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Introduction: Intestinal failure is being an entity with higher prevalence in the pediatric age, especially due to bowel resections causing the appearance of a short bowel syndrome., Objectives: To determine the prevalence and etiology of cases of short bowel syndrome (SIC) and Intestinal Failure (FI) existing in Andalusia. Analyze factors involved in evolution, the number of transplant patients and to know the time required to achieve enteral autonomy, studying whether there are differences in management between different participants., Methods: Multicenter retrospective descriptive observational study in which are collected data of patients diagnosed with short bowel syndrome or intestinal failure in 6 hospitals in Andalusia in the period from 1 January 2008 to 31 January 2014., Results: 25 patients. Average age at diagnosis 7.4 months. Average length of remnant intestine: 113.8 cm; 64% of patients with <75 cm length remaining intestine. We show that: the early introduction of enteral nutrition is a factor favoring the suspension of the NP (p = 0'033); and that the prevention of liver disease associated with parenteral nutrition (EHANP) is favored by: the use of fewer lipid Parenteral Nutrition (p = 0'008), a greater length of remaining intestine (p = 0'049 ), the early introduction of enteral nutrition (p = 0'009) and a lower gestational age (p = 0'006)., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

Dengue virus is the most significant virus transmitted by arthropods worldwide and may cause a potentially fatal systemic disease named dengue hemorrhagic fever. In this work, dengue virus serotype 4 was detected in the tissues of one fatal dengue hemorrhagic fever case using electron immunomicroscopy and molecular methods. This is the first report of dengue virus polypeptides findings by electron immunomicroscopy in human samples. In addition, not-previously-documented virus-like particles visualized in spleen, hepatic, brain, and pulmonary tissues from a dengue case are discussed.

Background: Malnutrition among hospitalized patients has clinical implications, and interest has arisen to find screening tools able to identify subjects under risk. At present, there is no consensus about the most suitable nutrition screening tool for pediatric patients., Aim: To validate STAMP (Screening Tool for the Assessment of Malnutrition in Pediatrics) pediatric screening tool in Spain., Methods: Descriptive cross-sectional study of patients admitted to a 3rd level children's hospital with both medical and surgical specialities. During the first 24 hours of admission, STAMP screening tool was applied. For its validation, results were compared with those obtained from a nutritional assessment performed by specialist staff, which included clinical, anthropometric and body composition data., Results: A sample of 250 children was studied. Nutritional assessment identified 64 patients (25.6%) under risk, 40 of whom were malnourished (16%). STAMP classified 48.4% of the patients as being under nutritional risk. This tool showed 75% sensitivity and 60.8% specificity when identifying patients under risk according to nutritional assessment. It showed 90% sensitivity and 59.5% specificity when identifying malnourished patients., Comments: Malnutrition was less frequent than that reported in other European countries, although diagnosis technique was different. STAMP is a simple and useful tool for nutritional screening, avoiding the need to assess all patients on admission in order to identify those under nutritional risk.

The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.

High-pressure homogenization-extrusion (HPHE) is a method that can be used for downsizing large lipid vesicles with commercially available instrumentation (e.g., from Avestin Inc., Canada), which covers a full range of processing capacities from laboratory (0.5-3.5 mL) to large-scale continuous (1-1000 L/h) production. Consequently, the feasibility (at the laboratory scale) of using HPHE for producing DNA-loaded liposomes by the conventional dehydration-rehydration method was explored. HPHE-generated small unilamellar vesicles had a mean size in the range of 27-76 nm depending on the number of processing cycles and lipid (PC:DOPE:DOTAP or PC:DOPE:Ethyl-DOPC, 1:0.5:0.5, mol/mol) formulation. The size could be further regulated by the pore size (50 or 100 nm) of the extrusion membrane. Using plasmids for the V3 loop of HIV-1, and the capsid, E1 and E2 of hepatitis C, entrapment yields of 72-98.2% into dehydrated-rehydrated vesicles (DRV) were obtained over a wide range (0.309-2.5 mg) of DNA quantities. Most of the plasmid DNA was retained by liposomes even in the presence of sodium dodecyl sulfate (from 0.05% to 0.3%) and efficiently protected from nuclease-mediated degradation. Although the encapsulation process slightly decreased (in the range of 42.8-65.7%) the relative abundance of plasmid super coiled isoforms, the transfection efficiency of monkey kidney COS-7 cells with the plasmid DNA extracted from liposomes (9+/-0.4%) was similar to that of the non-treated DNA (8.7+/-0.2%), using the commercial SuperFect(R) Transfection Reagent. Also, it was found that an appreciable loss of lipid mass-either associated with the HPHE or the dehydration-rehydration steps-occurs during the liposome manufacturing process. These results at the bench scale are a useful reference for planning pilot or large-scale manufacture of DNA vaccine-containing liposomes.

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.

Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly., (Copyright 2001 Academic Press.)

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli., (Copyright 2001 Academic Press.)

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.

A previously unknown disease, termed epidemic neuropathy (EN), occurred in Cuba between 1991 and 1993. When samples of cerebrospinal fluid (CSF) from 45 patients with EN and 11 controls were inoculated into cultures of VERO cells, almost all (93%) of the samples from the cases of EN but only one (9%) of the control samples produced a slowly progressing cytopathological effect (CPE). Although the results of other studies indicated the presence of a picornavirus-like virus in CSF samples from EN cases, the CPE and other physico-chemical characteristics observed were not those expected of picorn-viruses. Several aetiological factors may have contributed to EN but at least one virus could have played a major role.

Policosanol is a new cholesterol-lowering drug isolated and purified from sugar-cane wax, which prevents the development of lipofundin-induced lesions and foam-cell formation in New Zealand rabbits and Wistar rats. This study was conducted to examine the effects of policosanol on foam-cell formation in carrageenan-induced granulomas in rats. Eighteen Wistar rats were randomly distributed in three experimental groups which received orally for 20 days Tween 20 H2O as vehicle (control group) or policosanol at 2.5 or 25 mg kg-1. At the 11th day, lipofundin was injected intraperitoneally for 8 days to induce formation of foam cells in the granuloma. At day 13, carrageenan was injected subcutaneously for granuloma induction and seven days later animals were killed. A significant reduction of the foam-cell formation in granulomas of policosanol-treated rats was observed. It is concluded that policosanol prevents the development of foam cells in carrageenan-induced granulomas (extravascular medium) in rats.

A total of 115 samples of pharmaceutical raw materials (excipients) were analysed: 36 lactose, 27 talc, 19 corn starch, 18 arabic gum, 8 gelatin, 3 gelatinized starch, 3 cellulose and one tragacanth gum. 69.9% of the samples showed less than 10(2) bacteria/g (mean = 23.2 cfu/g) and 95.2% less than 10(2) fungi/g (mean = 4.92 cfu/g). Arabic and tragacanth gum were the most contaminated products by bacteria and fungi, respectively. Pregelatinized starch, cellulose and lactose were the least contaminated excipients. In none of the samples Escherichia coli or Salmonella-Shigella were detected; however, strains of Enterobacter, Serratia and Proteus were isolated from 10 samples of 5 different excipients. Only 5 samples did not comply with the microbiological standards as established by the European Pharmacopoeia and USP.

Policosanol is a mixture of higher aliphatic alcohols isolated from sugar cane wax, showing cholesterol-lowering effects and preventing the development of lipofundin-induced lesions in New Zealand rabbits. This study was conducted to determine whether policosanol orally administered to rats also protects against the development of lipofundin-induced atherosclerotic lesions. Fifty four male Wistar rats were randomly distributed amongst a negative control group, a positive control group intravenously injected with lipofundin for eight days, and four experimental groups also injected with lipofundin, but orally receiving policosanol at 0.5, 2.5, 5 and 25 mg kg-1, respectively. Policosanol treatment was orally administered once-a-day for eight days, while control groups similarly received equivalent amounts of vehicle. A significant reduction of the atherosclerotic lesions in the treated animals was observed. It is concluded that policosanol has a protective effect on lipofundin-induced aortic lesions in Wistar rats.

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10(3) cfu ml-1 and the number of sporulated bacteria less than 10(2) cfu ml-1. No significant differences were found in the counts obtained with Plate Count Agar (PCA) and PCA diluted 1:10 and incubated at 22 degrees, 37 degrees and 45 degrees C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85.4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.

The in vitro antimicrobial resistance of 391 bacterial strains isolated from 389 samples of oral and topical medicaments was examined. The numbers of strains isolated (and percentage of samples that present them) were: 234 Bacillus (32.1%), 79 Staphylococcus (13.6%), 46 Micrococcus (11.3%), nine Pseudomonas (1.5%), eight Acinetobacter (1.5%), five Enterococcus (1.2%), three Alcaligenes (0.8%), two Escherichia and Enterobacter (0.5%), one each Providencia, Serratia and Streptococcus (0.2%). Gram-positive bacteria were isolated from topical and oral medicaments and Gram-negative rods were detected only in topical medicaments. The 97.4% of Bacillus strains were resistant to lincomycin and B. cereus was resistant to beta-lactam and trimethoprim-sulphamethoxazole. Staphylococcus spp. showed a high percentage of resistant strains to ampicillin (51.8%), tetracycline (40.5%) and trimethoprim-sulphamethoxazole (48.1%). Staphylococcus epidermidis had the highest number of multiresistant strains. The 23.9% of Micrococcus strains were resistant to colistin. Enterococcus and Streptococcus strains showed multiresistance to penicillin G, aminoglycosides and erythromycin. The 61.5% of Gram-negative rod strains showed multiresistance to beta-lactam antibiotics and erythromycin; Pseudomonas spp. were the most resistant.

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.

The Bacillus and B. cereus spore populations of 102 samples of food (salad dressing, dried soups, sweet desserts, milk and milk products, rice dishes, pasta and flour), 93 collected from retail markets of Madrid and 9 from chinese restaurants have been studied. Bacillus spores were detected in 82.4% of the samples, while the incidence of B. cereus spores was 14.7%. In salad dressing and dried soups the contamination rate by species of Bacillus was 100% and also both showed the highest contamination of B. cereus spores (25% and 50% respectively). No samples of rice dishes and pasta exhibited B. cereus spore contamination although these were contaminated by other Bacillus species. All the samples studies showed less than 10 4 and 10 5 c.f.u./g or ml B. cereus and Bacillus spores respectively. Twenty-four strains of B. cereus isolated were characterized by morphology and biochemical properties and showed most of the characteristics of the type strain. Enterotoxin, phospholipase C and hemolysin production were present in 13 of the isolated strains showing different degrees of production. The vascular permeability reaction (VPR) was used for determining enterotoxin activity. The enterotoxigenic strains showed a positive VPR; 6 of them caused necrosis and 12 positive mouse lethal tests.

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.

The microbial contamination of 68 samples of topical and 324 samples of oral medicaments has been studied. The most common group of contaminants was members of the genus Bacillus (34.4%). Because of the pathogenic significance of B. cereus, 39 strains were characterized by morphology and biochemical properties. All except three showed most of the characteristics of the type strain. They were highly resistant to lincomycin, polymyxin B and penicillin G-cephalosporin and were susceptible to streptomycin, erythromycin and chloramphenicol. Enterotoxin, phospholipase C and haemolysin production were also studied: 33 strains gave a positive vascular permeability reaction, four of them causing necrosis, and 24 showed positive mouse lethal tests. All the strains had phospholipase activity. The majority also exhibited differing degrees of haemolysis. Permeability factor was related to mouse lethality and haemolytic activity. Phospholipase C was not related to any of the above activities.