Laparoscopic Liver Resection: A South American Experience with 2887 Cases.

BACKGROUND: Laparoscopic liver resections (LLR) have been increasingly performed in recent years. Most of the available evidence, however, comes from specialized centers in Asia, Europe and USA. Data from South America are limited and based on single-center experiences. To date, no multicenter studies evaluated the results of LLR in South America. The aim of this study was to evaluate the experience and results with LLR in South American centers. METHODS: From February to November 2019, a survey about LLR was conducted in 61 hepatobiliary centers in South America, composed by 20 questions concerning demographic characteristics, surgical data, and perioperative results. RESULTS: Fifty-one (83.6%) centers from seven different countries answered the survey. A total of 2887 LLR were performed, as follows: Argentina (928), Brazil (1326), Chile (322), Colombia (210), Paraguay (9), Peru (75), and Uruguay (8). The first program began in 1997; however, the majority (60.7%) started after 2010. The percentage of LLR over open resections was 28.4% (4.4-84%). Of the total, 76.5% were minor hepatectomies and 23.5% major, including 266 right hepatectomies and 343 left hepatectomies. The conversion rate was 9.7%, overall morbidity 13%, and mortality 0.7%. CONCLUSIONS: This is the largest study assessing the dissemination and results of LLR in South America. It showed an increasing number of centers performing LLR with the promising perioperative results, aligned with other worldwide excellence centers.

Phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase signaling pathways are required for the regulation of 5-aminolevulinate synthase gene expression by insulin.

Insulin regulates the expression of several hepatic genes. Although the general definition of insulin signaling has progressed dramatically, the elucidation of the complete signaling pathway from insulin receptor to transcription factors involved in the regulation of a specific gene remains to be established. In fact, recent works suggest that multiple divergent insulin signaling pathways regulate the expression of distinct genes. 5-Aminolevulinate synthase (ALAS) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. It has been reported that insulin caused the rapid inhibition of housekeeping ALAS transcription, but the mechanism involved in this repression has not been explored. The present study investigates the role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase pathways in insulin signaling relevant to ALAS inhibition. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in rat hepatocytes and HepG2 cells. Wortmannin and LY294002, PI3-kinase inhibitors, as well as lovastatin and PD152440, Ras farnesylation inhibitors, and MEK inhibitor PD98059 abolished the insulin repression of ALAS transcription. The inhibitor of mTOR/p70(S6K) rapamycin had no effect whatsoever upon hormone action. The overexpression of vectors encoding constitutively active Ras, MEK, or p90(RSK) mimicked the inhibitory action of insulin. Conversely, negative mutants of PKB, Ras, or MEK impaired insulin inhibition of ALAS promoter activity. Furthermore, inhibition of one of the pathways blocks the inhibitory effect produced by the activation of the other. Our findings suggest that factors involved in two signaling pathways that are often considered to be functionally separate during insulin action, the Ras/ERK/p90(RSK) pathway and the PI3K/PKB pathway, are jointly required for insulin-mediated inhibition of ALAS gene expression in rat hepatocytes and human hepatoma cells.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

Recent measles viral activity in Uruguai: serological and genetic approaches.

In the summer 1999, a measles outbreak occurred in Uruguai. During this outbreak 58 cases were recorded, 36 of which were laboratory confirmed as positive for measles virus (MV) IgM. The cases occurred in touristic places (Montevideo and Maldonado) predominantly among health facilities and tourist service personnel. Urine specimens collected between days 1 and 4 after the onset of the rash from seven cases were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR with primers specific for the carboxyl-terminal region of the nucleoprotein (N) gene. Three of these specimens/cases were positive for MV. Sequencing of 300 nucleotides (nt) of PCR products corresponding to a part of the carboxyl-terminal region of the MV N gene detected in these specimens MV of D6 genotype. The same nucleotide sequences and the same genotype were also previously observed for MV isolates from the 1997 epidemic in Brazil and the 1998 epidemic in Argentina, demonstrating that the D6 genotype was, and may be still circulating in South America.

Transcriptional regulation of 5-aminolevulinate synthase by phenobarbital and cAMP-dependent protein kinase.

5-Aminolevulinate synthase (ALA-S) is a mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of the heme biosynthesis. There are two ALA-S isozymes encoded by distinct genes. One gene encodes an isozyme that is expressed exclusively in erythroid cells, and the other gene encodes a housekeeping isozyme that is apparently expressed in all tissues. In this report we examine the mechanisms by which phenobarbital and cAMP regulate housekeeping ALA-S expression. We have determined that cAMP and phenobarbital effects are additive and the combined action is necessary to observe the cAMP effect on ALA-S mRNA in rat hepatocytes. The role of the cAMP-dependent protein kinase (PKA) has been examined. A synergism effect on ALA-S mRNA induction is observed in rat hepatocytes treated with pairs of selective analogs by each PKA cAMP binding sites. A 870-bp fragment of ALA-S 5'-flanking region is able to provide cAMP and phenobarbital stimulation to chloramphenicol O-acetyltranferase fusion vectors in transiently transfected HepG2 cells. ALA-S promoter activity is induced by cotransfection with an expression vector containing the catalytic subunit of PKA. Furthermore, cotransfection with a dominant negative mutant of the PKA regulatory subunit impairs the cAMP analog-mediated increase, but the phenobarbital-mediated induction is not modified. Our data suggest that the transcription factor cAMP-response element binding protein (CREB) is probably involved in PKA induction of ALA-S gene expression. Finally, heme addition greatly decreases the basal and phenobarbital or cAMP analog-mediated induction of ALA-S promoter activity. The present work provides evidence that cAMP, through PKA-mediated CREB phosphorylation, and phenobarbital induce ALA-S expression at the transcriptional level, while heme represses it.

Insulin inhibits delta-aminolevulinate synthase gene expression in rat hepatocytes and human hepatoma cells.

Insulin has been known to regulate intracellular metabolism by modifying the activity or location of many enzymes but it is only in the past few years that the regulation of gene expression is recognized to be a major action of this hormone. The present work provides evidences that insulin inhibits delta-aminolevulinate synthase (ALA-S) gene expression, the enzyme which governs the rate-limiting step in heme biosynthesis. The addition of 5 nM insulin to hepatocytes culture led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA in a dose-dependent manner, as measured by Northern and slot-blot analysis. Several clues as to how insulin regulates ALA-S transcription were determined. The inhibitory effect is achieved at physiological concentrations but much higher proinsulin doses are needed. Insulin's effect is rapid, quite specific, and protein synthesis is not required. Moreover, ALA-S mRNA half-life is not modified by the presence of the peptidic hormone. Our results demonstrate that the insulin effect is dominant; it overrides 8-CPT-cAMP plus phenobarbital-mediated induction. Also, insulin requires the activation of protein kinase C to exert its full effect. On the other hand, a 870-bp fragment of the ALA-S promoter region is able to sustain the inhibition of CAT expression in plasmid-transfected HepG2 cells. Thus, these results indicate that insulin plays an important role in regulating ALA-S expression by inhibiting its transcription.

Evidence that protein kinase C is involved in delta-aminolevulinate synthase expression in rat hepatocytes.

There are many factors that regulate the rate of synthesis of delta-aminolevulinate synthase (ALA-S), the enzyme which governs the rate-limiting step in heme biosynthesis. In rat hepatocytes, phenobarbital increases ALA-S gene transcription and dibutyryl cAMP potentiates this induction, whereas insulin and glucose have the opposite effect. The present report provides evidence that protein kinase C (PKC) activation negatively influences ALA-S mRNA levels, as measured by Northern and slot-blot analysis. The addition of 1,2-dioctanoyl-sn-glycerol (DOG) or 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator that mimics diacylglycerol function, to cultures led to a significant decrease of both basal and phenobarbital-induced ALA-S mRNA levels in a dose-dependent manner. This TPA effect depends on the specific activation of PKC because the analog 4 alpha-phorbol 12,13-diacetate, a nonstimulatory PKC phorbol ester, is unable to inhibit ALA-S mRNA. Furthermore, the effect of TPA is blocked by the PKC inhibitors staurosporine and calphostin C. Desensitization of the PKC pathway by prolonged exposure to TPA abolished the subsequent action of the phorbol ester. On the other hand, neither TPA nor DOG modified the half-life of ALA-S mRNA. The study of the combinatorial action of TPA and cAMP revealed that the inhibitory effect of TPA overcomes dibutyryl cAMP induction. Thus, these results indicate that PKC plays an essential role in regulating ALA-S expression, probably at a transcriptional level.

Glucose inhibits phenobarbital-induced delta-aminolevulinate synthase expression in normal but not in diabetic rat hepatocytes.

In the present work, we demonstrate the presence of a glucose inhibitory effect on the phenobarbital-mediated induction of the delta-aminolevulinate synthase mRNA in normal rat hepatocytes, consistent with the results obtained with the delta-aminolevulinate synthase activity previously reported. This "glucose effect" can be prevented by adding cAMP, adenylate cyclase activators, or a phosphodiesterase inhibitor. Delta-Aminolevulinate synthase mRNA half-life is not modified in the presence of phenobarbital or glucose. When the same experiments are performed using diabetic cells, no glucose effect is observed, even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study suggest that glucose decreases delta-aminolevulinate synthase biosynthesis by acting at a pretranslational step. Assuming that the glucose effect operates by a repression mechanism exerted by metabolites derived from or related to glucose, the present results may reflect a derangement in the formation of these metabolites as a result of the abnormal metabolism operating in the diabetic state.

cAMP regulation of phenobarbital-mediated induction of delta-aminolevulinate synthase mRNA in hepatocytes from normal and experimental diabetic rats.

We examined the mechanism underlying the effect of cAMP on delta-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of delta-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of delta-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates delta-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.

Regulation of phenobarbital-induced ferrochelatase mRNA activity by dibutyryl cAMP and glucose in normal and diabetic rat hepatocytes.

The induction of ferrochelatase activity by phenobarbital and its potentiation by dibutyryl cAMP assayed in normal rat hepatocytes are associated with increased activity of ferrochelatase mRNA. Glucose inhibits this stimulatory effect. This inhibition can be reversed with increasing concentrations of dibutyryl cAMP. The inducing effect exerted by phenobarbital on the activity of ferrochelatase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response in diabetic rat hepatocytes is neither potentiated by adding dibutyryl cAMP nor repressed by glucose. The absence of a glucose effect persists even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study are consistent with those reported in other published studies of ferrochelatase activity. This adds more experimental evidence to support the concept that ferrochelatase is inducible. The results obtained suggest that ferrochelatase is more susceptible to induction with phenobarbital in diabetic rat hepatocytes than in normal rat hepatocytes.

Identification of viruses in a study of acute respiratory tract infection in children from Uruguay.

For the purpose of identifying viral agents associated with acute respiratory tract infections (ARI) in children less than 5 years old, a longitudinal community study was undertaken in Montevideo, Uruguay, from May 1985 to December 1987. This report includes results obtained by cell culture and immunofluorescence techniques for detection of respiratory syncytial virus (RSV), influenza A and B viruses, parainfluenza 1 and 3 viruses, and adenovirus. Two populations were studied: children visited at home by pediatricians (group 1) and children with an ARI episode who attended an outpatient clinic (group 2). Nasopharyngeal aspirates were obtained at the time of an ARI episode: 858 from group 1 and 488 from group 2. Viruses were identified in 15.3% of group 1 specimens and in 17.6% of group 2 specimens. RSV was the most frequently recovered agent, accounting for 67.9% and 58.1%, respectively, of all viruses detected. The sensitivity and specificity of RSV isolation by cell culture are compared with detection by indirect immunofluorescence.

Studies on induction of delta-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin. Chlorpropamide, allylisopropylacetamide and lead in hepatocytes from normal and experimental diabetic rats.

The present work demonstrates that phenformin exerted an inducing effect on delta-aminolevulinic acid synthase (ALA-S) and ferrochelatase activities and on cytochrome P-450 content in isolated hepatocytes from rats with experimental diabetes. Similar results were obtained with respect to ALA-S activity and cytochrome P-450 content when chlorpropamide was used. The inducing effect exerted by allylisopropylacetamide (AIA) on ALA-S and ferrochelatase activities in diabetic hepatic cells was markedly greater than that observed in normal hepatocytes. This stimulatory response was not enhanced by adding dibutyryl cyclic AMP (cAMP). When phenformin was added to isolated rat hepatocytes of normal rats, induction of ALA-S and ferrochelatase activities and cytochrome P-450 content was observed only in the presence of added dibutyryl cAMP. Addition of chlorpropamide to this in vitro system did not exert an inducing effect on the same enzymes even in the presence of dibutyryl cAMP. The present results add more experimental evidence about the lability of the heme pathway of diabetic hepatocytes.

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of insulin.

In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of delta-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of delta-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of delta-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of cAMP.

In the present work we have been able to demonstrate the existence of some interrelationship between intracellular level of cAMP content and phenobarbital induction of delta-aminolevulinic acid synthase, ferrochelatase, and cytochrome P-450 biosynthesis in isolated rat hepatocytes. The increase of the level of intracellular cAMP produced by activators of adenylate cyclase, inhibitors of phosphodiesterase, or added cyclic nucleotides is reflected by an increase of the phenobarbital induction effect. The greater induction observed in hepatocytes of diabetic rats may be due to a higher level of the intracellular cAMP. The lack of potentiation of added cAMP in diabetic cells is mainly due to the fact that the maximum induction that could be attained is already achieved by the effect of the preexisting high level of the endogenous cAMP.

Evaluación de la cobertura del programa de vacunaciones, 1988 / Evaluation of the vaccination programm

Se presentan las coberturas del PAI en 1988, que llegan en BCG al 96,8%, en DPT y Polio al 95,2% y en sarampión al 94,3%. Se establece un ranking de cobertura óptimo, regular e insuficiente y el análisis de las 332 comunas del país: 63 comunas tuvieron una cobertura insuficiente, inferior al 80% para el sarampión y 58 comunas para DPT y polio. Se enumeran las causas locales del retardo

Comparación entre la inmunoflurescencia y el ensayo inmunoenzimático para el diagnóstico rápido del virus respiratorio sincicial en secreciones nasofaríngeas / Rapid detection of respiratory syncytial virus antigens in nasopharyngeal secretions: comparison of immunofluorescence and enzyme immunoassay

Un ensayo inmunoenzimático comercial Abboutt-RSV-EIA fue evaluado comprarándolo con la inmunofluorescencia indirecta. Aspirados nasofaríngeos de 95 niños con infección respiratoria aguda baja fueron procesados por inmunofluorescencia y por enzimoinmunoensayo para revelar la presencia de antígenos del virus respiratorio sincicial. De los 60 materiales positivos por inmunofluorescencia, 46 también lo fueron por enzimoinmunoensayo (sensibilidad 78,7%); de 35 negativos, 34 fueron también negativos por el Abbout-RSV-EIA (especificidad 97,1%). Según los resultados presentados, la evaluada es aceptable como una alternativa para el diagnóstico rápido de VRS en lugares donde no se cuente con otros recursos

Comparación entre la inmunoflurescencia y el ensayo inmunoenzimático para el diagnóstico rápido del virus respiratorio sincicial en secreciones nasofaríngeas / Rapid detection of respiratory syncytial virus antigens in nasopharyngeal secretions: comparison of immunofluorescence and enzyme immunoassay

Un ensayo inmunoenzimático comercial Abboutt-RSV-EIA fue evaluado comprarándolo con la inmunofluorescencia indirecta. Aspirados nasofaríngeos de 95 niños con infección respiratoria aguda baja fueron procesados por inmunofluorescencia y por enzimoinmunoensayo para revelar la presencia de antígenos del virus respiratorio sincicial. De los 60 materiales positivos por inmunofluorescencia, 46 también lo fueron por enzimoinmunoensayo (sensibilidad 78,7%); de 35 negativos, 34 fueron también negativos por el Abbout-RSV-EIA (especificidad 97,1%). Según los resultados presentados, la evaluada es aceptable como una alternativa para el diagnóstico rápido de VRS en lugares donde no se cuente con otros recursos (AU)

[Comparison between immunofluorescence and immunoenzymatic assay for the rapid diagnosis of the respiratory syncytial virus in nasopharyngeal secretions]. / Comparación entre la inmunofluorescencia y el ensayo inmunoenzimático para el diagnóstico rápido del virus respiratorio sincicial en secreciones nasofaríngeas.

An enzyme immunoassay, RSV-EIA Abbot, was evaluated by comparison with indirect immunofluorescence. Nasopharyngeal secretions obtained from 95 infants and young children with acute respiratory infections were examined for the presence of respiratory syncytial virus antigens with both methods. Specimens were stored at -70 degrees C before being tested by EIA. Out of 60 samples positive by indirect immunofluorescence, 46 were also positive by RSV-EIA (sensitivity 78.7%) and 34 out of 35 immunofluorescence negative specimens were negative by RSV-EIA (specificity 97.1%). Therefore, the EIA appears to be an acceptable test for the rapid detection of RSV as an alternative for indirect immunofluorescence.

Purification and characterization of ferro- and cobalto-chelatases.

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40,000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240,000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h, but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 microM for the metal and 30.3 microM for mesoporphyrin, and for cobaltochelatase activity, 27 and 45.5 microM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

Comparación entre la inmunofluorescencia y el ensayo inmunoenzimático para el diagnóstico rápido del virus respiratorio sincicial en secreciones nasofaríngeas. / [Comparison between immunofluorescence and immunoenzymatic assay for the rapid diagnosis of the respiratory syncytial virus in nasopharyngeal secretions]

An enzyme immunoassay, RSV-EIA Abbot, was evaluated by comparison with indirect immunofluorescence. Nasopharyngeal secretions obtained from 95 infants and young children with acute respiratory infections were examined for the presence of respiratory syncytial virus antigens with both methods. Specimens were stored at -70 degrees C before being tested by EIA. Out of 60 samples positive by indirect immunofluorescence, 46 were also positive by RSV-EIA (sensitivity 78.7

) and 34 out of 35 immunofluorescence negative specimens were negative by RSV-EIA (specificity 97.1

). Therefore, the EIA appears to be an acceptable test for the rapid detection of RSV as an alternative for indirect immunofluorescence.