RESUMEN
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.
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Introduction: About 80-95% of patients infected with the human immunodeficiency virus (HIV) develop skin manifestations, which are markers of the patients' immune status. Objective: To describe the dermatologic manifestations and the clinical and sociodemographic factors of hospitalized patients diagnosed with HIV and their correlation with CD4 T-lymphocyte count. Materials and methods: We conducted an observational, cross-sectional, and retrospective study of the medical records of 227 adult patients with HIV diagnosis evaluated by dermatology in a hospital in Medellín, Colombia. Results: We included 227 patient records with 433 dermatologic manifestations, 64.4% of them infectious. The most frequent manifestations were oral candidiasis, condylomata acuminata, and drug reactions. Moreover, a statistically significant relationship was found between disseminated herpes zoster virus and secondary syphilis with a CD4 count between 200-499 cells/mm3 (p=0.04 and 0.028, respectively). There was also a statistically significant relationship between oral candidiasis and a CD4 count of less than 100 cells/mm3 (p=0.008). Conclusions: The relationship between disseminated herpes zoster with CD4 between 200-499 cells/mm3 suggests that, despite having high CD4 counts, severe forms of the disease may occur due to possible T-cell dysfunction and depletion of the immune system. Additionally, the relationship between oral candidiasis and CD4 less than 100 cells/mm3 indicates the potential role of oral candidiasis as an essential marker of weakened immune status in HIV patients.
Introducción. Entre el 80 y el 95 % de los pacientes infectados por el virus de inmunodeficiencia humana (HIV) desarrollan manifestaciones en la piel que sirven como marcadores de su estado inmunológico. Objetivos. Describir las manifestaciones dermatológicas y los factores clínicos y sociodemográficos de los pacientes hospitalizados con diagnóstico de HIV y su correlación con el recuento de linfocitos T CD4. Materiales y métodos. Se hizo un estudio observacional de corte transversal y retrospectivo a partir del registro de las historias clínicas de 227 pacientes mayores de edad con diagnóstico de HIV, evaluados por dermatología en un hospital de Medellín, Colombia. Resultados. Los 227 registros daban cuenta de 433 manifestaciones dermatológicas, el 64,4 % de ellas infecciosas. Las tres manifestaciones más frecuentes fueron candidiasis oral, condilomas acuminados y reacciones a medicamentos. Se encontró una relación estadísticamente significativa entre el virus del herpes zóster (HZ) diseminado y la sífilis secundaria, con un recuento de CD4 entre 200 y 499 células/mm3 (p=0,04 y 0,028, respectivamente), y entre la candidiasis oral y un recuento de CD4 menor de 100 células/mm3 (p=0,008). Conclusiones. La relación entre el herpes zóster diseminado y un recuento de CD4 entre 200 y 499 células/mm3 sugiere que, a pesar de los recuentos altos, se pueden presentar formas graves de la enfermedad debido a una posible disfunción de las células T y el agotamiento del sistema inmunológico. La relación entre la candidiasis oral y un recuento de CD4 menor de 100 células/mm3 plantea la posibilidad de considerar esta infección micótica como un marcador importante de debilitamiento inmunológico de los pacientes con HIV.
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Candidiasis , Infecciones por VIH , Herpes Zóster , Colombia/epidemiología , Infecciones por VIH/epidemiología , Humanos , Estudios RetrospectivosRESUMEN
BACKGROUND: Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae. It is a polymorphic disease with a wide range of cutaneous and neural manifestations. Ulcer is not a common feature in leprosy patients, except during reactional states, Lucio's phenomenon (LP), or secondary to neuropathies. CASES PRESENTATION: We report eight patients with multibacillary leprosy who presented specific skin ulcers as part of their main leprosy manifestation. Ulcers were mostly present on lower limbs (eight patients), followed by the upper limbs (three patients), and the abdomen (one patient). Mean time from onset of skin ulcers to diagnosis of leprosy was 17.4 months: all patients were either misdiagnosed or had delayed diagnosis, with seven of them presenting grade 2 disability by the time of the diagnosis. Reactional states, LP or neuropathy as potential causes of ulcers were ruled out. Biopsy of the ulcer was available in seven patients: histopathology showed mild to moderate lympho-histiocytic infiltrate with vacuolized histiocytes and intact isolated and grouped acid-fast bacilli. Eosinophils, vasculitis, vasculopathy or signs of chronic venous insufficiency were not observed. Skin lesions improved rapidly after multidrug therapy, without any concomitant specific treatment for ulcers. CONCLUSIONS: This series of cases highlights the importance of recognizing ulcers as a specific cutaneous manifestation of leprosy, allowing diagnosis and treatment of the disease, and therefore avoiding development of disabilities and persistence of the transmission chain of M. leprae.
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Lepra Multibacilar/diagnóstico , Úlcera Cutánea/diagnóstico , Adolescente , Adulto , Anciano , Errores Diagnósticos , Humanos , Leprostáticos , Lepra Multibacilar/complicaciones , Masculino , Persona de Mediana Edad , Mycobacterium leprae/aislamiento & purificación , Piel/patología , Úlcera Cutánea/complicacionesRESUMEN
Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.
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OBJECTIVE: Identifying Fusarium isolates from mycosis symptomatic patients through molecular techniques as PCR and sequencing. METHODS: In this study, samples were taken from 101 mycosis symptomatic patients in-between 2004-2006. To determine isolates belonging to the Fusarium genus, the DNAr 28S region was amplified through PCR and specific PCR primers further confirmed their identity to the species level. Additionally, in order to confirm the identity of the species of the isolates, 75 isolates of these were analyzed by partial sequencing of the 28S rDNA and the TEF1-α gene. RESULTS: The 28S rDNA portion detected all 101 isolates as belonging to Fusarium and the PCR specific primers detected 52 and 29 isolates as F. oxysporum and F. solani, respectively; 34 and 41 of these, afterwards studied by partial sequencing of the 28S rDNA and TEF1- α genes respectively, were effectively identified by the technique. CONCLUSION: From all the molecular markers used to identify Fusarium isolates, the sequence of the TEF1-α gene provided the best resolution in the identification of species level; however it is possible to discriminate between F. oxysporum and F. solani isolates by PCR, in most of the cases, what is important considering the simplicity of the technique and a faster diagnosis.
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ADN de Hongos/análisis , Fusariosis/diagnóstico , Fusarium/clasificación , Tipificación Molecular/métodos , Colombia , Cartilla de ADN , Femenino , Fusariosis/microbiología , Fusarium/genética , Fusarium/aislamiento & purificación , Humanos , Masculino , Técnicas de Tipificación Micológica , Reacción en Cadena de la PolimerasaRESUMEN
Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.
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Iduronato Sulfatasa/genética , Iduronato Sulfatasa/metabolismo , Lisosomas/enzimología , Pichia/genética , Animales , Biomasa , Reactores Biológicos , Western Blotting , Células CHO , Codón , Cricetulus , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Fermentación , Células HEK293 , Semivida , Humanos , Concentración de Iones de Hidrógeno , Iduronato Sulfatasa/aislamiento & purificación , Oxígeno/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
ABSTRACT Objective Identifying Fusarium isolates from mycosis symptomatic patients through molecular techniques as PCR and sequencing. Methods In this study, samples were taken from 101 mycosis symptomatic patients in-between 2004-2006. To determine isolates belonging to the Fusarium genus, the DNAr 28S region was amplified through PCR and specific PCR primers further confirmed their identity to the species level. Additionally, in order to confirm the identity of the species of the isolates, 75 isolates of these were analyzed by partial sequencing of the 28S rDNA and the TEF1-α gene. Results The 28S rDNA portion detected all 101 isolates as belonging to Fusarium and the PCR specific primers detected 52 and 29 isolates as F. oxysporum and F. solani, respectively; 34 and 41 of these, afterwards studied by partial sequencing of the 28S rDNA and TEF1- α genes respectively, were effectively identified by the technique. Conclusion From all the molecular markers used to identify Fusarium isolates, the sequence of the TEF1-α gene provided the best resolution in the identification of species level; however it is possible to discriminate between F. oxysporum and F. solani isolates by PCR, in most of the cases, what is important considering the simplicity of the technique and a faster diagnosis.(AU)
RESUMEN Objetivo Identificar aislamientos de Fusarium en pacientes con micosis por medio de las técnicas moleculares de PCR y secuenciación. Métodos Se tomaron 101 muestras de pacientes con micosis sintomática, entre los años 2004 y 2006. Para la detección de aislamientos como pertenecientes al género Fusarium, se amplificó parcialmente por PCR la región 28S del DNAr; y posteriormente -para la detección de la especie de Fusarium- se utilizaron cebadores específicos para F. oxysporum y F. solani. La verificación de la identidad de la especie de los aislamientos se hizo por secuenciación parcial de los genes 28S DNAr y TEF1-α Resultados El total de 101 aislamientos fueron detectados como pertenecientes al género Fusarium utilizando un cebador universal de la region 28S DNAr; 52 y 29 aislamientos se detectaron como F. oxysporum o F. salani, respectivamente con los cebadores específicos, y la secuenciación parcial de los genes 28S rDNA o TEF1-α confirmó la identidad de las especies. Conclusión La secuenciación parcial del gen TEF1-α es aún el mejor marcador molecular para identificar aislamientos de Fusarium a nivel de especie. Sin embargo, en la mayoría de los casos es posible discriminar entre aislamientos de F. oxysporum y F. solani por PCR con cebadores específicos, lo que proporciona una ventaja importante considerando la simplicidad de la técnica y el rápido diagnóstico.(AU)
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Humanos , ADN Ribosómico , Reacción en Cadena de la Polimerasa/métodos , Fusarium/aislamiento & purificación , Micosis/diagnóstico , Manejo de Especímenes/métodos , ColombiaRESUMEN
Resumen Introducción: Los modelos de atención en artritis reumatoide consideran el abordaje sintomático y la intervención de discapacidad; sin embargo, es importante un modelo que integre los avances en el manejo farmacológico y las estrategias no farmacológicas. Materiales y métodos: Estudio retrospectivo, descriptivo, transversal, en el que incluimos a 640 pacientes con diagnóstico de artritis reumatoide que estaban participando en un programa de atención integral ambulatoria; tomamos las primeras valoraciones realizadas por terapia ocupacional con la evaluación funcional completa que incluían: índice de Barthel, escala Quick DASH y Health Assessment Questionnaire. El nivel de actividad de la enfermedad se calculó a través del DAS28. Resultados: Se evidenciaron puntuaciones más altas del HAQ a medida que el nivel de actividad de la enfermedad aumenta; no se encontraron diferencias significativas en cuanto al nivel de actividad de la enfermedad entre los sujetos que laboran y los que no. Encontramos una baja asociación entre el DAS28 y el Quick DASH en sus 2 módulos de evaluación (r = 0,399 para el instrumental y r = 0,291 para el módulo laboral; p < 0,005). De los 350 sujetos, el 66,7% presentó algún grado de actividad de la enfermedad y el porcentaje de pacientes que calificaron una limitación de moderada a severa en la función de su extremidad superior fue del 66,1% para el módulo laboral y del 84,75% para el módulo instrumental, con mayor limitación funcional a medida que aumenta el nivel de actividad de la enfermedad. Conclusiones: Este estudio ha permitido reevaluar dentro del programa la aplicación de escalas genéricas que abordan de forma general la funcionalidad.
Abstract Introduction: Although rheumatoid arthritis care models consider a symptomatic approach and intervention of disability, it is also important to have a model that integrates advances in the pharmacological management, as well as non-drug treatment strategies. Materials and methods: A descriptive cross-sectional retrospective study was conducted that included 640 patients diagnosed with rheumatoid arthritis and in an outpatient comprehensive care program. The first assessments made by occupational therapy with a full functional evaluation including the Barthel index, Quick DASH scale, and the Health Assessment Questionnaire. The activity of the disease was calculated using DAS28. Results: Higher HAQ scores were observed as the level of disease activity increased. There were no significant differences in the level of activity of the disease among subjects who worked and those who did not. A low correlation was found between the DAS28 and the Quick DASH in 2 evaluation modules (r=.399 for instrumental and r=.291 for the work module) (P<.005). Of the 350 subjects, 66.7% had some degree of disease activity and the percentage of patients shown to have a moderate to severe limitation in the functioning of the upper limb was 66.1% for the work module and 84.75% for the instrumental module, showing a greater functional limitation as the level of disease activity increased. Conclusion: This study has reassessed the applying of generic scales that deal with generally functionality, within the care program.
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Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Artritis Reumatoide , Terapia Ocupacional , Diagnóstico , Atención a la Salud , Estudios de la DiscapacidadRESUMEN
Previously, we demonstrated production of an active recombinant human N-acetylgalactosamine-6-sulfatase (rhGALNS) enzyme in Escherichia coli as a potential therapeutic alternative for mucopolysaccharidosis IVA. However, most of the rhGALNS produced was present as protein aggregates. Here, several methods were investigated to improve production and activity of rhGALNS. These methods involved the use of physiologically-regulated promoters and alternatives to improve protein folding including global stress responses (osmotic shock), overexpression of native chaperones, and enhancement of cytoplasmic disulfide bond formation. Increase of rhGALNS activity was obtained when a promoter regulated under σ s was implemented. Additionally, improvements were observed when osmotic shock was applied. Noteworthy, overexpression of chaperones did not have any effect on rhGALNS activity, suggesting that the effect of osmotic shock was probably due to a general stress response and not to the action of an individual chaperone. Finally, it was observed that high concentrations of sucrose in conjunction with the physiological-regulated promoter proU mod significantly increased the rhGALNS production and activity. Together, these results describe advances in the current knowledge on the production of human recombinant enzymes in a prokaryotic system such as E. coli, and could have a significant impact on the development of enzyme replacement therapies for lysosomal storage diseases.
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Condroitinsulfatasas/biosíntesis , Escherichia coli/metabolismo , Proteínas Recombinantes/biosíntesis , Biología Sintética/métodos , Disulfuros/metabolismo , Humanos , Ósmosis , Regiones Promotoras Genéticas/genéticaRESUMEN
Las fracturas de cadera son causa frecuente de consulta y cirugía en el Hospital Universitario del Valle. Anualmente ingresan cerca de 450 pacientes con fracturas de cadera, el 80% de los pacientes reciben tratamiento quirúrgico en nuestra institución y la mortalidad hospitalaria es del 11%. Durante el periodo entre Abril 1, 2004 y Junio 30, 2004, ingresaron a nuestro Servicio 86 pacientes mayores de 60 años con fracturas de la cadera, 74 pacientes recibieron tratamiento quirúrgico. El 60% de nuestros pacientes fueron de sexo femenino y el 50% no tenían ningún servicio médico al ingreso. El promedio de la estancia hospitalaria fue de trece días y el promedio para la cirugía fue 10,4 días. Se estudiaron los procesos que en la actualidad se realizan en el Hospital para la evaluación médica inicial, consecución del material de osteosíntesis y el tratamiento quirúrgico definitivo. Se diseñaron organigramas que en la actualidad se desarrollan y se estableció un plan de mejoramiento.