Latin American Association for the study of the liver (ALEH) practice guidance for the diagnosis and treatment of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) currently represents an epidemic worldwide. NAFLD is the most frequently diagnosed chronic liver disease, affecting 20-30% of the general population. Furthermore, its prevalence is predicted to increase exponentially in the next decades, concomitantly with the global epidemic of obesity, type 2 diabetes mellitus (T2DM), and sedentary lifestyle. NAFLD is a clinical syndrome that encompasses a wide spectrum of associated diseases and hepatic complications such as hepatocellular carcinoma (HCC). Moreover, this disease is believed to become the main indication for liver transplantation in the near future. Since NAFLD management represents a growing challenge for primary care physicians, the Asociación Latinoamericana para el Estudio del Hígado (ALEH) has decided to organize this Practice Guidance for the Diagnosis and Treatment of Non-Alcoholic Fatty Liver Disease, written by Latin-American specialists in different clinical areas, and destined to general practitioners, internal medicine specialists, endocrinologists, diabetologists, gastroenterologists, and hepatologists. The main purpose of this document is to improve patient care and awareness of NAFLD. The information provided in this guidance may also be useful in assisting stakeholders in the decision-making process related to NAFLD. Since new evidence is constantly emerging on different aspects of the disease, updates to this guideline will be required in future.

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza.

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

MEKC-UV as an effective tool for the separation and identification of explosives, high explosives, and their degradation products in environmental samples.

MEKC has been used in conjunction with UV detection for identification and quantitation of high explosives in environmental samples. To ensure the compatibility of the technique with ESI-MS, perfluorooctanoic acid (PFOA), a volatile micelle, was used. Separation of EPA Method 8330 Mixes A and B using various concentrations of the micelle showed that the 80 mM solution of PFOA was the optimum concentration for the separation of the explosives. MEKC analysis of explosives with ESI-MS under optimum micelle concentration provided excellent results indicating the compatibility of the method with ESI-MS. Finally, the MEKC-UV method was applied to the detection and quantitation of explosives in various environmental samples including water, sand, and soil. The results demonstrate that the MEKC method described herein is a viable technique for detection of explosives in environmental samples using UV detection, while maintaining the compatibility of the technique with MS detection without any modification to the separation method, if laboratories decided to pursue this route in the future.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice.

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.

Poroqueratosis de Mibelli. Presentación de caso / Mibelli's porokeratosis. Presentation of case

RESUMEN Se realizó la presentación de dos pacientes con poroqueratosis de Mibelli por ser una dermatosis poco frecuente, por lo que debe ser de interés para los profesionales de diferentes disciplinas, bien por relacionarse con el cáncer de piel o con disímiles enfermedades. Los pacientes estudiados fueron: paciente femenino de 62 años de edad, piel blanca y ocupación ama de casa. Fue remitida por presentar piel con fototipo cutáneo II, una lesión en placa de color amarillento, bordes elevados, hiperqueratósicos, centro atrófico de 1 cm de diámetro; localizada en cara ántero-externa de pierna derecha. El otro paciente, de 59 años, sexo masculino, piel blanca, y ocupación pescador. Solicitó los servicios por presentar piel fototipo cutáneo II lesión en placa de color amarillento, bordes elevados, hiperqueratósicos, centro atrófico de 2cm de diámetro, localizada en cara póstero-externa de antebrazo derecho. Se les realizó biopsia de ambas lesiones. El diagnóstico fue la poroqueratosis de Mibelli para la prevención del cáncer de piel. Ambos pacientes presentaron en común fototipo cutáneo II y otras manifestaciones cutáneas inducidas por la exposición solar. La terapéutica utilizada fue la extirpación quirúrgica, que resultó también el proceder diagnóstico Se orientaron medidas para la prevención del cáncer de piel con bloqueadores solares, y protegerse de los rayos solares con ropa adecuaday otros accesorios (AU).

ABSTRACT Two patients with Mibelli´s porokeratosis were presented because it is a few frequent dermatosis that might be of interest for professionals of different disciplines since it is related to skin cancer and to several other diseases. The first studied patients was 62 years old, female, white, and a housewife, remitted due to presenting skin with cutaneous phototype II, a yellowish lesion in plaque, risen, hyperkeratoid borders and atrophic center of 1 cm diameter, located in the anterior-external face of the right leg. The other patient was 59 years old, male, white, and a fisherman. He assisted the service presenting skin with cutaneous phototype II, a yellowish lesion in plaque, risen, hyperkeratoid borders and atrophic center of 2 cm diameter, located in the posterior-external face of the right forearm. A biopsy of both lesions was carried out. The diagnosis was Mibelli´s porokeratosis. Both patients presented a common cutaneous phototype II and skin manifestations induced by the sun exposition. The used therapeutic was the surgical removal, that also was the diagnosis procedure. Preventing skin cancer with sun blockers was oriented and also wearing adequate clothes and other accessories (AU).

Comportamiento del tabaquismo en los adolescentes del Reparto Fructuoso Rodríguez, Cárdenas / Behavior of smoking in teenagers of the Fructuoso Rodriguez Neighborhood. Cardenas

Introducción: la Organización Mundial de la Salud reporta al tabaquismo como la principal causa prevenible de enfermedad, discapacidad y muerte prematura, y ha señalado que mueren aproximadamente 14 000 personas al día, por alguna de las enfermedades atribuibles a este. Objetivo: caracterizar el comportamiento del tabaquismo en adolescentes. Materiales y métodos: la investigación fue observacional, descriptiva, transversal, en un universo de 51 adolescentes de los consultorios 24, 25 y 26 del Policlínico Universitario “Héroes del Moncada”, de Cárdenas, que cumplieron los siguientes criterios de inclusión: tener edad comprendida entre 10 y 19 años, ser fumador activo, independientemente del tiempo con el hábito, la cantidad de cigarrillos que fume, y con consentimiento asistido por los padres de participar en el estudio durante el período de enero a junio de 2013. Resultados: el 11,4 % de los adolescentes son fumadores, y del sexo masculino 78,4 %. Predominó el grupo de 17 a 19 años, con un 58,8 %. El 92,2 % comenzó a fumar a los 12 años, el 56,9 % lo hace diariamente, y compran personalmente el cigarro en las tiendas el 43,8 %. Todos fuman públicamente, y en casa lo hace el 62,7 %. Conclusiones: predominaron los fumadores varones entre 17 y 19 años de edad. La mayoría de los adolescentes fuman diariamente en lugares públicos, compran personalmente los cigarros en las tiendas y comenzaron a fumar con más de 12 años de edad.

Background: the World Health Organization reports smoking as the main preventable cause of disease, disability and premature death, and has pointed out that around 14 000 persons per day die due to any of the diseases attributed to it. Aim: characterizing smoking behavior in teenagers. Material and Methods: the research was observational, descriptive, cross-sectional, in a universe of 51 teenagers belonging to the family physician consultations 24, 25 and 26 of the Teaching Policlinic “Heroes del Moncada”, of Cardenas, fulfilling the following inclusion criteria: aged 10-19 years, being active smokers without taking into account the time they have being smoking, the quantity of cigarettes they smoke, and having their relatives´ informed consent for participating in the research in the period from January to June 2013. Outcomes: 11.4 % of the teenagers were smokers, and 78.4 % were male. There it was a predominance of the 17-19 years-old group, for 58.8 %. 92.2 % of them began smoking at the age of 12, 56.9 percent smokes every day, and 43.8 % buys cigarettes in the stores by themselves. All of them smoke in public, and 62.7 % smokes at home. Conclusions: there it is a predominance of male teenagers aged 17-19 years. Most of the teenagers smoke every day in public places, buy personally cigarettes in the stores and began smoking when they were more than 12 years old.

Compatibilidad química por calorimetría diferencial de barrido y termogravimetría del auranofin tabletas 3 mg / Chemical compatibility of 3 mg Auranofin tablets demonstrated by differential scanning calorimetry and thermogravimetry

Como parte de la pre-estabilidad de la preformulación de auranofin tabletas, se realizó un estudio de compatibilidad química, para lo cual se emplearon técnicas de análisis térmico como la calorimetría diferencial de barrido y la termogravimetría. Previo a dichos estudios se caracterizó térmicamente por calorimetría diferencial de barrido el principio activo y cada uno de los excipientes. Posteriormente se procedió a la realización del estudio de compatibilidad química, mediante la preparación de mezclas físicas binarias entre el principio activo y cada uno de los excipientes. Se detectó por ambos métodos que el principio activo tuvo una transición física de fusión, no reportada en la literatura, lo que permitió poder calcular su pureza por calorimetría diferencial de barrido. Mediante la técnica calorimétrica fue posible inferir la ausencia de incompatibilidad química entre el principio activo y los excipientes estudiados. Además, mediante el cálculo de la energía de activación se estableció el siguiente orden de estabilidad térmica: auranofin:PVP> auranofin:lactosa> auranofin:explotab> auranofin:estearato> auranofin:aerosil> auranofin:celulosa, por lo que se recomienda el uso de estos excipientes en la elaboración de la formulación farmacéutica

As part of the pre-stability study of the Auranofin tablet pre-formulation, a chemical compatibility study was conducted using thermal analysis techniques such as the differential scanning calorimetry and the thermogravimetry. Prior to these studies, the active principle and each of the excipients were thermally characterized with the aid of the differential scanning calorimetry. Then, there proceeded to carry out the chemical compatibility study by preparing binary physical mixtures between the active principle and each of the excipients. Both methods showed that the active principle had a melting physical transition, not reported in the literature, which allowed calculating its purity aided by the differential scanning calorimetry. It was possible to infer from the calorimetry technique that there was not chemical incompatibility between the active principle and the studied excipients. By means of the activation energy estimation, the following order of thermal stability was set: Auranofin:PVP> Auranofin: lactose> Auranofin:explotab> Auranofin:estearate> Auranofin:aerosil> Auranofin:celullose. The use of these excipients was recommended for the preparation of the pharmaceutical formulation

Plasmaféresis en el tratamiento de la Miastenia Gravis: presentación de caso / Plasmapheresis in the treatment of the Miastenia Gravis: a case presentation

Se presentó una paciente de19 años de edad, raza blanca, femenina, con antecedentes de padecer de miastenia gravis desde hace aproximadamente 2 años, que acudió al cuerpo de guardia en plena crisis de su enfermedad de base; este cuadro progresó tomando los músculos respiratorios por lo que necesitó de ventilación mecánica artificial, cuadro clínico que llevó a realizar tres sesiones de plasmaféresis como tratamiento de la crisis aguda de la miastenia gravis con toma de la musculatura respiratoria, se efectuaron en días consecutivos con un promedio de cuatro horas y media, la albúmina humana como solución de reemplazo del plasma separado de la sangre de la paciente para eliminar los mediadores que llevaron a este cuadro a la paciente, acto seguido a la última sesión se separó del respirador artificial.

A nineteen years patient, white race, female with antecedents of suffering miastenia gravis since two years approximately, arrived to the emergency department in full crisis of her basic disease, guardhouse, this profile progressed taking respiratory muscles, reason why she needed artificial mechanical ventilation, this clinical profile carried out three sessions of plasmapheresis as acute crisis treatment of miastenia gravis with taking of respiratory muscle, they took place in consecutive days with an average of four hours and a half, human albumin as replaced solution plasma separated from the patient blood to eliminate the mediators that carried out this patient to this clinical profile, immediately after wards to the last session she was apart from the artificial respirator.

An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification.

The use of transgenic plants for the production of pharmaceutical compounds has received increasing attention in the last few years. However, many technological and regulatory issues regarding the practical exploitation of this alternative system of production remain to be solved; a situation that explains the lack of commercial products derived from such a system. This paper reports the expression in transgenic plants and cells of a single-chain antibody variable-region fragment (scFv) and a mouse monoclonal antibody to the hepatitis B virus surface antigen (HBsAg). The large-scale purification of the scFv from plants and its use for immunopurification of HBsAg are also described, together with elements concerning regulatory issues and technologies for compliance with good manufacturing and agricultural practices.

Expression of a single-chain Fv antibody fragment specific for the hepatitis B surface antigen in transgenic tobacco plants.

An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F I transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94-95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15-20 microg of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.

Expression and characterization of an anti-(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco (Nicotiana tabacum) plants and its use in the immunopurification of its target antigen.

Transgenic plants expressing recombinant immunoglobulins have arisen as an alternative technology for the large-scale production of antibodies useful in therapeutics and in industrial processes. In the present paper we report the expression in transgenic tobacco ( Nicotiana tabacum ) of an anti-HBsAg [anti-(hepatitis B virus surface antigen)] mouse IgG1 mAb (monoclonal antibody), currently used for the industrial purification of the recombinant vaccine antigen. Using the sweet potato sporamin signal peptide, a KDEL (Lys-Asp-Glu-Leu) ER (endoplasmic reticulum) anchorage domain, and a heavy- and light-chain gene tandem construction, we generated F1 plants in which the expression of the antibody accounted for 0.5% of the total soluble proteins. The 'plantibody' (functional IgG antibody produced in plants) was easily purified by Protein A-Sepharose chromatography with a yield of approximately 35 microg/g of fresh leaf material, and its glycosylation indicated that, irrespective of the KDEL signal, the molecule is modified in both the ER and Golgi. Finally, a successful comparison of the plantibody with the ascites-derived mAb in the immunoaffinity purification of the vaccine recombinant HBsAg was performed. Taken as a whole, our results show that the large-scale production of this antibody of industrial relevance in transgenic tobacco is feasible.

Actividad molusquicida del Paraiso (Melia azedarach L.) (Meliaceae) sobre Lymnaea cubensis, molusco vector de Fasciolosis / Molluscicidal activity of Paraiso (Melia azedarach L.) (Meliaceae) on Lymnaea cubensis, host snail of Fasciolosis

A Fasciolosis constitui em diversas regiöes fonte importante de perdas econômicas, e quando näo se adotam medidas de controle de seus hospedeiros (moluscos), junto a condiçöes ecológicas favoráveis, pode ocorrer o aparecimento de casos isolados de Fasciolosis humana. Dentro dos métodos alternativos para o seu controle está o uso de extratos vegetais e se tem pretendido avaliar o provável emprego do suco extraíso do fruto e sementes do Paraiso (Melia azedarach L.), no controle de Lymnaea cubensis, principal vetor da Fasciolosis em Cuba. Diferentes concentraçöes do suco extraído do fruto e semente do Paraiso (Melia azedarach L.) foram testadas para determinar as doses médias letais (DL50) e máxima (DL90) usando um programa computadorizado PROBIT-LOG. Sete séries experimentais foram realizadas, usando 72 moluscos em cada uma. Para determinar a influência sobre a freqüência cardíaca foram testados três grupos de 10 moluscos, dois foram tratados com as CL50=0,88627 e CL90=1,7641, respectivamente, enquanto que o terceiro foi considerado como controle. Observou-se uma marcada influência de ambas as doses sobre a freqüência cardíaca do molusco estudado. Os resultados foram alentadores, pois demonstraram um potencial uso dessa planta no controle de moluscos näo desejados

Un diagnóstico difícil: absceso hepático o carcinoma hepato-celular / A difficult diagnostic: hepatic abscess or hepato-cellular carcinoma

El diagnóstico diferencial entre el absceso hepático (AH) y el carcinoma hepatocelular (CHC) es difícil, ya que en el 20% de los CHC pueden ser diagnosticados clínicamente como AH. Nosotroas hicimos el diagnóstico clínico, valor predictivo y eficiencia (VPE) 84% y 78% respectivamente. La elevación del hemidiafragma derecho, dio un VPE de 84% en AH y 78% en CHC. La Hemoglobina P> 0,70; Hto P> 0,70; leucocitos P> 0,70 y hemograma P> 0,60 no fueron útiles para el diagnóstico. Los tests hepáticos: GTP P> 0,10; TGO P> 0,10; coeficiente TGO/TGP P> 0,40 y LDH P> 0,10 tampoco dieron resultados estadísticamente significativos. La única prueba útil en el diagnóstico entre AH y CHC fue la determinación sérica de IgG con un promedio de 2.315 ñ 658 DS en AH y 1.492 ñ 539 DS en CHC P> 0.02; la IgA y la IgM aunque están elevados sus valores no son estadísticamente significativos. El U.S. demostró un VPE de 84% para AH y 61% en CHC. La laparoscopia tiene un VPE de 81% en CHC y la biopsia más laparoscopia de 100%. Podemos concluir que la determinación de inmunoglobulinas (IgG) juegan un papel importante en el diagnóstico diferencial en los casos de "lesiones ocupantes de espacio" del hígado fundamentalmente en el AH y CHC