Search

Background: Stimulation of hepatic hypertrophy is a useful aid to accomplish hepatic resections when the future liver remnant (FLR) is small. Although inflow occlusion, especially through portal flow, has been extensively studied, the role of outflow modulation has not yet been described., Methods: Description of outflow modulation to tailor hypertrophy of future liver remnant in the context of bilobar metastatic disease. A patient with small FLR (segments I and IV) was managed with a two-stage procedure. The first stage consisted of a right hepatectomy and modulation of the left hepatic vein outflow through reduction of its diameter, with macroscopic congestion of segments II-III. The second stage consisted of a left lateral sectionectomy six weeks later. Postoperative courses were uneventful without any sign of liver failure., Results: Following the first stage procedure computed tomography revealed distinct hypertrophy rates between sections. The non-congested area had an increase of 156% in the volume of segment IV (from 137 to 351 cm(3)) and 100% in the volume of segment I (from 20 to 40 cm(3)). The congested area, segments II-III, increased only 24% (from 205 to 253 cm(3))., Conclusion: Modulation of liver outflow allows maintenance of function in the segments to be resected while avoiding their hypertrophy. This process prevents liver failure and optimizes regeneration of hepatic territories to be preserved., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The development of atherosclerosis is caused by the accumulation of lipid, inflammatory cytokine production, and the large amount of inflammatory cells in the arterial wall. It is now established that the presence of oxidized low-density lipoproteins (ox-LDL) has an important role in the pathogenesis of the disease. There are many scavenger receptors for ox-LDL, among which LOX-1 seems to be important for the induction of endothelial dysfunction and the other subsequent events that lead to the formation of atheromatous plaque. Our findings indicate the presence of a regulatory role induced by the presence of ox-LDL on LOX-1 through the amplification of IL-6 synthesis. This mechanism contributes to the upregulation of the ORL-1 gene expression in presence of risk factors. Many authors have shown the possibility to use LOX-1 as a good marker for the diagnosis and prognosis of coronary artery disease because it is easy to measure and more sensitive than other markers commonly used in the routine of laboratory medicine.

The use of near-infrared spectroscopy (NIRS) is proposed in this study for the characterization of the quality parameters of a smoked and dry-cured meat product known as Bauernspeck (originally from Northern Italy), as well as of some technological traits of the pork carcass used for its manufacturing. In particular, NIRS is shown to successfully estimate several key quality parameters (including water activity, moisture, dry matter, ash and protein content), suggesting its suitability for real time application in replacement of expensive and time consuming chemical analysis. Furthermore, a correlative approach based on canonical correlation analysis was used to investigate the spectral regions that are mostly correlated to the characteristics of interest. The identification of these regions, which can be linked to the absorbance of the main functional chemical groups, is intended to provide a better understanding of the chemical structure of the substrate under investigation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mate (Ilex paraguariensis A. St.-Hil) is a native species of South America used to prepare traditional beverages. Recently a possible effect of its infusion on oxidative stress found in dyslipidemias has been reported. The main compounds related to these activities are phenolic compounds derived from chlorogenic acid. This study aimed to determine the anticholesteremic effect of the hydroethanolic extract (HEIP) and its n-butanolic fraction (n-BFIP), with standardized content of phenolic compounds derived from chlorogenic acid, in rats treated with high-fat diet (HFD). The contents of these compounds in the ethanol extract and n-butanol fraction were respectively two and three times higher than in traditional infusion with predominance of dicaffeoylquinic derivatives. The extracts were able to reduce serum triglycerides and cholesterol and decrease the atherogenic index in treated animals. These results support a potential effect of the mate extract in cardiovascular disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study aims to investigate the effects of supercritical carbon dioxide (SC-CO₂) treatment on the inactivation of the natural microbial flora in cubed cooked ham. Response surface methodology with a central composite design was applied to determine the optimal process conditions and investigate the effect of three independent variables (pressure, temperature and treatment time). Additionally, analyses of texture, pH and color together with a storage study of the product were performed to determine its microbial and qualitative stability. Response surface analysis revealed that 12 MPa, 50 °C, 5 min were the optimal conditions to obtain about 3.0, 1.6, and 2.5 Log(CFU/g) reductions of mesophilic aerobic bacteria, psychrophilic bacteria and lactic acid bacteria respectively. Inactivation to undetectable levels of yeasts and molds and coliforms was also obtained. A storage study of 30 days at 4 °C was carried out on the treated product (12 MPa, 50 °C, 5 min) monitoring microbial growth, pH, texture, and color parameters (L*, a*, b* and ΔE). Microbial loads slightly increased and after 30 days of storage reached the same levels detected in the fresh product. Color parameters (L*, a*, b*) showed slight variations while pH and texture did not change significantly. On the basis of the results obtained, SC-CO₂ can be considered a promising technique to microbiologically stabilize cubed cooked ham and, in general, cut/sliced meat products without affecting its quality attributes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Abnormal angiogenesis is implicated in a number of human diseases and endothelial growth inhibition represents a common approach in tumor therapy. Recently itraconazole, frequently used in humans as antifungal drug, which blocks the biosynthesis of cholesterol, has been found to be antiangiogenic in primary umbilical vein endothelial cells. However, the exact antiangiogenic mechanisms remain largely unknown. In this paper, we studied the effect of itraconazole in human dermal microvascular endothelial cells (HMEC-1), an immortalized cell line to study adult angiogenesis. A 50% reduction of microtubule formation was observed after itraconazole treatment which was partially rescued by cholesterol addition. We found that itraconazole inhibits angiogenesis markers such as VEGF, AAMP and e-NOS. mTOR and ERK1/2 phosphorylation as well as the expression of Gli1, one of the main controllers of the Shh pathway, were also inhibited by itraconazole. Cholesterol addition did not completely rescue inhibition of these pathways, suggesting that the itraconazole antiangiogenic activity could be due to multiple mechanisms. Our results may contribute to novel approaches to block angiogenesis with therapeutic application., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The objective of the present study was the evaluation of the effectiveness of supercritical carbon dioxide (SC-CO(2)) as a nonthermal technology for the pasteurization of fresh-cut coconut, as an example of ready-to-eat and minimally processed food. First, the inactivation kinetics of microbiota on coconut were determined using SC-CO(2) treatments (pressures at 8 and 12 MPa, temperatures from 24 to 45 °C, treatment times from 5 to 60 min). Second, the effects of SC-CO(2) on the hardness and microstructure of fresh-cut coconut processed at the optimal conditions for microbial reduction were investigated. SC-CO(2) treatment of 15 min at 45 °C and 12 MPa induced 4 log CFU/g reductions of mesophilic microorganisms, lactic acid bacteria, total coliforms, and yeasts and molds. The hardness of coconut was not affected by the treatment but the samples developed an irregular and disorderly microstructure. Results suggested the potential of SC-CO(2) in preserving fresh-cut fruits and ready-to-eat products., (© 2012 Institute of Food Technologists®)

Context: TSH, which acts via specific cell membrane TSH receptors (TSHR), is regarded as a thyroid-specific growth factor. Recently, the presence of TSHR has been reported in extrathyroid tissues, but the role of TSH in nonthyroid tissue is unknown., Objective: Our objective was to study the direct effect of TSH on angiogenesis in a human dermal microvascular endothelial cell line (HMEC-1). Parallel experiments were conducted with human primary cardiac microvascular endothelial cells (HMVEC-C)., Design: TSHR in HMEC-1 was measured by immunofluorescence, Western blot, and RT-PCR and its functional activity by variation of intracellular cAMP concentrations. The expression of some angiogenic genes and angiogenic signaling pathways was also evaluated after TSH treatment. Assays of cell proliferation and capillary network formation on collagen or Matrigel were performed in HMEC-1 cells and HMVEC-C., Results: We showed the presence of TSHR in HMEC-1 cells. Increased intracellular cAMP concentrations after TSH treatment indicated the TSHR to be functional. TSH enhanced proliferation and stimulated capillary network formation in HMEC-1, whereas antibodies against vascular endothelial growth factor (VEGF) and TSHR abolished this effect. TSH increased AAMP, VEGF, and eNOS expression. TSH induced phosphorylation of protein kinase S6K1, whereas TSHR blocking antibodies inhibited the phosphorylation of the protein kinase S6K1. A similar effect of TSH on capillary network formation was observed in HMVEC-C., Conclusion: Our findings provide strong evidence for a direct effect of TSH on angiogenesis through its receptor, via cAMP-mammalian target of rapamycin signaling and indicate that this effect is VEGF dependent.

We previously reported the presence in the membrane erythrocyte of a TSH receptor (TSHR), a G-protein coupled receptor, which responds to TSH with increased cAMP level. Since there is evidence for a role of G protein receptors as oxygen sensor(s) implicated in cell volume regulation, we hypothesized that erythrocyte TSHR, by TSH stimulation, could modify the erythrocyte volume and the oxygenation state of erythrocytes. We determined the effect of TSH on the gas analysis in 35 thyroidectomized patients for stage I differentiated thyroid cancer enrolled for recombinant human thyroid-stimulating hormone (rhTSH) test during chronic treatment with synthetic l-thyroxine. Moreover, we explored the influence of TSH on the shape of erythrocytes. Venous blood-gas analysis before and after TSH were determined with a pH/blood gas electrolyte and 682 CO-Oxymeter. In a subgroup of subjects (n=10), the isolated red blood cells (RBC) were analyzed by flow cytometry for morphological changes. After TSH stimulation, we found a significant decrease in PCO(2) (P<0.001), an increase in pH (P<0.01) and an increase of % O(2)-Hb (P<0.05) and pO(2) (P<0.05). By flow cytometry, the erythrocytes after TSH showed a significant enrichment on the mean number in the selected region R1 corresponding to bigger volumes (P<0.05, n=10). Finally, by contrast phase microscopy, when the cell area was measured, a mean increased volume was observed in erythrocytes after TSH compared to the basal before TSH (P<0.05). In conclusion, our results indicate that acute stimulation of TSH by rhTSH modifies the oxygenation state and volume of erythrocyte., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Background: The new global epidemic, overweight and obesity, has a significant role in the etiology of liver tumors. However, the impact of body weight on the outcome after liver resection is unknown., Methods: We carried out a prospective study of 684 patients who underwent liver resections. Patients were stratified according their body mass index (BMI) as follows: normal (<25 kg/m(2)) (52%), overweight (25-29 kg/m(2)) (34%), and obese (≥30 kg/m(2)) (14%), and according to the extent of resection, as either minor or major hepatectomy. Preoperative and intraoperative characteristics and outcomes were prospectively studied. The Dindo-Clavien classification of morbidity was used., Results: Overall postoperative morbidity and morbidity rates were not influenced by BMI. Pulmonary complications were significantly more frequent in obese patients irrespective of the extent of resection. During major resection obese had longer pedicular clamping and more frequently required blood transfusion. After major resection, major morbidity (Dindo-Clavien grade III or more) was more frequent in obese (57%) and overweight (54%) patients than in patients of normal body weight (35%; P < 0.05), including a higher rate of respiratory complications and ascites and longer intensive care unit (ICU) and hospital stays. Obesity and overweight were independent predictors of major morbidity (OR 2.6, 95% CI 1.2-5.8 and OR 1.9, 95% CI 1.2-3.2, respectively), and obesity was a predictor of the need for blood transfusion (OR 3.3, 95% CI 1.4-7.9) after major resections., Conclusions: Obese and overweight patients are at increased risk of potentially life-threatening morbidity after major hepatic resections. Because the risk of mortality is not increased significantly, there is no justification for a compromise in the indication or extent of surgery.

The aim of this study was to apply biometric measurements and analyses of proximate composition, fatty acid composition, and ratios of stable isotopes of carbon (δ(13)C) and nitrogen (δ(15)N) in muscle tissue to reliably differentiate between wild and farmed European sea bass (Dicentrarchus labrax). Farmed (n = 20) and wild (n = 19) European sea bass were purchased between March and May 2008 and used as standard samples. In the same months, a survey was conducted to evaluate the truthfulness of the statements on the labels of European sea bass sold in retail markets (declared farmed n = 34 and declared wild n = 33). In addition, data from the literature (reference) were employed to build the profile type of wild and farmed European sea bass. Primarily, an exploration and comparison of the analytical data of the standard data set based on principal component analysis and permutation test were performed. Afterward, an inferential statistical approach based on nonparametric combination test methodology (NPC) was applied on standard samples to check its suitability in discriminating the production method. This multivariate statistical analysis selected 30 variables on a total of 36 available. The validation of standard fish data set was accomplished by a novel nonparametric rank-based method according to profile type (just 1 misclassification over 39 samples). Both the NPC test and nonparametric rank-based method were then applied to survey fishes using the selected variables with the aim to classify the individual European sea bass as "true farmed" or "true wild". The former test segregated 10 fishes over 33 declared wild, whereas the results obtained by the nonparametric rank-based method showed that 11 of 33 declared wild European sea bass samples could be unquestionably attributed to the wild cluster. Moreover, considering the comparative contribution of profile type, a few surveyed farmed samples were ascribed to the wild cluster.

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra-thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K-ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K-pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K-pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co-immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K-ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co-localization of TSHR with Na/K-ATPase and co-immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K-ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control., ((c) 2009 John Wiley & Sons, Ltd.)

The presence of thyrotropin receptors (TSHR) has been reported in some extrathyroidal tissues but its physio-pathological role still remains unclear. TSH (seems to) affects the human erythrocytes Na(+)/K(+)-ATPase in vitro, however receptors on erythrocytes have not yet been described. In this work the effect of recombinant human TSH (rhTSH) on sites number and activity of erythrocyte Na(+)/K(+)-ATPase was investigated in a group of thyroidectomized patient enrolled for rhTSH test. As detected by (3)H-ouabain binding, rhTSH administration induced a significant increase in the number of sites (p=0.005) and in the Kd (p=0.006) of Na(+)/K(+)-ATPase. rhTSH did not induce significant difference in Na(+)/K(+)-ATPase activity measured by (86)Rb uptake. (125)I-TSH binding studies and Western blotting data showed the existence of TSHR in the erythrocytes of healthy donors. In conclusion The TSH action on Na(+)/K(+)-ATPase of human erythrocytes can be explained by the presence of TSHR.

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.

Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Bacterial translocation (BT) is a phenomenon in which live bacteria or its products cross the intestinal barrier. Gut translocation of bacteria has been shown in both animal and human studies. BT and its complications have been shown clearly to occur in animal models, but its existence and importance in humans has been difficult to ascertain. We review the mechanisms of BT and its clinical impact based on the current literature.

Objective: To standardize the definition of postoperative liver failure (PLF) for prediction of early mortality after hepatectomy., Summary Background Data: The definition of PLF is not standardized, making the comparison of innovations in surgical techniques and the timely use of specific therapeutic interventions complex., Methods: Between 1998 and 2002, 775 elective liver resections, including 69% for malignancies and 60% major resections, were included in a prospective database. The nontumorous liver was abnormal in 43% with steatosis >30% in 14%, noncirrhotic fibrosis in 43%, and cirrhosis in 12%. The impact of prothrombin time (PT) <50% and serum bilirubin (SB) >50 micromol/L on postoperative days (POD) 1, 3, 5, and 7 was analyzed., Results: The lowest PT level was observed on postoperative day (POD) 1, while the peak of SB was observed on POD 3. These 2 variables tended to return to preoperative values by POD 5. The median interval between hepatectomy and postoperative death was 15 days (range, 5-39 days). Postoperative mortality significantly increased in patients with PT <50% and SB >50 microml/L. The conjunction of PT <50% and SB >50 micromol/L on POD 5 was a strong predictive factor of mortality. In patients with significant morbidity, this "50-50 criteria" was met 3 to 8 days before clinical evidence of complications., Conclusions: The association of PT <50% and SB >50 microml/L on POD 5 (the 50-50 criteria) was a simple, early, and accurate predictor of more than 50% mortality rate after hepatectomy. This criteria could be identified early enough, before clinical evidence of complications, for specific interventions to be applied in due time.

The location of a pseudocyst (PC) in the liver is an exceptional event, and intrahepatic PCs are mainly located in the left lobe. We report here a case of right intrahepatic PC following acute pancreatitis associated with cystic (aberrant pancreatic) dystrophy of the duodenal wall (CDDW) and chronic pancreatitis. Morphological assessment (ultrasound, computed tomography [CT] scan, and cholangio-magnetic resonance imaging [MRI]) revealed a 10-cm right intrahepatic collection and rupture of the main pancreatic duct. Percutaneous puncture permitted us to detect a high level of amylase in the collection, confirming the diagnosis of intrahepatic PC. Surgical drainage concomitant with pancreatico-duodenectomy for the treatment of CDDW resulted in disappearance of the collection. The mechanism involved in this patient was rupture of the pancreatic duct in the retroperitoneal cavity and erosion reaching the right hepatic parenchyma. Although intrahepatic PCs are rare, the diagnosis of intrahepatic PC complicating acute pancreatitis can be confirmed by a high level of amylase in the collection. Asymptomatic intrahepatic PCs can be treated conservatively, and symptomatic intrahepatic PCs can be managed either transcutaneously or surgically.

In animal models the function of the sodium pump (sodium/potassium-adenosine triphosphatase [Na+/K(+)-ATPase]) is enhanced by 3,5,3'-triiodothyronine (T3) and inhibited by the antiarrhythmic agent amio. However, it is still unclear whether the effect of the drug on Na+/K(+)-ATPase depends on the interference with thyroid hormone action. We evaluated the interaction of T3 with amiodarone on Na+/K(+)-ATPase activity and site number in human myocardium. Right atrial slices were cultured with (T3+) and without (T3-) 3 nM T3 in presence and absence of amiodarone at therapeutical dose (1.5 microM). When compared to T3+, T3- preparations showed decreased 3H-ouabain binding (p < 0.004) and lower 20-minute and 45-minute 86Rb-uptake (p < or = 0.004). Amiodarone caused an average 49% reduction of the T3-dependent 3H-ouabain binding and decreased the Western blot signal for the Na+/K(+)-ATPase alpha1 subunit. The drug also inhibited T3-dependent increase in 86Rb-influx at 20 and 45 minutes by 66% and 42%, respectively, without affecting the affinity of the pump for K+. No differences were found in the 3H-ouabain binding and 86Rb-uptake of T3-, T3- amio and T3(+)-amio. In conclusion, T3 stimulates the Na+/K(+)-ATPase in human atrial myocardium by increasing the number of ouabain-binding sites, whereas amiodarone decreases the sodium pump function secondarily to the antagonism with thyroid hormone.

The split-liver technique is an important means to alleviating donor shortage. Its development is, at least in part, hindered by the risk of biliary complications, particularly when splitting is performed ex situ. We present a simple technique aimed at improving the identification of the biliary anatomy at the hilar level and the safety of the procedure.

Endogenous ouabain-like factor (OLF) has been detected in mammalian plasma, adrenal gland, and hypothalamus. We investigate whether cardiac tissue may also produce OLF. HPLC chromatographic separation of cardiac extracts showed that RIA-determined OLF activity coincided with the elution profile of exogenous ouabain and with the ability to inhibit 86Rb uptake in human erythrocytes. OLF activity was remarkably higher in excised hearts (3.94 +/- 0.84 pmol/g wet weight by RIA) than in rat blood (0.05 +/- 0.02 pmol/ml). Similar values were obtained in perfused working hearts, without significant changes over time from 5 to 30 minutes of aerobic perfusion. Significant OLF release in the perfusion buffer was also observed (0.54 +/- 0.05 pmoles over 30 minutes). In hearts subjected to 15 minutes of aerobic perfusion followed by 15 minutes of global myocardial ischemia OLF concentration was remarkably increased (8.59 +/- 1.13 versus 4.58 +/- 0.57 pmol/g wet weight by RIA, P < 0.01; an increase after ischemia was confirmed by the assay of 86Rb uptake). Our findings suggest that the rat heart is able to produce OLF, and that its concentration increases during ischemia. Myocardial OLF might modulate the Na/K-ATPase, producing relevant effects on ionic homeostasis and/or gene transcription.

3H-ouabain is useful to evaluate the tissue localization of Na,K-ATPase. In this work we determined the distribution of 3H-ouabain in rabbit tissue by digital radioautography. Using this method, we were able to obtain a comparison of various organs in a relatively short time (6.5 days), while with traditional radioautography, only the kidney was detectable after seven months of film exposure. The kidney has the highest intensity (concentration of 3H-ouabain), followed by the heart, liver, muscle, lung, spleen and finally brain, which was almost undetectable. In kidney the activity was higher in the cortex, while the other tissues displayed a more uniform intensity, suggesting that Na,K-ATPase was evenly distributed.

Nonparasitic splenic cysts are uncommon, with only around 800 cases described in the literature. Posttraumatic splenic pseudocysts constitute most such cases and require surgical treatment when symptomatic or voluminous. Recent studies have provided a better understanding of splenic tissue function and the consequent risks of complete resection of the spleen. Hence surgeons should make every possible effort to preserve splenic tissue. Several spleen-conserving surgical treatments have been proposed, especially for treatment of splenic posttraumatic pseudocysts. The authors report the case of a 13-year-old girl who had a posttraumatic splenic cyst with progressive growth. The diameter of the cyst at surgery was 15 cm, and partial splenectomy was performed. The most common spleen-conserving surgical techniques are briefly reviewed.

Objective: To explore the relation of patient age, status of liver parenchyma, presence of markers of active hepatitis, and blood loss to subsequent death and complications in patients undergoing a similar major hepatectomy for the same disease using a standardized technique., Summary Background Data: Major liver resection carries a high risk of postoperative liver failure in patients with chronic liver disease. However, this underlying liver disease may comprise a wide range of pathologic changes that have, in the past, not been well defined., Methods: The nontumorous liver of 55 patients undergoing a right hepatectomy for hepatocellular carcinoma was classified according to a semiquantitative grading of fibrosis. The authors analyzed the influence of this pathologic feature and of other preoperative variables on the risk of postoperative death and complications., Results: Serum bilirubin and prothrombin time increased on postoperative day 1, and their speed of recovery was influenced by the severity of fibrosis. Incidence of death from liver failure was 32% in patients with grade 4 fibrosis (cirrhosis) and 0% in patients with grade 0 to 3 fibrosis. The preoperative serum aspartate transaminase (ASAT) level ranged from 68 to 207 IU/l in patients with cirrhosis who died, compared with 20 to 62 in patients with cirrhosis who survived., Conclusion: A major liver resection such as a right hepatectomy may be safely performed in patients with underlying liver disease, provided no additional risk factors are present. Patients with a preoperative increase in ASAT should undergo a liver biopsy to rule out the presence of grade 4 fibrosis, which should contraindicate this resection.

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

Considerable, but as yet still controversial evidence indicates the presence, in mammalian tissues of endogenous digitalis-like factors (EDLFs) which inhibit cell membrane Na+, K(+)-adenosine triphosphatase (Na+, K(+)-ATPase) and which may cross-react with anti-digitalis antibodies. The aim of this study was to evaluate the effect of antibodies against cardiac glycosides on Na+, K(+)-ATPase in human erythrocytes. For this purpose, we measured the effect of antibodies against two different cardiac glycosides (anti-ouabain rabbit antiserum and anti-digoxin Fab fragments) on the activity of the Na+, K(+)-ATPase, as measured by erythrocyte rubidium-86 (86Rb) uptake, in subjects who had never come into contact with exogenous cardiac glycosides, and compared these results with the effect of two control rabbit sera: a normal serum and an antiserum to a non-related antigen. Anti-ouabain rabbit antiserum and anti-digoxin Fab fragments induced a significantly greater percentage change in 86Rb uptake in the erythrocytes than the two control sera (ANOVA followed by multiple comparison by the Games-Howell test). The average percentage change was +11.8 +/- 16.3% (n = 19) (mean +/- SD) for anti-ouabain antiserum +10.8 +/- 15.6% (n = 23) for anti-digoxin Fab fragments, -1.68 +/- 11.2% (n = 11) for anti-rhGM-CSF antiserum, and -5.8 +/- 11.7 (n = 10) for normal control serum. In a subgroup of ten subjects in whom the 3 antisera were tested simultaneously, the stimulation of erythrocyte 86Rb uptake induced by the two antidigitalis antibodies correlated significantly (r = 0.906, p = 0.001, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

The identification in human plasma of ouabain as an endogenous digitalis-like factor (EDLF) claimed by Hamlyn et al. has recently been contradicted by two studies which failed to demonstrate endogenous ouabain-like immunoreactivity in HPLC fractions in which exogenous ouabain was eluted. In this paper we report the results obtained on the cross-reactivity with antiouabain antibodies of an EDLF purified by us from human newborn cord plasma. We found that this EDLF coeluted with ouabain on HPLC and cross-reacted both with rabbit anti-ouabain antiserum and with the purified antibodies, which excluded possible interferences due to antibodies directed against non-ouabain portions of the immunogen. Similar but not identical slopes of the ouabain and EDLF displacements curves were observed in all competition ELISA experiments. The inhibitory effect of EDLF on erythrocyte 86Rb uptake was reversed by antiouabain antiserum and antibodies. The concentration of EDLF in newborn plasma, in the four different purifications studied ranged from 30 to 380 pM ouabain equivalents (o.e.) by ELISA and from 100 to 300 pM o.e. by 86Rb uptake. Our data thus support the existence, in human newborn plasma, of a factor with both biological and immunological ouabain-like properties, although not necessarily identical to ouabain.

Several lines of evidence suggest the existence, in mammalian body fluids and tissue extracts, of an as yet unidentified endogenous digitalis-like factor with potential cross-immunoreactivity with digitalis. Using anti-digoxin antibodies, we found preliminary immunohistochemical evidence of digoxin-like immunoreactivity in several human tissues. Strong reactivity was found in the adrenal cortex, which may thus represent a site of production of this endogenous factor.

We have previously shown that antidigoxin antibodies may neutralize partially purified endogenous digitalis like factor(s) present in newborn (umbilical cord) plasma. We here report on the preparation of an immunoaffinity chromatographic system (high affinity digoxin-binding antibodies (Fab fragments) bound covalently to Sepharose) for the purification of endogenous digitalis like factor(s). Neonate plasma extract loses all its biological digitalis-like activity (erythrocyte 86Rb uptake inhibition) after absorption on Sepharose coupled to Fab fragments but not after absorption on uncoupled Sepharose. Endogenous digitalis like factor(s) absorbed to Sepharose coupled to Fab fragments can be eluted by methanol. Subsequent HPLC separation indicate that at least two molecular species with digitalis-like properties are retained by antibodies bound to Sepharose and can be recovered with methanol.

Increasing evidence indicates the presence of endogenous digitalis-like compound(s) in human body fluids. In this preliminary report, we describe a study of the partial purification by HPLC of these compounds in the plasma of neonates (who have particularly high concentrations of this substance) and adults. Plasma samples from neonates (cord blood) and adults, lyophilized and extracted with methanol, were applied on a 300 x 3.9 mm C18 Nova Pak column and eluted with a mobile phase of acetonitrile/methanol/water (17/17/66 or 14/14/72 by vol) and, after 30 min, with 100% methanol. We assayed eluted fractions for inhibitory activity of 86Rb uptake and for digoxin-like immunoreactivity. The elution profile revealed a first peak of inhibitory activity of 86Rb uptake at the beginning of the chromatography; another peak was eluted with the 100% methanol. The two peaks also cross-reacted with antidigoxin antibodies. Because the second peak could possibly reflect the nonspecific interference of various lipophilic compounds, we focused our attention on the first peak. For these fractions dose-response curves for 86Rb uptake and for displacement of digoxin were parallel, respectively, to those of ouabain and digoxin, suggesting similarities of digoxin-like immunoreactive substance to cardiac glycosides. Similar chromatographic profiles were also obtained for plasma from adults, suggesting that the endogenous glycoside-like compound(s) in the neonate may be the same as those in the adult.

Increasing evidence indicates the existence of endogenous digitalis like factor(s) (EDLF). We recently reported on the partial purification of an EDLF from newborn (cord) blood which possesses both digoxin-like immunoreactivity and the ability to inhibit the cell membrane sodium pump measured as the inhibitory activity on erythrocyte 86Rb uptake. We here report that high affinity digoxin-binding antibodies (Fab fragments; Digibind, Burroughs Wellcome Co.) are capable of neutralizing the inhibitory activity on erythrocyte Rb uptake not only of digoxin but also of ouabain and of partially purified newborn EDLF. These results provide, to our knowledge for the first time, direct evidence that antidigitalis antibodies may cross-react with one or more circulating substances which share antigenic determinants with digoxin and ouabain and possess endogenous digitalis-like properties, strongly suggesting that these antibodies may be useful tools both for the assay of EDLF and for the study of its biological effects.

Endogenous factors with biological and immunological activity similar to cardiac glycoside drugs (endogenous digitalis-like factors; EDLF) have been found in several tissues and body fluids of animals and humans. Detectable EDLF concentrations were found in blood and urine extracts of adults (normal healthy controls, hypertensive patients and salt-loaded healthy subjects), while higher levels were generally observed in plasma samples of pregnant women, newborns, and patients with renal insufficiency. The chemical characteristics of this endogenous factor are, at present, unknown, although it has been suggested that EDLF could be a substance with low molecular weight. Experimental studies and theoretical considerations suggest that EDLF, in addition to the ability to react with antibodies, might also bind to the specific cellular receptor of the cardiac glycosides and thus inhibit the membrane Na+/K(+)-ATPase (sodium pump). Therefore, it has been suggested that EDLF is an endogenous modulator of the membrane sodium-potassium pump, and that it could play a role in the regulation of fluids and electrolytes, in the myocardial muscular tone and also in the pathogenesis of hypertension.

We assayed digoxin-like immunoreactivity in the serum of 19 healthy neonates (blood sample from umbilical cord), not receiving digoxin therapy, using a solid-phase radioimmunological method. In addition, urinary concentrations of a digoxin-like immunoreactive substance (DLIS) were assayed throughout the first 10 weeks of extrauterine life in 50 healthy infants. The mean (+/- SD) serum DLIS concentration obtained was 211 +/- 60 ng/l digoxin equivalents (d.e.) while, in the urinary samples of the first day of life, the mean concentration was 818 +/- 560 ng/l d.e. (n = 20). These DLIS values in infants were higher (about 5- to 10-fold) than those previously observed in adults. In addition, urinary DLIS values were very high in the first week of life and fell quite rapidly in the next weeks of life. We used serum and urine pools from neonates for gel filtration experiments using a Sephadex G 25 column. In the urine sample we found some immunoreactive peaks eluted at the end and after the region of salts, while the chromatographic profiles of the serum pool showed only a major peak eluted with the peak of proteins. After boiling, the same serum pool showed an elution pattern characterized by a smaller immunoreactive peak eluted with that of proteins and by other peaks similar to those observed for the urine sample. Our study demonstrated that a substance or more substances which cross-react with digoxin-specific antibodies are present in blood and urine of healthy infants. In addition, our chromatographic studies indicate that DLIS is probably a low molecular weight substance(s) which circulate(s) in blood bound to a higher molecular weight molecule.

The existence of endogenous cardiac glycoside-like compounds and their property of being recognized by anti-digoxin antibodies is still a matter of controversy. In order to investigate this problem, endogenous digoxin-like immunoreactivity (measured by RIA) and digitalis-like radioreceptor activity (measured by displacement of 3H-ouabain from erythrocyte membranes) were assessed in plasma extracts of normal adults, pregnant women and newborns. These three groups were chosen because of their known widely different levels of digoxin-like immunoreactivity. Compared to adults, newborns and pregnant women had significantly higher levels not only of immunoreactivity but also of displacement of 3H-ouabain binding, the latter being due, according to Scatchard analysis, to a decrease of the affinity of ouabain to its cellular receptor rather than to its maximal binding capacity. Furthermore, immunoreactivity and binding displacement correlated significantly. Our data indicate that one (or more) compounds with cardiac glycoside-like properties (both immunological and at the receptor level) are present in the plasma of newborns and pregnant women, and confirm the idea that radioimmunological methods may be useful in studying endogenous inhibitors of the sodium pump.

The presence of an endogenous digoxin-like immunoreactive substance(s) (DLIS) has been reported in the plasma and urine of experimental animals and humans. This substance might have a role in arterial hypertension. Although the chemical structure of DLIS is at present unknown, several studies indicate that DLIS has a low molecular weight and is reversibly bound in serum to carrier proteins. We have investigated the carrier protein of DLIS in chromatographic studies, using neonate plasma eluted on a Sephadex G200 column. The chromatographic profile of digoxin-like immunoreactivity showed a major peak with a molecular weight of approximately 60,000 daltons and a smaller peak eluted after the salt region. When the major peak was chromatographed on a Sephadex G100 column only one single peak was obtained, which closely coincided with the elution peak of albumin. Chromatographic separation of neonate plasma on a Sephadex G25 revealed a major post-salt immunoreactive peak. When these fractions were incubated with purified human albumin and separated again on the Sephadex G25, a large immunoreactive peak corresponding to albumin was again found while the immunoreactivity after salt completely disappeared. Our data therefore strongly suggest that the endogenous digitalis-like factor is carried in plasma by albumin.

Evidence has been provided on the increased presence, in essential hypertension, of endogenous digitalis-like factor(s) [DLIS, digoxin-like immunoreactive substance(s)] able to cross-react with antidigoxin antibodies and to inhibit the membrane-bound sodium-potassium pump. An inhibition of the sodium pump could lead, in smooth muscle cells, to an increase of intracellular calcium ions and to an increase of total peripheral resistances. In this study the relation between plasma levels of DLIS and the acute hypotensive effect of a calcium antagonist (nifedipine) has been evaluated in a group of borderline to severe hypertensive patients and in a control group of normotensive subjects. The results obtained confirm that the hypotensive effect of nifedipine is related to pretreatment blood pressure and show, only in hypertensive patients, a significant relation of DLIS with both pretreatment blood pressure and blood pressure decrement induced by nifedipine. These findings are compatible with a possible role of DLIS in modulating cellular calcium handling.

The chromosomal number in relation with the age of suspension culture of nicotiana glauca (NG) and the non tumorous N. glauca x N. langsdorffii hybrid has been studied. In both species, an higher variability of chromosome number is present in younger culture. Aged cultures show a stabilization, for NG, around the hexaploid number (3n = 72) and for NNT, which is an amphidiploid specie, on the tetraploid level (4n = 84). Both species show, with the age of culture, a decrease in plant regeneration capacity, which is not due to chromosomal variability, since young cultures are much more variable than older one.