Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris

Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein. Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (α-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein.

Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies

Background: The elastase inhibitor alpha-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-beta-sheets, nine-alpha-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into beta-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris. Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region.

Instilación intratraqueal de elastasa en la rata: cambios electroforéticos de la alfa1-AT-antitripsina en el lavado broncoalveolar / Intratracheal instillation of elastase in the rat: electrophoretical changes of alphal-antitrypsin in bronchoalveolar lavage fluid

Introduction: Endogenous alphal-antitrypsin alpha is the main inhibitor of the intratracheally instilled elastase in experimental animals. Objective: To evaluate by electrophoresis and immunodetection using western blot analysis, the different forms of alpha1-AT in bronchoalveolar lavage fluid (BALF) of Sprague Dawley rats after intratracheal instillation of elastase, with the hypothesis that the previously observed increment in antielastase activity is due to high levels of active alpha1-AT. Results: In the first hours after elastase instillation the concentration of alpha1-AT increases more than seven times due to an increase in alveolar-capillary permeability. Alpha 1-AT in BAIF is found as the native protein (~ 52 kDa), as complexes of different molecular sizes (> 75 kDa and > 100 kDa) and as a proteolytic product (< 40 kDa). Conclusion: In spite of a high proportion of alpha1-AT in the inactive form as part of different complexes, the increase in alveolar-capillary permeability after elastase treatment contributes to maintain high levels of active alpha. These results could be of importance in other inflammatory lung processes.

Introducción: la antiproteasa alfa 1-antitripsina alfa constituye el principal inhibidor endógeno de la elastasa instilada por vía intratraqueal en modelos experimentales. Objetivo: Evaluar mediante electroforesis e inmunodetección por western blot, las distintas formas en que se encuentra la alfa1-AT en el lavado broncoalveolar (IBA) de ratas Sprague Dawley después de la instilación de elastasa, con la hipótesis de que el aumento en la actividad antielastasa previamente encontrada se acompaña de niveles altos de alfa1-AT activa. Resultados: En las primeras horas post-elastasa la concentración de alfa1-AT en el IBA aumenta más de 7 veces, debido al aumento de la permeabilidad alvéolo-capilar, encontrándose tanto como proteína nativa (~ 52 kDa), como parte de complejos de mayor tamaño (> 75 kDa y > 100 kDa) y como producto de proteólisis (< 40 kDa). Conclusión: A pesar de existir una alta proporción de alfa1-AT inactiva formando complejos, el aumento de la permeabilidad alvéolo-capilar contribuye a mantener niveles altos de alfa1-AT activa. Estos resultados podrían ser extrapolables a distintos procesos inflamatorios pulmonares.

Deficiência de alfa 1-antitripsina na infância / Alpha-1-antitrypsin deficiency in children

A alfa-antitripsina é a principal glicoproteína circulante com a funçäo de inibir a açäo das proteases séricas, tais como a elastase produzida pelos glóbulos brancos e a tripsina pelo pâncreas. A quantidade produzida pelo fígado é controlada pro fenômenos inflamatórios e hormonais, com o intuito de balancear a açäo das proteases e seus inibidores em nível vascular e extravascular. A deficiência de alfa 1AT é geralmente designada pelo fenótipo anormal desta glipcoproteína polimórfica. Em torno de 2 a 30 por cento dos indivíduos de certas populaçöes apresentam outro fenótipo Pi (inibidor de protease) que näo o MN, que é o mais comum. A maioria dos fenótipos apresenta níveis...

Clearance fecal de Ó1 antitripsina: valores normales en niños de la ciudad de Córdoba / Faecal clearence of the Ó1-antitrypsin: values normal in children of the Cordoba city

En las enteropatías perdedoras de proteínas (EPP) el incremento de la permeabilidad de las paredes del TGI causa exudación proteica superior a la normal que se manifiesta por elevada excreción proteica fecal. El estudio del turnover metabólico, mediante la inyección intravenosa de proteínas marcadas con radioisótopos del yodo, provee información sobre el metabolismo proteico, pero no es útil para medir pérdida proteica gastrointestinal pues el yodo libre atraviesa con facilidad la mucosa gástrica en ambos sentidos, lo que conduce a una valoración errónea de la excreción proteíca en la materia fecal (MF). La albúmina marcada con 51Cr permite valorar la pérdida proteíca gastrointestinal caracterizada por un patron de pérdida al bulto. Los mencionados son metodos de referencia, invasores, costosos y su empleo está reglamentado por la legislación especial pues es material radioactivo. El clearance fecal de Ó1 antitripsina es inocuo y accesible a laboratorios clínicos y posee eficacia clínica probada para valorar la excreción proteica gastrointestinal. Se ha implementado la metodología y establecido valores normales de referencia para la población infantil de la ciudad de Córdoba. La muestra estuvo conformoda por 30 niños clínicamente sanos de ambos sexos, cuyas edades estuvieron comprendidas entre los 14 y 120 meses; en el protocolo de estudio de identificación se registró la edad, sexo y análisis de laboratorio. La determinación de Ó1AT sérica y fecal se realizó por inmunodifusión radial simple (IDRS). En las enteropatías puede ocurrir malabsorción proteíca, causante de hipoalbuminemia, o pérdida proteica gastrointestinal (en enteropatías exudativas), la cual afecta las fracciones albúmina y globulinas. El clearance fecal de Ó1AT indica los mililitros de plasma depurados en 24 horas por el tracto gastrointestinal y provee orientación diagnóstica útil para el uso clínico de rutina. Los valores de referencia hallados en la población infantil de Córdoba son: -clearance fecal de Ó1AT= desde no determinable hasta 10.8ml/24h; -concentración de Ó1AT sérica = 118 a 396mg por ciento

Evaluation of factors predisposing to arterial thrombosis in coronary artery disease.

Estimation of antithrombin III, alpha 2 macroglobulin and alpha 1 antitrypsin in patients with stable and unstable angina and acute myocardial infarction (15 cases each) were carried out. Twenty age, sex and weight matched healthy subjects were included as controls. Mean platelet factor 4(PF4) levels measured in 10 cases of each subgroup were significantly elevated in myocardial infarction (MI) (48.4 +/- 15.16 ng/ml) and III unstable angina patients (44.7 +/- 15.9 ng/ml) as compared to controls (25.42 +/- 12.47 ng/ml; P less than 0.01). Mean antithrombin III (AT III) levels were markedly reduced in all patients with MI (39.65 +/- 12.8% of normal pooled plasma) and unstable angina (37.9 +/- 16.6% of normal pooled plasma) and in 9 patients with stable angina. Alpha I antitrypsin and alpha 2 macroglobulin levels in these cases showed no significant difference compared to normals. Reduced AT III in coronary artery disease suggests a prethrombotic tendency in these patients. Raised PF4 levels in acute phase of the disease suggests heightened platelet activation.

Clearance fecal de alfa-1-antitripsina en enteropatias perdedoras de proteínas en pediatría / Fecal alpha 1-antitrypsin clearance in protein-losing enteropathies in pediatrics

El clearance fecal de alfa-1-antitripsina fue realizado en 47 pacientes de edad pediátrica que presentaron diversas enfermedades digestivas: 6 con colitis ulcerosa, 5 con enfermedad celíaca, 6 con intolerancia a la proteína de la leche de vaca, 1 con linfagiectasia intestinal, 1 con giardiasis, 1 con colitis inespecífica, 1 con resección del íleon terminal, y 1 con talasemia mayor y dolor abdominal. Quince pacientes presentaron diarrea crónica inespecífica. Diez niños sin enfermedad digestiva fueron incorporados como grupo control. El 1er. grupo de niños presentó valores significativamente mayores (p < 0.05) del clearance fecal de alfa-1-antitripsina con respecto a los pacientes con diarrea crónica inespecífica y del grupo control. Solo un niño con intolerancia a la proteína de la leche de vaca tuvo un valor inferior a la media + 2 Desvío estandar. Todos los niños con diarrea crónica inespecífica presentaron valores similares a los del grupo control, con excepción de 1 que tuvo un resultado ligeramente aumentado, no hallándose diferencia significativa. El paciente con talasemia mayor y dolor abdominal presentó un valor muy elevado, desconociéndose el motivo de este hallazgo. El clearance fecal de alfa-1-antripsina es un método útil, simple, menos costoso y no invansivo que las técnicas tradicionales para el diagnóstico de enteropatía perdedora de proteínas en la infancia

Depuración plasmática de alfa-1-antitripsina, método simple para el estudio de la pérdida proteica intestinal / Plasma clearance of alpha 1-antitrypsin, a simple method for the study of intestinal protein losing

Tratamos de corroborar el valor del clearence de alfa-antitripsina como una técnica eficaz para objetivar la pérdida de proteínas a través del tubo digestivo y su aplicación como método de diagnóstico en tal patología. Se estudiaron 22 individuos; 11 con patología intestinal capaz de producir pérdida proteica y 11 controles sin daño de la mucosa intestinal. Se determinó la concentración de alfa-1-antitripsina tanto en suero como en materia fecal en días consecutivos y pesando la materia fecal de 24 horas se pudo obtener el clearence intestinal de dicha proteína. En los pacientes con enfermedad perdedora de proteínas el valor del clearence fue siempre patológico mientras que en los controles se obtuvieron valores normales. Este método es útil para diagnosticar la pérdida proteica intestinal y tiene la ventaja de no utilizar material radioactivo para su determinación