Liquid chromatography coupled to high-resolution mass spectrometry metabolomics: A useful tool for investigating tumor secretome based on a three-dimensional co-culture model.

Three-dimensional (3D) cell culture technologies, which more closely mimic the complex microenvironment of tissue, are being increasingly evaluated as a tool for the preclinical screening of clinically promising new molecules, and studying of tissue metabolism. Studies of metabolites released into the extracellular space (secretome) allow understanding the metabolic dynamics of tissues and changes caused by therapeutic interventions. Although quite advanced in the field of proteomics, studies on the secretome of low molecular weight metabolites (< 1500 Da) are still very scarce. We present an untargeted metabolomic protocol based on the hybrid technique of liquid chromatography coupled with high-resolution mass spectrometry for the analysis of low-molecular-weight metabolites released into the culture medium by 3D cultures and co-culture (secretome model). For that we analyzed HT-29 human colon carcinoma cells and 3T3-L1 preadipocytes in 3D-monoculture and 3D-co-culture. The putative identification of the metabolites indicated a sort of metabolites, among them arachidonic acid, glyceric acid, docosapentaenoic acid and beta-Alanine which are related to cancer and obesity. This protocol represents a possibility to list metabolites released in the extracellular environment in a comprehensive and untargeted manner, opening the way for the generation of metabolic hypotheses that will certainly contribute to the understanding of tissue metabolism, tissue-tissue interactions, and metabolic responses to the most varied interventions. Moreover, it brings the potential to determine novel pathways and accurately identify biomarkers in cancer and other diseases. The metabolites indicated in our study have a close relationship with the tumor microenvironment in accordance with the literature review.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1ß. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Laparoscopic transhiatal subtotal esophagectomy for the treatment of advanced megaesophagus.

BACKGROUND: Chagas' disease affects about 5 to 8 million individuals in Brazil, with 5% to 8% of them developing megaesophagus. In view of the transformation of the esophagus into an inert tube unable to propel food to the stomach, and in order to prevent complications, the elected treatment for advanced megaesophagus is subtotal esophagectomy. We evaluate here the outcome of laparoscopic transhiatal subtotal esophagectomy in the treatment of advanced megaesophagus. METHODS: Thirty patients with advanced esophagopathy, 26 with chagasic and 4 with idiopathic megaesophagus, were submitted to transhiatal subtotal esophagectomy without thoracotomy through laparoscopy and left cervicotomy. Contrast exams of the esophagus, stomach, and duodenum (ESD), upper digestive tract endoscopy (UDE), esophageal electromanometry, and 24-hour pHmetry were performed during the preoperative and postoperative period. With respect to the surgical technique, pyloroplasty was not performed. The cervical esophagus was dissected through a left cervicotomy and the esophagogastric anastomosis was performed between the cervical segment of the esophagus and the posterior wall of the stomach. RESULTS: No death or conversion to open surgery occurred in the present series. Complications were observed in 8 patients (26.7%): 6 cases of pneumothorax (20%), 2 of cervical fistulas (6.7%), 7 of transient dysphonia (23.3%), and 1 of anastomotic esophagogastric stenosis (3.3%). One (3.3%) of the patients developed dysphagia for solid food after 36 months despite normal ESD, UDE, electromanometry, and 24-hour pHmetry. CONCLUSIONS: The present results show that laparoscopic transhiatal subtotal esophagectomy is a feasible and safe procedure with an excellent postoperative outcome.

Análise comparativa de três tipos de válvulas anti-refluxo associada à cirurgia de heller: estudo experimental em cães / Comparative analysis between three types of anti-reflux valves associated with heller's surgery: experimental study in dogs

OBJETIVO: Comparar experimentalmente, em cães, três técnicas de esofagogastrofundoplicatura - anterior, posterior e lateral - após cardiomiotomia ampla. MÉTODO: Os animais foram separados aleatoriamente em quatro grupos: Grupo A: Válvula anti-refluxo anterior (n=5); Grupo P: válvula anti-refluxo posterior (n=5); Grupo L: Válvula anti-refluxo lateral (n=5); Grupo C: Grupo controle, no qual não foi realizado nenhum tipo de fundoplicatura após a cardiomiotomia (n=10). A avaliação pré-operatória foi realizada através de endoscopia, biópsia da mucosa gastroesofágica, pHmetria e manometria esofágica. Após cinco semanas, realizou-se reavaliação, baseada em dados endoscópicos, pHmétricos, manométricos e histopatológicos. Os animais foram mortos e os segmentos esofagogástricos retirados para exame histopatológico RESULTADOS: Ao exame histopatológico detectou-se esofagite em todos os casos do grupo controle e em nove casos dos grupos com válvulas, e nestes, quando presente, a esofagite foi de menor extensão e de grau menos avançado do que no grupo controle. Através da manometria pré e pós-operatória, observou-se que: 1) o grupo controle apresentou uma redução na pressão na ZAP de 39,2mmHg para 23,4mmHg; 2) os grupos A, P, e L apresentaram uma variação da ZAP de 39,7, 39,9 e 39,7mmHg para 40,9, 44,8 e 41,3mmHg, respectivamente. CONCLUSÕES: 1) A cardiomiotomia ampla provoca refluxo gastroesofágico e conseqüente esofagite; 2) As três válvulas anti-refluxo estudadas restauram o segmento de alta pressão e diminuem a incidência de refluxo e de esofagite.

OBJECTIVE: To compare three techniques of esophagogastrofundoplication - anterior, posterior and lateral-post cardiomyotomy, in dogs. METHOD: Pre-operative assessment included esophagoscopy plus biopsy of the mucosa, pH determination and manometry. The animals were divided into four groups: Group A: anterior anti-reflux valve (n=5); group P: posterior valve (n=5); Group L: lateral valve (n=5); Group C: control group, no fundoplication (n=10). After five weeks, the animals were submitted to endoscopy plus biopsy, pHmetric and manometric examinations and histopathological analysis. RESULTS: Histophatological examination detected esophagitis in every case of the control group and in nine cases of the groups with fundoplication. When present, esophagitis was milder in the valve groups than in the control group. The average value of the pressure in high pressure zone (HPZ) detected by manometric examination in the control group, was significantly reduced after cardiomyotomy (39,2 to 23.4mmHg). In the groups with fundoplication, there was no significant difference between preoperative and postoperative mean values of the HPZ pressure. The values detected preoperatively in groups A, P and L were 39.7, 39.9, and 39.7mmHg respectively and 40.9, 44.8 and 41.3 mmHg postoperatively. CONCLUSIONS: 1) cardiomyotomy causes gastroesophageal reflux and esophagitis; 2) any type of fundoplication valve, added to the myotomy, restores the lenght and the pressure of the HPZ=ZAP and reduces the incidence of reflux and esophagitis.