Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Glycerol as a substrate for actinobacteria of biotechnological interest: Advantages and perspectives in circular economy systems.

Actinobacteria represent a ubiquitous group of microorganisms widely distributed in ecosystems. They have diverse physiological and metabolic properties, including the production of extracellular enzymes and a variety of secondary bioactive metabolites, such as antibiotics, immunosuppressants, and other compounds of industrial interest. Therefore, actinobacteria have been used for biotechnological purposes for more than three decades. The development of a biotechnological process requires the evaluation of its cost/benefit ratio, including the search for economic and efficient substrates for microorganisms development. Biodiesel is a clean, renewable, quality and economically viable source of energy, which also contributes to the conservation of the environment. Crude glycerol is the main by-product of biodiesel production and has many properties, so it has a commercial value that can be used to finance the biofuel production process. Actinobacteria can use glycerol as a source of carbon and energy, either pure o crude. A circular economy system aims to eliminate waste and pollution, keep products and materials in use, and regenerate natural systems. Although these principles are not yet met, some approaches are being made in this direction; the transformation of crude glycerol by actinobacteria is a process with great potential to be scaled on an industrial level. This review discusses the reports on glycerol as a promising source of carbon and energy for obtaining biomass and high-added value products by actinobacteria. Also, the factors influencing the biomass and secondary metabolites production in bioreactors are analyzed, and the tools available to overcome those that generate the main problems are discussed.

Proteomic analysis of secretomes from Bacillus sp. AR03: characterization of enzymatic cocktails active on complex carbohydrates for xylooligosaccharides production.

Bacillus sp. AR03 have been described as an important producer of carbohydrate-active enzymes (CAZymes) when growing in a peptone-based medium supplemented with simple sugars and/or carboxymethyl cellulose (CMC) as carbon sources. This work aimed to identify the extracellular enzymatic cocktails through shotgun proteomics. The proteomic analysis showed that enzymes involved in cellulose and xylan degradation were among the most abundant proteins. These enzymes included an endo-glucanase GH5_2 and a glucuronoxylanase GH30_8, which were found in all conditions. In addition, several proteins were differentially expressed in the three evaluated culture media, indicating microbial metabolic changes due to the different supplied carbon sources, particularly, in the presence of CMC. Finally, the capability of the crude enzymatic cocktails from culture media to degrade birchwood xylan was assessed, which produced mostly xylooligosaccharides containing among 3-5 xylose units. Consequently, this work shows the potential of the extracellular enzymes from Bacillus sp. AR03 for producing emergent prebiotics.

Bioremediation of lindane-contaminated soils by combining of bioaugmentation and biostimulation: Effective scaling-up from microcosms to mesocosms.

The scaling-up of lindane-contaminated soils bioremediation from microcosms to mesocosms bioaugmentated with an actinobacteria quadruple culture and biostimulated with sugarcane filter cake (SCFC) was surveyed. Mesocosms of silty loam soil, clayey soil, and sandy soil were polluted with the pesticide, bioaugmented with the mixed culture, biostimulated with adequate amounts of 0.5 mm SCFC particles, and assessed during 63 days maintaining environmental parameters with minimal intervention. Samples were taken to determine residual lindane, heterotrophic microorganisms, enzymatic activities, and bioremediation effectiveness using ecotoxicity tests with Raphanus sativus, Lactuca sativa, and Lycopersicon esculentum. The bioaugmentation and biostimulation of the three soils improved lindane removal, microbial counts, and enzymatic activities, and reduced pesticide T1/2, regarding the values obtained in non-bioremediated controls. The removal process was significantly affected by the soil type, and the highest pesticide dissipation (82.6%) was detected in bioremediated sandy soil. Ecotoxicity tests confirmed the bioremediation success through a rise in the vigor index of seedlings compared to non-treated soils (R. sativus: 12-22%; L. sativa: 12-20%; L. esculentum: 30-45%). Finally, scanning electron microscopy corroborated soil colonization by actinobacteria. Successful scaling-up of the combined application of an actinobacteria quadruple culture and SCFC as an appropriate strategy for restoring lindane-polluted soils at mesocosms-scale was confirmed.

Enhanced bioremediation of lindane-contaminated soils through microbial bioaugmentation assisted by biostimulation with sugarcane filter cake.

Lindane is a toxic and persistent organochlorine pesticide, whose extensive use generated its accumulation in different environmental matrices. Bioremediation is a promising technology that can be used combining bioaugmentation and biostimulation processes to soil restoration. The aim of the present work was to determine the conditions of maximum lindane removal by bioaugmentation with an actinobacteria consortium and biostimulation with sugarcane filter cake (SCFC). The assays were carried out on lindane-contaminated silty loam (SLS), clayey (CS), and sandy (SS) soils. Through complete factorial designs, the effects of three abiotic factors (moisture content, proportion and size of SCFC particles) were evaluated on lindane removal. In addition, a response optimizer determined the optimal conditions for pesticide removal in bioaugmented and biostimulated soils, in the range of levels studied for each factor. In these conditions, bioaugmentation of biostimulated soils increased the pesticide removal (SLS: 61.4%, CS: 70.8%, SS: 86.3%), heterotrophic microbial counts, and soil enzymatic activities, and decreased lindane T1/2, regarding the non-bioaugmented biostimulated controls, after 14 days of assay. The values of these parameters confirmed the efficiency of the bioremediation process. Finally, the viability of the four strains was demonstrated at the end of the assay. The results indicate that the simultaneous application of bioaugmentation with the actinobacteria consortium and biostimulation with SCFC constitutes a promising tool for restoring soils contaminated with lindane, by using the optimal conditions obtained through the factorial designs.

Coupling of bioaugmentation and biostimulation to improve lindane removal from different soil types.

Lindane is an organochlorine pesticide that, due to its persistence in the environment, is still detected in different matrices. Bioremediation using actinobacteria consortia proved to be promising for the restoration of contaminated soils. Another alternative to remove xenobiotics is to use agricultural residues, which stimulates microbial activity, increasing its capacity to degrade organic pollutants. The present work studies the coupling of sugarcane bagasse biostimulation and bioaugmentation with the actinobacteria consortium composed of Streptomyces sp. A2, A5, A11 and M7 on lindane removal in different soil types. In this sense, factorial designs with three factors (proportion and size of sugarcane bagasse particles, and moisture content) were employed. A response optimizer identified the combination of factors levels that jointly allowed obtaining the maximum lindane removal in the evaluated conditions. In the optimal conditions, the effect of the bioremediation process on soil microbiota was studied by evaluating different parameters. The highest lindane removal percentages were detected in biostimulated microcosms bioaugmented with the microbial consortium, which were accompanied by a decrease in lindane half-life respect to the controls. Also, the bioaugmentation of biostimulated microcosms increased the microbial counts and enhanced soil enzymatic activities, corroborating the bioremediation process efficiency. The survival of the four actinobacteria at the end of the assay confirmed the ability of all Streptomyces strains to colonize amended soils. Bioremediation by simultaneous application of biostimulation with sugarcane bagasse and bioaugmentation with the actinobacteria consortium, in the optimized conditions, represents an efficient strategy to restore lindane contaminated soils.

Lindane dissipation in a biomixture: Effect of soil properties and bioaugmentation.

The biomixture is the major constituent of a biopurification system and one of the most important factors in its efficiency; hence the selection of the components is crucial to ensure the efficient pesticides removal. Besides, bioaugmentation is an interesting approach for the optimization of these systems. A mixed culture of the fungus Trametes versicolor SGNG1 and the actinobacteria Streptomyces sp. A2, A5, A11, and M7, was designed to inoculate the biomixtures, based on previously demonstrated ligninolytic and pesticide-degrading activities and the absence of antagonism among the strains. The presence of lindane and/or the inoculum in the biomixtures had no significant effect on the development of culturable microorganisms regardless the soil type. The consortium improved lindane dissipation achieving 81-87% of removal at 66 d of incubation in the different biomixtures, decreasing lindane half-life to an average of 24 d, i.e. 6-fold less than t1/2 of lindane in soils. However, after recontamination, only the bioaugmented biomixture of silty loam soil enhanced lindane dissipation and decreased the t1/2 compared to non-bioaugmented. The biomixture formulated with silty loam soil, sugarcane bagasse, and peat, inoculated with a fungal-actinobacterial consortium, could be appropriate for the treatment of agroindustrial effluents contaminated with organochlorine pesticides in biopurification systems.

Petroleum oil removal by immobilized bacterial cells on polyurethane foam under different temperature conditions.

In this work, a mixed biofilm composed by Pseudomonas monteilii P26 and Gordonia sp. H19 was formed using polyurethane foam (PUF) as immobilization support, for crude oil removal from artificial sea water. Fresh immobilized cells and immobilized cells that were stored at 4°C for two months before use were assessed. The oil removal assays were carried out at microcosm scale at 4, 15 and 30°C. A viability loss of P. monteilii P26 was observed after the storage. The highest removal value (75%) was obtained at 30°C after 7days using fresh immobilized cells on PUF. Enhanced oil bioremoval was obtained at 4°C and 15°C with the previously stored immobilized cells compared to the fresh immobilized cells. Crude oil sorption on the different systems was responsible for the removal of 22-33% oil at the different temperatures. In conclusion, an economic tool for petroleum bioremediation is proposed.

Removal of a mixture of pesticides by a Streptomyces consortium: Influence of different soil systems.

Although the use of organochlorine pesticides (OPs) is restricted or banned in most countries, they continue posing environmental and health concerns, so it is imperative to develop methods for removing them from the environment. This work is aimed to investigate the simultaneous removal of three OPs (lindane, chlordane and methoxychlor) from diverse types of systems by employing a native Streptomyces consortium. In liquid systems, a satisfactory microbial growth was observed accompanied by removal of lindane (40.4%), methoxychlor (99.5%) and chlordane (99.8%). In sterile soil microcosms, the consortium was able to grow without significant differences in the different textured soils (clay silty loam, sandy and loam), both contaminated or not contaminated with the OPs-mixture. The Streptomyces consortium was able to remove all the OPs in sterile soil microcosm (removal order: clay silty loam > loam > sandy). So, clay silty loam soil (CSLS) was selected for next assays. In non-sterile CSLS microcosms, chlordane removal was only about 5%, nonetheless, higher rates was observed for lindane (11%) and methoxychlor (20%). In CSLS slurries, the consortium exhibited similar growth levels, in the presence of or in the absence of the OPs-mixture. Not all pesticides were removed in the same way; the order of pesticide dissipation was: methoxychlor (26%)>lindane (12.5%)>chlordane (10%). The outlines of microbial growth and pesticides removal provide information about using actinobacteria consortium as strategies for bioremediation of OPs-mixture in diverse soil systems. Texture of soils and assay conditions (sterility, slurry formulation) were determining factors influencing the removal of each pesticide of the mixture.

Signaling crosstalk between TGFß and Dishevelled/Par1b.

Crosstalk of signaling pathways is critical during metazoan development and adult tissue homeostasis. Even though the transforming growth factor-beta (TGFß) transduction cascade is rather simple, in vivo responsiveness to TGFß ligands is tightly regulated at several steps. As such, TGFß represents a paradigm for how the activity of one signaling system is modulated by others. Here, we report an unsuspected regulatory step involving Dishevelled (Dvl) and Par1b (also known as MARK2). Dvl and Par1b cooperate to enable TGFß/bone morphogenetic protein (BMP) signaling in Xenopus mesoderm development and TGFß responsiveness in mammalian cells. Mechanistically, the assembly of the Par1b/Dvl3/Smad4 complex is fostered by Wnt5a. The association of Smad4 to Dvl/Par1 prevents its inhibitory ubiquitination by ectodermin (also known as transcriptional intermediary factor 1 gamma or tripartite motif protein 33). We propose that this crosstalk is relevant to coordinate TGFß responses with Wnt-noncanonical and polarity pathways.

APAP titration in patients with mild to moderate OSAS and periodic limb movement syndrome.

AIMS: 1) to evaluate automatic positive airway pressure (APAP) titration in a partially attended setting; 2) to verify whether APAP performance depends on the apnea-hypopnea and periodic limb movement indexes (PLMI). METHODS: 65 CPAP naïve subjects with a sleep disorder of breathing and daytime sleepiness underwent a standard polysomnography (first night), APAP titration (second night, partially attended), and a standard polysomnography using continuous positive airway pressure (CPAP) at the effective pressure (Peff) established from the APAP titration (third night) in a sleep disorder laboratory in a 400-bed community hospital. We examined the apnea-hypopnea index (AHI), sleep stages, arousals induced by respiratory events (RESPa) and PLM (PLMa), and oxygen saturation during the first and third nights on CPAP at the Peff. Patients were divided into three groups according to their AHI and PLMI. RESULTS: At the Peff defined using APAP on the third night, the mean AHI dropped from 29.6 +/- 21.8 to 3.1 +/- 3.4, and the RESPa index from 16.5 +/- 16.2 to 1.7 +/- 2.6. No differences emerged in sleep stages or spontaneous arousals (first vs third night). Overall, 92% of the patients met the standard for an acceptable outcome of positive pressure titration. Baseline AHI and PLMI did not affect the outcome of titration. CONCLUSIONS: In patients with mild to moderate OSAS and PLMS, APAP titration enables the optimal fixed pressure for CPAP home therapy to be determined in at least 90% of patients.

Hemodynamic evaluation of the addition of isosorbide-5-mononitrate to nadolol in cirrhotic patients with insufficient response to the beta-blocker alone.

The association beta-blockers plus isosorbide-5-mononitrate (I5M) has been proposed for the treatment of portal hypertension in patients with insufficient response to beta-blockers alone, according to hemodynamic criteria. The mechanism of action in these patients is not clearly defined. Fifteen patients with cirrhosis and esophageal varices were evaluated by hepatic venous pressure gradient (HVPG) measurement and duplex-Doppler ultrasonography before and after 1 month of treatment with nadolol. Nine patients who did not exhibit a decrease in HVPG to 12 mm Hg or a percent decrease greater than 20% were classified as poor responders, and were studied again with the same methodology after 3 months of chronic administration of nadolol + I5M 20 mg twice per day. In poor responders, mean HVPG decrease after nadolol was 8.9% +/- 2.8%, and after the combination, it was 25.7% +/- 1.7% (P = .004). All patients except one became good responders to the association. Portal blood flow (PBF) decreased significantly after nadolol (P = .004), and remained unchanged after the addition of nitrates. Resistance to portal blood flow (RPBF) increased after nadolol (P = .02) and returned to baseline values during combined treatment (P = .03). In good responders, an adequate decrease in HVPG was associated with a decrease in PBF (P = .06) but no change in RPBF. A wide spectrum of combined changes in PBF and in RPBF after nadolol was observed in poor responders, ranging from no change in either parameter to a marked decrease in PBF counterbalanced by a marked increase in RPBF. The addition of I5M was followed in most cases by larger effects on resistance than on flow. Doppler parameters were not significantly correlated with the HVPG response to nadolol alone or associated with I5M. It is concluded that good hemodynamic responders to nadolol differ from poor responders in the lack of increase in RPBF after the drug. The addition of nitrates to nadolol is effective in decreasing portal pressure in most poor responders to nadolol alone. A decrease in outflow resistance is the main mechanism involved.

Effects of chronic therapy with nadolol on portal hemodynamics and on splanchnic impedance indices using Doppler sonography: comparison between acute and chronic effects.

BACKGROUND/AIMS: Beta-blockers are currently used for chronic therapy of portal hypertension. Duplex Doppler ultrasonography has been proposed for non-invasive evaluation of splanchnic pharmacodynamics, but the chronic effects of beta-blockers on portal hemodynamics and on splanchnic arterial impedance indices have not been analyzed with this method. This was the aim of the study. METHODS: The effects of acute (80 mg p.o.) and chronic (2 months at a dosage sufficient to reduce heart rate by at least 25% in respect of basal values) nadolol administration on portal blood flow velocity and volume, and on splanchnic and renal arterial impedance indices [pulsatility index = (peak systolic velocity-minimum velocity)/mean velocity] were evaluated in patients with cirrhosis. Twenty-eight patients with cirrhosis and portal hypertension were investigated. Nineteen patients received nadolol, and nine received placebo. RESULTS: Placebo caused no significant hemodynamic change. Portal blood flow mean velocity decreased after chronic therapy (11.7 +/- 2.9 cm/s to 9.1 +/- 2.3, p < 0.001). In the 16 patients with acute and chronic evaluation, portal blood flow mean velocity decreased after acute therapy (11.8 +/- 3.0 cm/s to 10.4 +/- 3.0, p < 0.01), and even more so after chronic therapy (to 9.2 +/- 2.4, p < 0.01). No parallel was found between acute and chronic effects. Hepatic, mesenteric and splenic pulsatility indices increased after chronic therapy (1.26 +/- 0.33 to 1.39 +/- 0.28, p < 0.02; 2.04 +/- 0.41 to 2.50 +/- 0.61, p < 0.01; 0.92 +/- 0.22 to 1.18 +/- 0.27, p < 0.001 respectively); renal pulsatility index increased (1.12 +/- 0.20 to 1.40 +/- 0.28, p < 0.001). CONCLUSIONS: Chronic therapy with nadolol decreased portal blood flow velocity and volume, and increased splanchnic and renal impedance indices. Chronic effects of nadolol on portal inflow cannot be predicted from its acute effects. Evaluation of the effect of nadolol on portal blood velocity and volume should be performed after chronic therapy. Duplex Doppler ultrasonography allows venous and arterial splanchnic pharmacodynamics to be studied separately.

Randomised trial of nadolol alone or with isosorbide mononitrate for primary prophylaxis of variceal bleeding in cirrhosis. Gruppo-Triveneto per L'ipertensione portale (GTIP)

BACKGROUND: The risk of having a first cirrhosis-associated variceal bleed is lowered by about 50% by beta-blockers. Use of beta-blockers is currently recommended for patients with cirrhosis and oesophageal varices that are at risk of bleeding. We aimed to test the effectiveness of isosorbide mononitrate as an adjunct to the beta-blocker nadolol in the prophylaxis of first variceal bleeding in these patients. METHODS: We did a randomised multicentre study to compare the non-selective beta-blocker, nadolol, with nadolol plus isosorbide mononitrate in 146 relatively well (Child-Pugh score < or = 11) patients who had oesophageal varices at risk of bleeding. Patients on nadolol alone received a single oral 40 mg daily dose. Every second day the dose was titrated to achieve 20-25% decrease in resting heart rate (maximum dose 160 mg daily). Patients receiving both drugs received nadolol as above then isosorbide mononitrate was added starting with 10 mg orally twice daily, which was increased to 20 mg unless hypotension or severe headache occurred. The main endpoint was the occurrence of variceal bleeding of any severity. Patients were followed up for up to 40 months. FINDINGS: During the study period 11 of 74 patients from the nadolol alone group and four of 72 from the nadolol plus isosorbide mononitrate group had variceal bleeding (log-rank test p = 0.03). Cumulative risk of variceal bleeding was 18% in the nadolol group and 7.5% in the combined treatment group (95% CI for difference 1-25%). Two patients in each group had a non-variceal bleed related to portal hypertension. 14 patients from the nadolol only group and eight from the combined treatment group died during the study period (log-rank test p = 0.09). Four and eight patients, respectively, had to discontinue one of the drugs because of side-effects. INTERPRETATION: Nadolol plus isosorbide mononitrate is significantly more effective than nadolol alone in the primary prophylaxis of variceal bleeding in relatively well patients with cirrhosis, and has few side-effects.

The course of galactose elimination capacity in patients with alcoholic cirrhosis: possible use as a surrogate marker for death.

There is increasing interest for the use of surrogate end points in the evaluation of treatments in patients with liver disease, but adequate validation is seldom available. This study aimed to describe the different course of galactose elimination capacity in patients with alcoholic cirrhosis who continued to drink or abstained from alcohol consumption during follow-up, and to validate changes in galactose elimination as a surrogate end point for death from liver-related causes. Forty-five patients with alcoholic cirrhosis (22 who continued drinking throughout the study period, and 23 who stopped drinking and were abstinent throughout the study period) were retrospectively selected among patients who had galactose elimination capacity measured at 6-month intervals. During follow-up 10 drinkers and 3 abstainers died of liver-related causes (P = .025). Abstainers showed a transient improvement in galactose elimination capacity, followed by a decrease. Continuous drinkers showed a reduction from the beginning. According to Cox's regression analyses, persistent alcohol abuse and galactose elimination capacity were separately related to the risk of death, but, when a time-dependent model was fitted containing galactose elimination capacity and persistent alcohol abuse, only the former remained significant. This implies that variations in the risk of death occurring as a consequence of abstinence from alcohol consumption may be predicted from changes in galactose elimination capacity, and that the mechanisms through which abstinence influences survival are strictly linked to the mechanisms responsible for the changes in the test. Because of the strict association of decrease in galactose elimination capacity and short survival, as proved in several series, this observation represents adherence to the criteria requested for adequacy of a surrogate end point. In conclusion, in alcoholic cirrhosis the decrease in galactose elimination capacity is an adequate surrogate end point for death from liver-related causes, which is worth testing in other conditions and in response to other treatments.

Short-term effects of transcatheter arterial chemoembolisation on metabolic activity of the liver of cirrhotic patients with hepatocellular carcinoma.

BACKGROUND: Transcatheter arterial chemoembolisation, a procedure for the treatment of hepatocellular carcinoma, provokes a pronounced but transient increase in hepatic cytolysis parameters. A definite evaluation of the impairment of liver function after this treatment, performed by adequate techniques, is still lacking. AIMS: To assess and quantify the impairment of liver metabolic activity after arterial chemoembolisation in patients with cirrhosis. The variations of hepatic vein pressure gradient provoked by this procedure were evaluated. PATIENTS: 15 patients with cirrhosis (Child's class A and B) and hepatocellular carcinoma. METHODS: 17 transcatheter arterial chemoembolisations with epirubicin, iodised oil, and gelfoam were performed; liver function was assessed before, the following day, and after seven days measuring galactose elimination capacity; aminopyrine breath test was also performed in six patients before the procedure and seven days after. In 10 patients intrinsic hepatic clearance of indocyanine green and hepatic vein pressure gradient were measured by hepatic vein catheterisation before and 30 minutes after chemoembolisation. RESULTS: Intrinsic hepatic clearance of indocyanine green decreased significantly from (mean (SEM)) 355 (140) ml/min to 277 (98) ml/min after the procedure (p = 0.0007). Galactose elimination capacity did not show significant changes, being 4.00 (0.90) mg/min/kg body weight at baseline, 4.20 (0.90) mg/min/kg body weight after one day, and 3.95 (0.87) mg/min/kg body weight seven days after chemoembolisation. Aminopyrine breath test was 2.31 (1.09)% and remained unchanged after treatment, being 2.39 (2.04)% at day 7. Baseline hepatic vein pressure gradient was 17.0 (5.5) mm Hg, and 14.4 (3.7) mm Hg 30 minutes after chemoembolisation (p = 0.09). CONCLUSIONS: A single transcatheter chemoembolisation in cirrhotic patients was detected by galactose elimination capacity and aminopyrine breath test one and seven days after the procedure. Therefore it can be considered a safe therapeutic tool for hepatocellular carcinoma in Child's class A and B cirrhotic patients.

Optimizing the time-frame for the definition of bleeding-related death after acute variceal bleeding in cirrhosis.

OBJECTIVE: To identify the best time-frame for defining bleeding-related death after variceal bleeding in patients with cirrhosis. DESIGN: Prospective long-term evaluation of a cohort of 155 patients admitted with variceal bleeding. SETTING: Eight medical departments in seven hospitals in north-eastern Italy. METHODS: Non-linear regression analysis of a hazard curve for death, and Cox's multiple regression analyses using different zero-time points. RESULTS: Cumulative hazard plots gave two slopes, the first corresponding to the risk of death from acute bleeding, the second a baseline risk of death. The first 30 days were outside the confidence limits of the regression curve for the baseline risk of death. Using Cox's regression analysis, the significant predictors of overall mortality risk were balanced between factors related to severity of bleeding and those related to severity of liver disease. If only deaths occurring after 30 days were considered, only predictors related to the severity of liver disease were found to be of importance. CONCLUSION: Thirty days after bleeding is considered to be a reasonable time-frame for the definition of bleeding-related death in patients with cirrhosis and variceal bleeding.

Long-term effect of nadolol or nadolol plus isosorbide-5-mononitrate on renal function and ascites formation in patients with cirrhosis. GTIP Gruppo Triveneto per l'Ipertensione Portale.

The association beta-blockers plus nitrates has been reported to impair renal function and renal sodium handling, leading to increased risk of development of ascites, or worsening of a preexisting ascites, or increase in the requirements of diuretic agents. In 81 patients with cirrhosis and esophageal varices, participating in a multicenter controlled clinical trial of prophylaxis of variceal bleeding comparing nadolol (NAD) plus isosorbide-5-mononitrate (I5M) with NAD alone, renal function, presence of ascites, and diuretic requirements were assessed at inclusion and after 6 months of follow-up. No significant variation in s-urea or s-creatinine was observed in either group, Three patients in the nadolol group and two in the NAD plus I5M developed ascites at 6 months (P = .70), and a need to increase diuretic regimen was observed in four and three patients, respectively (P = .76). Decrease in heart rate and in mean arterial pressure was similar in the two groups. There was a significant correlation between increases in s-creatinine and decrease in mean arterial pressure in the whole series (P = .015). Only in patients treated with the association was there a significant larger proportion of patients ascitic who became anascitic, than of patients anascitic who became ascitic (P = .03). In patients treated with the association, there was a significantly larger decrease in hepatic venous pressure gradient (P = .05). It is concluded that patients treated with the association NAD plus I5M are not at increased risk of developing renal dysfunction or worsening of ascites compared with patients treated with NAD alone.(ABSTRACT TRUNCATED AT 250 WORDS)