RESUMO
BACKGROUND: Diamine Oxidase (DAO) has an essential role for degradation of exogenous histamine in the intestine; thus, histamine intolerance (HI) mainly has been correlated to a low concentration and/or activity of this enzyme. The objective of the study was to standardize a colorimetric technique to measure the enzymatic activity (function) of hDAO to then apply it to a series of 22 patients with a clinical diagnosis of HI. METHODS: For the standardization variables such as volume and type of sample, incubation time, wavelength of maximum absorption, types of substrates, and concentration of oxidized ascorbate were evaluated. Then the activity and concentration of DAO was determined in 22 patients diagnosed with HI and 22 healthy subjects. RESULTS: The mean of serum DAO concentration in the 22 patients was of 9.268 ± 1.124 U/mL. The mean of serum DAO concentration in the 22 controls was of 20.710 ± 2.509 U/mL, being significantly higher (P value 0.0002) the mean of the samples. The mean of serum DAO activity of the patients was of 1.143 ± 0.085 U/L and the controls was 1.533 ± 0.119 U/L, significantly greater than the patients (P value 0.011). In addition, the sensitivity of both techniques was 0.63. In the measuring of DAO concentration the specificity was 0.9, constituting a good diagnostic test, especially to rule out the true negatives. The determination of DAO activity had a specificity of 0.68. CONCLUSIONS: Although we used a small number of patients and controls and the absorbance values were lower than expected, statistically significant differences were found in the levels of concentration and DAO activity between the patients with histamine intolerance and the controls. Therefore, the measuring of DAO concentration and DAO activity is a good diagnostic strategy for study suspect cases of HI. The simultaneous use of both assays allows to reduce positive and negative false results, for example, patients with normal DAO levels that could present a dysfunction in the activity of this enzyme.
RESUMO
Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.
