Ubiquitin of Entamoeba histolytica induces antibody response in patients with invasive amoebiasis.

Entamoeba histolytica causes amoebic liver abscess (ALA) in humans. The injury of target cells by E. histolytica includes processes controlled by the ubiquitin Ehub. Previously, we found immunodominance of Ehub glycan moieties using immunized rabbits. In this work, we analysed dominance of antibodies to the glycoprotein Ehub in the sera from 52 patients with ALA. Controls were sera from 20 healthy people living in endemic areas with a high seroprevalence of antibodies to amoebas, and 20 patients with alcoholic hepatitis (AH) to rule out the cross-reaction of Ehub with autoantibodies induced by liver damage. Antigens were trophozoite extract, glycoprotein Ehub and the recombinant protein E. histolytica recombinant ubiquitin (rEhub). The sera from healthy volunteers and patients with AH do not have antibodies to glycoprotein Ehub. Surprisingly, only the antibodies from patients with ALA recognized the glycoprotein Ehub, and some sera gave a faint reaction with the recombinant protein, especially because evolutionarily, the ubiquitin is conserved between species. This is the first report demonstrating that antibodies to ubiquitin Ehub are induced exclusively in patients with invasive amoebiasis, and the antibody response is mainly to the glycoprotein, indicating glycans are immunodominant. Inhibitors of the Ehub glycans could be potential treatment for amoebiasis by selectively damaging trophozoites.

Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Salivary stimulation by prolonged release of pilocarpine using films in diabetic rats / Estimulación salival mediante biopelículas de liberación prolongada de pilocarpina en ratas diabéticas

The local use of prolonged drug delivery in the oral cavity provides many advantages, i.e., it increases pharmacologic actions in the desired local site, allows smaller doses and reduces adverse effects. Pilocarpineis a cholinergic drug approved by the FDA for treating glandular hypofunction; however, the adverse effects associated with it limit its use. Objective: To evaluate cytotoxicity of films in adherent fibroblasts and their ability to release pilocarpine in vivo for a prolonged time in the oral cavity of diabetic rats and its effect on salivary flow. Methods: Chitosan and Hydroxypropylmethylcellulose (Methocel K4MCR) films were prepared in 1 percent acetic acid and pilocarpine was added under magnetic stirring. Cytotoxicity of films was evaluated in adherent fibroblasts HS27 and assessed by neutral red technique. The sialogogue effect of films was evaluated on the floor of the mouth of diabetic rats. Later, histopathological analysis was performed using hematoxylin and eosin and Masson’s trichrome stains. Results: Films were biocompatible and had 96 percent cell viability. It was possible to increase stimulation of salivary flow in diabetic rats (6.36+/-0.987mg/hr) compared to the control group (0.5+/-0.06mg/hr). The histopathological analysis did not show inflammatory infiltrate in the area where films were placed. Conclusion: Films were biocompatible and had high cell viability. Also, they considerably increased salivary flow in diabetic rats, without triggering an inflammatory infiltrate in the area which indicates that it is a biocompatible product for sustained release and safe for pilocarpine administration...

El uso local de administración prolongada de fármacos en la cavidad oral proporciona múltiples ventajas, aumentando la acción farmacológica en el sitio de acción, reducción de la dosis usual y disminución de los efectos adversos. La pilocarpina es una droga aprobada por la FDA para el tratamiento del deterioro de las glándulas salivales, sin embargo sus efectos adversos limitan su uso. Objetivo: Evaluar la citotoxicidad de las biopelículas en fibroblastos adherentes y su capacidad in vivo de liberar pilocarpina prolongadamente en la cavidad oral de ratas diabéticas y su efecto en el flujo salival. Metodología: Se elaboraron biopelículas de Quitosán y HPMC (Methocel K4MCR) adicionadas con pilocarpina en una solución de ácido acético 1 por ciento. Fue evaluada la citotoxicidad de las biopelículas en fibroblastos adherentes HS27 mediante la técnica de rojo neutro, además de ser evaluado el efecto sialogogo de las biopelículas en el piso de boca en ratas diabéticas Wistar, realizando después un análisis histopatológico de la zona de colocación, mediante la tinción de Hematoxilina y Eosina y Tricrómico de Masson. Resultados: Las biopelículas resultaron ser biocompatibles con un 96 por ciento de viabilidad celular. Se logró aumentar la estimulación del flujo salival en las ratas diabéticas (6.36+/-0.987 mg/hr), a comparación del grupo control (0.5+/-0.06 mg/hr). El análisis histopatológico no reveló un infiltrado inflamatorio presente en la zona de aplicación de las biopelículas. Conclusión: Las biopelículas resultaron biocompatibles con alta viabilidad celular, además que lograron aumentar considerablemente el flujo salival en ratas diabéticas, sin desencadenar un infiltrado inflamatorio en la zona de aplicación, indicando que es un producto de liberación prolongada biocompatible y seguro para la administración de pilocarpina...

Physicochemical and antimicrobial evaluation of chitosan and hydroxypropyl methylcellulose films forprolonged release of pilocarpine / Evaluación fisico-química y antimicrobiana de biopelículas de quitosán e hidroxipropilmetilcelulosa para liberación prolongada de pilocarpina

The use of prolonged local drug delivery to the oral cavity offers multiple benefits, such as increasing the pharmacological action in the desirable local site and reducing the usual dose and the adverse effects. Pilocarpine is a cholinergic drug approved by the FDA for the treatment of glandular hypofunction; however, the extent of its adverse effects limits its use. Objective: The main aim of this study was to analyze the physical and chemical properties of films, including pH, thickness, solubility, consistency and the ability to release pilocarpine for a prolonged time. Additionally, theantimicrobial activity in two opportunistic pathogens in hyposialia (Streptococcus mutans and Candida albicans) was also assessed. Methodology: Chitosan and HPMC (Methocel K4M CR) films were prepared in 1 percent acetic acid and pilocarpine was added under magnetic stirring. PH, thickness and time of solubility in artificial saliva, as well as diffusion and drug release kinetics per cm2 (OD=420nm) were assessed by spectrophotometry. The antimicrobialactivity was tested by disk diffusion test against St. mutans ATCC 700610 and C. albicans ATCC 90029 at concentrations of hyposalivation (1.44x1.2x106 CFU and 103 CFU, respectively). Results: All the films, except for Hydroxypropyl methylcellulose / Pilocarpine formulation, were found to have optimal physical-chemical properties for handling, maintaining drug diffusion in 76 percent per cm2 for four hours extended-release without showing antimicrobial activity at concentrations of hyposalivation. Conclusion: The films had optimum handling properties and a constant drug release; however, antimicrobial activity was not found...

El uso local de administración prolongada de fármacos en la cavidad oral proporciona múltiples ventajas, aumentando la acción farmacológica en el sitio local deseable, reducción de la dosis usual y disminución de los efectos adversos. La pilocarpina es una droga colinérgica aprobada por la FDA para el tratamiento de la hipofunción glandular, sin embargo la amplitud de sus efectos adversos limitan su uso. Objetivo: Con el objetivo de analizar las propiedades físico-químicas de las biopelículas se evaluó el pH, grosor, solubilidad, uniformidad y la capacidad de liberar prolongadamente pilocarpina, así como su actividad antimicrobiana ante los dos microorganismos patógenos oportunistas en la hiposialia (Streptococcus mutans y Candida albicans). Metodología: Se elaboraron biopelículas de Quitosán e Hidroxipropilmetilcelulosa (Methocel K4MCR) en ácido acético al 1 por ciento, adicionadas con pilocarpina bajo agitación magnética, evaluando el pH, grosor y el tiempo de solubilidad en saliva artificial, así como la uniformidad de difusión y cinética de liberación de la droga por cm2 mediante espectrofotometría (OD=420nm). Mediante difusión en disco se evaluó la actividad antimicrobiana ante Streptococcus mutans ATCC 700610 y Candida albicans ATCC 90029 en concentraciones encontradas en hiposalivación (1.44 x 106 UFC y 1.2 x 103 UFC respectivamente). Resultados: Todas las biopelículas, a excepción de la formulación Hidroxipropilmetilcelulosa e Hidroxipropilmetilcelulosa/ Pilocarpina resultaron tener las propiedades físico-químicas óptimas de manipulación, manteniendo una uniformidad de difusión de la droga en 76 por ciento por cm2 con liberación prolongada por 4 horas, sin mostrar actividad antimicrobiana en concentraciones de hiposalivación. Conclusión: Las películas obtuvieron las propiedades óptimas de manipulación, y una constante liberación del fármaco, sin embargo, ninguna formulación presentó actividad antimicrobiana...

Hypocholesterolemia in patients with an amebic liver abscess.

BACKGROUND/AIMS: Many parasites induce changes in the lipid profiles of the host. Cholesterol increases the virulence of Entamoeba histolytica in animal models and in vitro culture. This study aimed to determine, in patients with an amebic liver abscess, the correlation between cholesterol and other features, such as the size and number of abscesses, standard hematological and serum chemistry profiles, liver tests, and duration of hospital stay. METHODS: A total of 108 patients with an amebic liver abscess and 140 clinically healthy volunteers were investigated. Cholesterol and triglycerides were measured in the sera. The data from medical observations and laboratory tests were obtained from the clinical records. RESULTS: A total of 93% of patients with an amebic liver abscess showed hypocholesterolemia not related to any of the studied parameters. Liver function tests correlated with the size of the abscess. The most severe cases of amebic liver disease or death were found in patients whose cholesterol levels continued to decrease despite receiving antiamebic treatment and hospital care. CONCLUSIONS: Our results show that the hypocholesterolemia observed in patients with an amebic liver abscess is not related to any of the clinical and laboratory features analyzed. This is the first study relating hypocholesterolemia to severity of hepatic amebiasis.