Setting of Methods for Analysis of Mucosal Antibodies in Seminal and Vaginal Fluids of HIV Seropositive Subjects from Cambodian and Italian Cohorts

International audienceBACKGROUND: Genital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA. METHODOLOGY: Genital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared. PRINCIPAL FINDINGS: The optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction. CONCLUSIONS/SIGNIFICANCE: Specific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV

Longitudinal Tracking of Human Fetal Cells Labeled with Super Paramagnetic Iron Oxide Nanoparticles in the Brain of Mice with Motor Neuron Disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival

Statistical analysis of Modes and Additives in immunoglobulin recovery from vaginal fluids.

<p>Modes: Jo, joint; I, Immediate; D, delayed. Additives legends: Null, cryopreservation only; EDTA: ethylenediaminetetraacetic acid; IP, protease inhibitors; AB, Antibiotics. Only significant P values (before and after adjustment, Basic and Adj, respectively) were included in the Table.</p

Affinity purification of immunoglobulins.

<p>Comparison of the four chromatographic methods for immunoglobulin isolation that were evaluated in the study.</p

Cross-reactivity of commercial anti-human IgA and IgG reagents used in sandwich ELISA assays.

<p>Dilutions: 1∶1 = 4 microg/mL; 1∶10 = 400 ng/mL; 1∶10² = 40 ng/mL; 1∶10³ = 4 ng/mL; 1∶10⁴ = 400 pg/mL; 1∶10⁵ = 40 pg/mL. Response variability was also reported in the Table.</p><p>Results are expressed in OD values.</p

Statistical analysis of Modes and Additives in immunoglobulin recovery from seminal fluids.

<p>Modes: I, immediate; D, delayed. Basic, basic model; Adj, adjusted values. Additives legends: Null, cryopreservation only; EDTA: ethylenediaminetetraacetic acid; IP, protease inhibitors; AB, Antibiotics. Only significant P values were included in the Table; asterisks indicated P values maintaining their significativity after model adjustment (Adj).</p