Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Predictive factors that influence the survival rates in liver cirrhosis patients with spontaneous bacterial peritonitis

BACKGROUND/AIMS: Spontaneous bacterial peritonitis (SBP) has been known to greatly influence the survival rate of patients with liver cirrhosis. However, the factors that affect the survival rate in patients with SBP need to be clarified. METHODS: This study enrolled 95 liver cirrhosis patients diagnosed with SBP. The laboratory findings of their serum and ascitic fluid were examined and the characteristics of the isolated microorganisms in their peritoneal fluid were analyzed. RESULTS: The proportion of patients with culture-positive SBP was 41.1%, and 47 microorganisms were isolated from the ascitic fluid. The proportions of cultured bacteria that were Gram negative and Gram positive were 57.4% and 40.4%, respectively. The proportions of Escherichia coli, Klebsiella species, and Streptococcus species were 25.5%, 19.1%, and 19.1%, respectively. Enterococcus species represented 12.8% of the microorganisms cultured. The overall survival rates at 6, 12, and 24 months were 44.5%, 37.4%, and 32.2%, respectively. There was no relationship between the bacterial factors and the survival rate in SBP. Multivariate analysis revealed that the presence of hepatocellular carcinoma (HCC; P=0.001), higher serum bilirubin levels (> or =3 mg/dL, P=0.002), a prolonged serum prothrombin time (i.e., international normalized ratio >2.3, P1.3 mg/dL, P<0.001), and lower glucose levels in the ascitic fluid (<50 mg/dL, P<0.001) were independent predictive factors of overall survival rate. CONCLUSIONS: HCC, higher serum bilirubin levels, a prolonged serum prothrombin time, renal dysfunction, and lower ascitic glucose levels are associated with higher mortality rates in cirrhotic patients with SBP.

Evaluation of anti-allergic activity of gossypin and suramin in mast cell-mediated allergy model.

The mast cell-mediated allergic reactions are involved in many allergic diseases, such as asthma, allergic rhinitis and sinusitis. Stimulation of mast cells initiates the process of degranulation, resulting in the release of mediators such as histamine and an array of inflammatory cytokines. In this report, we investigated the effect of gossypin (a biflavonoid) and suramin (a synthetic polysulphonated naphtylurea) on the mast cell-mediated allergy model, and studied the possible mechanism of their action. Both gossypin and suramin inhibited (P<0.001) compound 48/80-induced systemic anaphylaxis reactions, antiprurities (P<0.001) and reduced the histamine release in rats. Further, both showed significant (P<0.001) protection against rat peritoneal mast cells activated by compound 48/80. Thus, our findings provide evidence that gossypin and suramin inhibit mast cell-derived allergic reactions.

Avaliação do fluido peritoneal de asininos / Evaluation of peritoneal fluid from asinines

The peritoneal fluid from six female health donkeys was evaluated. In each animal, it was carried out eight abdominocentesis with 40x12 needles, at 72-hour intervals. All animals presented clinical changes, and there was higher frequency of yellow-tinged fluid with turbidity aspect. Means, and respective standard deviations, for cytology and protein concentration were: erythrocytes, 24.7±11.7x10³/mm³; leukocytes, 15.8±6.79x10³/mm³; mesothelial cells, 3.85±1.30x10³/mm³; neutrophils, 10.9±4.51x10³/mm³; lymphocytes, 1.41±0.51x10³/mm³; macrophages, 0.98±0.55x10³/mm³; eosinophils, 2.48±1.36x10³/mm³, and total proteins, 1.97±0.31 g/dl.

Positive correlation between serum and peritoneal fluid CA-125 levels in women with pelvic endometriosis / Correlação positiva entre os níveis séricos e no fluido peritonial de CA-125 em mulheres com endometriose pélvica

CONTEXT AND OBJECTIVE: One of the diagnostic markers of endometriosis is CA-125, and elevated levels of this are caused by high concentrations in the ectopic endometrium. The objective of this study was to correlate CA-125 levels in serum and peritoneal fluid from women with and without pelvic endometriosis. DESIGN AND SETTING: This was a prospective, cross-sectional, controlled study of consecutive pa-tients undergoing laparoscopy for infertility, pelvic pain or tubal ligation, during early follicular phase, at the university hospital of Faculdade de Medicina de Ribeirão Preto. METHODS: Fifty-two patients were divided into two groups: endometriosis group, consisting of 35 patients with biopsy-confirmed pelvic endometriosis, and control group, consisting of 17 patients without endometriosis. CA-125 levels in serum samples and peritoneal fluid were determined by chemiluminescence. RESULTS: CA-125 levels in serum and peritoneal fluid were higher in patients with advanced pelvic endometriosis (means of 39.1 ± 45.8 U/ml versus 10.5 ± 5.9 U/ml in serum, p < 0.005; 1,469.4 ± 1,350.4 U/ml versus 888.7 ± 784.3 U/ml in peritoneal fluid, p < 0.05), and showed a positive correlation between each other (correlation coefficient (r) = 0.4880). Women with more advanced degrees of endometriosis showed higher CA-125 levels in both serum and peritoneal fluid (p = 0.0001). CONCLUSION: There is a positive correlation between serum and peritoneal fluid values of CA-125 in women with and without endometriosis, and their levels are higher in peritoneal fluid. Advanced endometriosis is related to higher levels in both serum and peritoneal fluid.

CONTEXTO E OBJETIVO: Um dos marcadores diagnósticos de endometriose é o CA-125, e seus níveis elevados são devidos à alta concentração no endométrio ectópico. O objetivo deste estudo foi correlacionar os níveis de CA-125 no soro e fluido peritonial de mulheres com e sem endometriose pélvica. TIPO DE ESTUDO E LOCAL: Estudo prospectivo, longitudinal, controlado, de pacientes consecutivas submetidas a laparoscopia por infertilidade, dor pélvica ou laqueadura tubária, durante a fase folicular precoce no Hospital Universitário da Faculdade de Medicina de Ribeirão Preto. MÉTODOS: Cinqüenta e duas pacientes foram divididas em dois grupos: grupo endometriose, com 35 pacientes com biópsia confirmada de endometriose pélvica, e grupo controle, com 17 pacientes sem endometriose. Níveis de CA-125 em amostras no soro e fluido peritonial foram determinadas por quimiluminescência. RESULTADOS: Os níveis de CA-125 no soro e fluido peritonial foram mais altos nas pacientes com endometriose pélvica avançada (média 39,1 ± 45,8 U/ml versus 10,5 ± 5,9 U/ml no soro, p < 0,005, 1469,4 ± 1350,4 U/ml versus 888,7 ± 784,3 U/ml no fluido peritonial, p < 0,05), e o estudo mostrou uma correlação positiva entre eles (coeficiente de correlação = 0,4880). Mulheres com estágios mais avançados de endometriose mostraram níveis de CA-125 maiores em ambos soro e fluido peritonial (p = 0,0001). CONCLUSÃO: Há uma correlação positiva entre os valores de CA-125 no soro e no fluido peritonial em pacientes com e sem endometriose e seus níveis são maiores no fluido peritonial. Endometriose avançada é relacionada com níveis mais altos de CA-125 em ambos soro e fluido peritonial.

Mechanism of elevated vascular endothelial growth factor levels in peritoneal fluids from patients with endometriosis / 华中科技大学学报（医学）（英德文版）

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.

Measurement of Peritoneal Fluid pH in Patients with Non-Serosal Invasive Gastric Cancer

The accurate pH range of peritoneal fluid is clinically valuable for the evaluation of some pathological conditions of the body, however, it is not easy to measure in healthy individuals. The aim of this study was to measure; pH, pCO2, pO2, Na+, K++, Ca++, HCO3-, and O2 saturation of the peritoneal fluid in patients with non-serosal invasive gastric cancer. One hundred and thirty four patients (86 men and 48 women), ranging in age from 24 to 91 years were enrolled in this study. After opening the abdominal wall, the probe of a portable pH meter was placed in the peritoneal fluid in the subhepatic space. In addition, I collected the peritoneal fluid from the subhepatic space to measure, pH, pCO2, pO2, Na+, K++, Ca++, HCO3-, and O2 saturation using an autoanalyzer. The pHs of the peritoneal fluids tested has a mean of 7.73 (range 7.46 - 8.10), and the other parameters were pCO2, 22.81 mmHg; pO2, 136.49 mmHg; Na+, 146.57 mmol/L; K++, 4.80 mmol/L; Ca++, 0.89 mmol/L; HCO3-, 30.54 mmol/L, and O2 saturation, 99.74%. This study describes a practical method of measuring the pH of peritoneal fluid. The result obtained reflects the normal adult peritoneal pH value, which I propose as a reference value.

Detection of laminin in serum and ascites from patients with epithelial ovarian tumor / 华中科技大学学报（医学）（英德文版）

The change in serum laminin (LN) level and its clinical significance in epithelial ovarian tumor were investigated. The LN levels in serum and ascites samples from 69 patients with epithelial ovarian tumor and 42 cases as control group before and after operation were analyzed by radioimmunoassay. The results showed that the serum LN levels in the patients with malignant tumors (157.85 +/- 14.37 ng/ml) were significantly higher than that in the control group (125.14 +/- 7.03 ng/ml) and in the patients with benign tumors (128.36 +/- 8.75 ng/ml) (both P < 0.01) before operation. The serum LN levels in the malignant group were decreased significantly after operation as compared with those before operation (P < 0.05). The serum LN levels in low-differentiated tumors was higher than those in moderate-differentiated tumors and high-differentiated tumors (P < 0.05). The LN levels in ascites (172.94 +/- 15.26 ng/ml) was significantly higher than in serum (161.34 +/- 6.59 ng/ml) (P < 0.05) in malignant tumors. The serum LN levels in the patients with lymph node metastasis (165.41 +/- 19.91 ng/ml) was obviously higher than those without lymph node metastasis (152.35 +/- 10.34 ng/ml) (P < 0.05). It was concluded that LN levels in serum and acistis were remarkably increased in malignant epithelial ovarian tumors, suggesting that LN might be one of important diameters reflecting tumor biological characteristics.

Estudo de proteínas do fluido peritoneal pela técnica de eletroforese em mulheres inférteis com endometriose / Study of proteins within the peritoneal fluids through electrophopresis technique in infertile women with endometriosis

Estudou-se a concentraçäo de proteínas no "pool" de fluidos peritoniais de grupos de mulheres férteis (5),mulheres inférteis de causa desconhecida (5) e de mulheres inférteis com endometriose (5). O fluido peritoneal foi coletado através de laparoscopia. O grupo de mulheres com endometriose e infertilidade apresentava os estágios I e II da classificaçäo revisadada "American Ferlitity Society". As proteínas identificadas pela técnica de eletroforese unidimensional com o gel poliacrilamida 10 por cento foram a de peso molecular 21-kd, 51-kd, 107-kd e 127-kd. Näo houve diferença significativa entre as concentraçöes de proteínas nos diferentes grupos, sendo que o maior porcentual de concentraçäo proteica foi a de 51kd e a menor de 21kd, ambos no "pool" de pacientes inférteis com endometriose. Necessitam de maior confirmaçäo as evidências encontradas de que a infertilidade dos grupos estudados possa estar correlacionada com as alteraçöes do perfil de proteínas do líquido peritoneal. As concentraçöes proteicas no pool dos fluídos peritoniais dos grupos estudados säo sugestivas de que a infertilidade de causa desconhecida e a infertilidade com a endometriose sejam o espectro da mesma doença. Outras pesquisas säo necessárias para confirmar ou infirmar esta suposiçäo.

Avaliaçäo dos efeitos da paracentese total em pacientes cirróticos / Evaluation of the effects of total paracentesis in cirrhotic patients

Os autores propoem-se a estudar os efeitos da paracentese terapêutica em pacientes cirróticos com ascite refratária ou recidivante. Para tanto, analisam sua repercussao na mobilizaçao da ascite, na funçao hemodinâmica e renal e os eventuais efeitos colaterais presentes. Foram estudados prospectivamente 11 pacientes, tendo sido a ascite considerada refratária em sete e recidivante em quatro doentes. A paracentese terapêutica foi realizada com retirada de todo o fluido peritoneal em uma única sessao, sendo administrados 6g de albumina por litro de ascite drenado. A medida do volume de ascite através da cintilografia com tecnécio-fitato evidenciou, em média, 8.845ml de líquido ascítico e o volume médio drenado foi de 8.268ml, mostrando ser a paracentese total um método efetivo em mobilizar a ascite da cavidade peritoneal.Nao houve alteraçoes estatisticamente significativas antes e após a realizaçao da paracentese total, quando se avaliou o sistema hemodinâmico através do débito cardíaco, do índice cardíaco e do volume sistólico, bem como do sistema renina-angiotensina-aldosterona. Nao houve diferença estatística antes e após a paracentese, quando foi analisada a funçao renal através do volume urinário, da uréia, da creatinina, da depuraçao da creatinina endógena e da taxa de filtraçao glomerular utilizando (51)Cr-EDTA. Nao houve complicaçoes decorrentes do procedimento e a sobrevida observada foi de 73 por cento em 17 meses.

Papel do gradiente de pH sangue arterial-ascite no dignóstico diferencial dos derrames peritoneais / Arterial-ascitic fluid pH gradient role in the differential deagnosis of ascites

Os autores avaliam o papel do gradiente de pH sangue arterial-ascite no diagnóstico da carcinomatose peritoneal. Para tanto, analisam de forma prospectiva 362 pacientes com ascite de diversas etiologias. Na ascite decorrente de carcinomatose peritoneal, o gradiente de pH foi superior a 0,10 em 44,74 por cento dos casos, com especificidade de 90,56 por cento. Concluem que embora este parâmetro nao apresente alta sensibilidade no diagnóstico do comprometimento neoplásico da membrana peritoneal, é bastante específico para tal, podendo orientar o clínico na investigaçao propedêutica dos derrames peritoneais.

An experience with diagnostic paracentesis in 100 cases of acute abdomen.

Prognosis of acute surgical conditions of abdomen of both traumatic and non-traumatic origin depends on accurate diagnosis and early surgical intervention. However, the diagnosis in acute abdomen is quite difficult. The problem becomes more complex when 24 hours services of radiology and laboratories are not available. Abdominal tap gives a valuable clue to diagnosis. A 10 ml syringe fitted with a 20 gauge intravenous needle was used for the purpose. Both the flanks and the four quadrants of the abdomen were selected for site of the tap according to the suspected pathology. Results were indicated as positive when abnormal fluid (clear, turbid, purulent, bloody, serosanguinous, bile stained and urine, etc) were aspirated. Accurate diagnosis were made in 84.3% in blunt abdominal trauma and 76.47% in non-traumatic acute abdomen. High incidence of accurate results were obtained in gastroduodenal perforations (92%), ruptured ectopic gestation (100%) and burst amoebic liver abscess (100%). The procedure is very simple which could be done at bed side without much disturbance to the severely ill patients. It is safe and free from any complications even if the bowel is also punctured during the tap.