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.

Background/AimsMany 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.MethodsA 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.ResultsA 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.ConclusionsOur 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.

Abstract:Introduction: 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. Pilocarpine is 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% 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% 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.

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.

Introduction: 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, the antimicrobial 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% 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 cm 2 (OD=420nm) were assessed by spectrophotometry. The antimicrobial activity was tested by disk diffusion test against St. mutans ATCC 700610 and C. albicans ATCC 90029 at concentrations of hyposalivation (1.44x1.2x10 6 CFU and 10 3 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% per cm 2 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.