[Human Metapneumovirus (hMPV) associated to severe bronchial asthmatic crisis]. / Metapneumovirus humano (hMPV) asociado con exacerbación de asma aguda bronquial severa.

Human Metapneumovirus (hMPV) is a recently reported agent of acute infection in the respiratory tract. It has been found in children as well as in young adults and elders. The clinical manifestations produced by hMPV are indistinguishable from those by common respiratory virus, and can evolve from asymptomatic infection into severe pneumonia. On the other hand, some authors have described cases of bronchial asthma exacerbation associated with hMPV infection. In this work we report a case of a child who presented a severe bronchial asthmatic crisis with a suspected viral associated infection. Immunofluorescence tests yielded negative results for sincitial respiratory virus, adenovirus, a-b influenza virus and parainfluenza 1, 2, 3, virus. In an attempt to detect the presence of hMPV, a RT-PCR was carried out to amplify sequences from both N and F genes. Using this approach, a positive result for hMPV was obtained. To our knowledge, this is the first description of a case of asthma exacerbation associated to hMPV in our region. In addition, these results are similar to previous reports where it was hypothesized that, like RSV, hMPV can trigger a respiratory chronic disease as asthma.

Parameters affecting the adsorption of ligands to polyvinyl chloride plates in enzyme immunoassays.

In the present work, we studied the efficacy of three blocking agents (HSA, BSA and OVA) in the inhibition of non-specific binding to PVC plates. According to the inhibition data, 1% OVA was the most effective blocking agent. On the other hand, the presence of detergents in all of the blocking solutions drastically decreased the percent inhibition of the non-specific binding. Furthermore, the effect of ligand concentration on adsorption and the kinetics of ligand adsorption to PVC plates were also investigated. Ligand adsorption is a linear function of input up to a limit (around 8.70 ng/mm2) where saturation is reached. The rate of adsorption of pure human IgG to PVC plates was proportionally increased with the temperature, as shown by proportional rate constants almost 2 times faster at 37 degrees C than at 4 degrees C. These results have practical implications for investigators using PVC for immunoassays and should be taken into consideration when designing such assays.

Differential reactivity of Agaricus bisporus lectin with human IgA subclasses in gel precipitation.

The interaction between purified Agaricus bisporus lectin and several human proteins was studied using the Ouchterlony double diffusion and immunoelectrophoresis techniques. Only one precipitation line was observed with normal human serum, normal human colostrum, IgA1 myeloma serum, both serum monoclonal and secretory IgA1 and monoclonal IgD. No reaction was observed with monoclonal and secretory IgA2, IgG, IgM, alpha 2 macroglobulin or pregnancy-associated alpha 2 glycoprotein. These results were confirmed by hemagglutination inhibition assays when IgA1, IgA2 and IgD were tested. On the basis of this reactivity, ABL could be a useful tool for distinguishing and isolating human IgA subclasses.

A procedure of silver staining for nucleic acids in agarose gels without pretreatment or drying steps.

In this paper, we report a fast, simple, and reproducible staining protocol for nucleic acids in agarose gels with a sensitivity in the order of 10 pg/mm2. It took only three steps: fixation, incubation with silver ions, and development of the gels (total time 50 min). The resulting calibration curves (area vs ng of loaded DNA) after a densitometric scanning of agarose gels stained with this procedure were linear up to 50 ng of double-stranded DNA. We found this method suitable for routine laboratory use and especially appropriate for densitometric analysis due to homogeneous background development. Furthermore, it avoids the pretreatment and/or drying steps proposed by other authors for agarose gels.

Agaricus bisporus lectin binds mainly O-glycans but also N-glycans of human IgA subclasses.

The primary interaction between purified Agaricus bisporus lectin (ABL) and human IgA subclasses was studied by ABL-affinity chromatography, dot blot assay and competitive enzyme-lectin assay, considering that ABL could be an alternative tool for detection of IgA1 O-glycans. Both secretory IgA subclasses bound to ABL-Sepharose and the IgA2 subclass (which contains only N-glycans) was recovered with a high degree of purity when NH4OH was used as eluent. ABL-Ig interaction was also observed by dot blot assays using ABL-peroxidase against monoclonal IgA1 k Pan, IgA2m(1)k Gir, IgA2m(2)k Bel, secretory IgA2 and normal IgG (also contains only N-glycans). When these immunoglobulins were enzymatically treated with peptide N-glycosidase F (N-glycan hydrolysis), the ABL-IgA2 and -IgG interaction did not occur while IgA1 maintained a high degree of interaction with ABL. Also, the ABL-IgA interaction was observed by competitive enzyme-lectin assay, and when IgA1 subclass was treated with endo-alpha-N-acetylgalactosaminidase for O-glycans hydrolysis, the reactivity with ABL was very low. We conclude that the complementary use of ABL and peptide N-glycosidase F could be a useful tool to assess the O-glycosylation state of human IgA1 subclass, which is of relevant importance in the effector functions of immunoglobulins.