand, facilitates leukocyte recruitment and reactive oxygen species (ROS) generation. We have shown in a rat model of adhesion formation that intraperitoneal administration of a NK-1R antagonist at the time of abdominal operation reduces postoperative adhesion formation. Thus we determined the effects of NK-1R antagonist administration on peritoneal leukocyte recruitment and oxidative stress within 24 h of surgery. Adhesions were induced in Wistar rats randomly assigned to receive the antagonist or vehicle intraperitoneally. Peritoneal tissue was isolated at 2, 4, 6, and 24 h after surgery for analysis of the oxidative stress biomarkers 8-isoprostane (8-IP), protein carbonyl, NADPH oxidase, myeloperoxidase (MPO), and ICAM-1 and VCAM-1 mRNAs. Total antioxidant capacity of peritoneal fluid was also determined. MPO, NADPH oxidase, 8-IP, and protein carbonyl were elevated (P < 0.05) by 6 h. ICAM-1 mRNA was elevated (P < 0.05) by 2 h, whereas VCAM-1 levels decreased (P < 0.05) at 24 h. The NK-1R antagonist delayed the MPO rise and reduced (P < 0.05) 8-IP levels by 6 h and ICAM-1 mRNA, VCAM-1 mRNA, and protein carbonyl at 2 h. The antagonist also increased (P < 0.05) the antioxidant capacity of peritoneal fluid at all time points. These data further support a role for oxidative stress in adhesion formation and suggest that the NK-1R antagonist may limit adhesions, in part, by reducing postoperative oxidative stress through an inhibition of neutrophil recruitment and an increase in peritoneal fluid antioxidant capacity.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Fármacos Gastrointestinales/farmacología , Antagonistas del Receptor de Neuroquinina-1 , Infiltración Neutrófila/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Enfermedades Peritoneales/prevención & control , Peritoneo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Dinoprost/análogos & derivados , Dinoprost/metabolismo , Modelos Animales de Enfermedad , Fármacos Gastrointestinales/uso terapéutico , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Laparotomía , Masculino , NADPH Oxidasas/metabolismo , Enfermedades Peritoneales/enzimología , Enfermedades Peritoneales/metabolismo , Peritoneo/enzimología , Peritoneo/metabolismo , Peritoneo/cirugía , Peroxidasa/metabolismo , Carbonilación Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Factores de Tiempo , Adherencias Tisulares/prevención & control , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Asunto(s)

Antígeno Carcinoembrionario/fisiología , Adhesión Celular , Neoplasias Colorrectales/patología , Células Endoteliales/fisiología , Macrófagos/fisiología , Línea Celular , Selectina E/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Receptores de Superficie Celular/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Atherothrombotic cardiovascular disease associated with hyperhomocysteinemia has been proposed to result, at least in part, from increased vascular oxidative stress. Here we characterize one mechanism by which homocyteine may induce a vascular cell type-specific oxidative stress. Our results show that L-homocysteine at micromolar levels stereospecifically increases lipid peroxidation in cultured endothelial cells, but not in vascular smooth muscle cells or when medium is incubated in the absence of cells. Consistent with these observations, homocysteine also increases the formation of intracellular reactive oxygen species. The pro-oxidant effect of homocysteine can be fully replicated by an equivalent concentration of homocystine (i.e., an oxidized form of homocysteine), but not with cysteine or glutathione. Homocyst(e)ine-dependent lipid peroxidation is independent of H(2)O(2) and alterations in glutathione peroxidase activity, but dependent on superoxide. Mechanistically, the pro-oxidant effect of homocysteine appears to involve endothelial nitric oxide synthase (eNOS), as it is blocked by the eNOS inhibitor L-N(G)-nitroarginine methyl ester. Thus, homocyst(e)ine actively promotes oxidative stress in endothelial cells via an eNOS-dependent mechanism.

Asunto(s)

Células Endoteliales/enzimología , Homocisteína/farmacología , Homocistina/farmacología , Hiperhomocisteinemia/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Bovinos , Células Cultivadas , Cisteína/metabolismo , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III

RESUMEN

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. An increasing body of evidence has implicated oxidative stress as being contributory to homocysteine's deleterious effects on the vasculature. Elevated levels of homocysteine may lead to increased generation of superoxide by a biochemical mechanism involving nitric oxide synthase, and, to a lesser extent, by an increase in the chemical oxidation of homocysteine and other aminothiols in the circulation. The resultant increase in superoxide levels is further amplified by homocysteine-dependent alterations in the function of cellular antioxidant enzymes such as cellular glutathione peroxidase or extracellular superoxide dismutase. One direct clinical consequence of elevated vascular superoxide levels is the inactivation of the vasorelaxant messenger nitric oxide, leading to endothelial dysfunction. Scavenging of superoxide anion by either superoxide dismutase or 4,5-dihydroxybenzene 1,3-disulfonate (Tiron) reverses endothelial dysfunction in hyperhomocysteinemic animal models and in isolated aortic rings incubated with homocysteine. Similarly, homocysteine-induced endothelial dysfunction is also reversed by increasing the concentration of the endogenous antioxidant glutathione or overexpressing cellular glutathione peroxidase in animal models of mild hyperhomocysteinemia. Taken together, these findings strongly suggest that the adverse vascular effects of homocysteine are at least partly mediated by oxidative inactivation of nitric oxide.

Asunto(s)

Endotelio Vascular/metabolismo , Homocisteína/sangre , Hiperhomocisteinemia/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/complicaciones , Oxidación-Reducción , Especies Reactivas de Oxígeno/sangre , Enfermedades Vasculares/sangre , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo

RESUMEN

Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1(-/-)). Mesenteric arterioles of GPx-1(-/-) mice demonstrated paradoxical vasoconstriction to beta-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1(-/-) mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1(-/-) mice. We observed an increase of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in the plasma and aortas of GPx-1(-/-) mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1(-/-) mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.

Asunto(s)

Endotelio Vascular/fisiopatología , Glutatión Peroxidasa/deficiencia , Estrés Oxidativo/fisiología , Animales , Bradiquinina/farmacología , GMP Cíclico/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Cloruro de Metacolina/farmacología , Ratones , Ratones Noqueados , Microcirculación/efectos de los fármacos , Microcirculación/fisiología , Modelos Animales , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , Circulación Esplácnica/fisiología

RESUMEN

Previous in vitro experiments have shown that hyperhomocysteinemia leads to oxidative inactivation of nitric oxide, in part by inhibiting the expression of cellular glutathione peroxidase (GPx-1). To elucidate the role of intracellular redox status on homocysteine-induced endothelial dysfunction and oxidant stress, heterozygous cystathionine beta-synthase-deficient (CBS(-/+)) and wild-type (CBS(+/+)) mice were treated with the cysteine donor L-2-oxothiazolidine-4-carboxylic acid (OTC). CBS(-/+) mice had significantly lower GPx-1 activity compared with their CBS(+/+) littermates, and OTC treatment led to a modest increase in tissue GPx-1 activity and significant increases in total thiols and in reduced glutathione levels in both CBS(+/+) and CBS(-/+) mice. Superfusion of the mesentery with beta-methacholine or bradykinin produced dose-dependent vasodilation of mesenteric arterioles in CBS(+/+) mice and in CBS(+/+) mice treated with OTC. In contrast, mesenteric arterioles from CBS(-/+) mice manifested dose-dependent vasoconstriction in response to both agonists. OTC treatment of CBS(-/+) mice restored normal microvascular vasodilator reactivity to beta-methacholine and bradykinin. These findings demonstrate that mild hyperhomocysteinemia leads to endothelial dysfunction in association with decreased bioavailable nitric oxide. Increasing the cellular thiol and reduced glutathione pools and increasing GPx-1 activity restores endothelial function. These findings emphasize the importance of intracellular redox balance for nitric oxide bioactivity and endothelial function.

Asunto(s)

Glutatión Peroxidasa/metabolismo , Hiperhomocisteinemia/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo , Vasodilatación/fisiología , Animales , Bradiquinina/farmacología , Cistationina betasintasa/deficiencia , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Homocisteína/sangre , Hiperhomocisteinemia/fisiopatología , Hígado/metabolismo , Mesenterio/irrigación sanguínea , Cloruro de Metacolina/farmacología , Ratones , Oxidación-Reducción , Ácido Pirrolidona Carboxílico , Compuestos de Sulfhidrilo/sangre , Tiazoles/farmacología , Tiazolidinas , Vasodilatación/efectos de los fármacos