Phytohemagglutinin (pha)

PHA blasts from the patient and an age matched control were generated stimulating 1 × 10⁶ PBMC with PHA (Gibco, diluted 1/50 in RPMI 20% ABS Penicillin/Streptomycin/Glutamine) plus recombinant IL2 (Roche, 50 IU) during 48 h. After two washes with RPMI, mediumcells were divided in two aliquots and incubated either with RPMI 2% FCS (basal tube) or 0.1 µg/ml recombinant IL12 R&D (stimulated tube) for another 24 h. Then, both basal and stimulated tubes were re-stimulated with PMA 100 ng/ml and ionomycin (1 µg/ml) in RPMI 20% ABS P/S/G for 5 h in the presence of 10 µg/ml Brefeldin A (Sigma). Cells from basal and simulated tubes were then washed twice, lysed with Becton Dickinson (BD) FACS lysing, and permeabilized with BD FACS permeabilizing solution 2. Permeabilized cells were stained with IFN-γ FITC, CD3 PerCP, and CD8 PE (BD) for 30 min; washed twice with PBS /BSA; and acquired in a FACS Canto II. Percentages of IFN-γ positive cells from basal and IL12 stimulated blast cells were analyzed on gated CD3 + CD8 + and CD3 + CD8- populations on FlowJo.

The CBMN assay was performed accordingly to Fenech [38 (link)], with slight modifications. Cultures of whole blood in duplicate (0.5 mL each) were set up. Culture medium was composed by RPMI-1640 medium (Gibco, Billings, USA) containing 10% of fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 1 mM sodium pyruvate (Invitrogen). Phytohemagglutinin (Invitrogen, Carlsbad, USA) was added at 202 μg/mL to stimulate cell division at time 0 h. Cultures were incubated at 37°C and 5% of CO₂ in a humidified incubator. Cytochalasin B (Sigma, St. Louis, USA) was added at 6 μg/mL after 40 h of culture, and cells were harvested 24 h later by density centrifugation with Ficoll (GE, Chicago, USA). Cells were transferred to slides by cytocentrifugation, fixed, and stained using Panoptic staining (New Prov, Pinhais, BR). Slides were labeled and scored blindly. From each subject, 400 cells were evaluated for mono-, bi-, multinuclear cells, apoptosis, and necrosis frequencies. Subsequently, 1000 binucleated (BN) cells were scored to determine the frequency of BN cells with one or more micronucleus (MN), nuclear plasmatic bridges (NPB), and nuclear buds (NBUD), to evaluate genome damage [38 (link)].

Primary lymphocytes cultures were obtained from heparinised whole blood samples processed within 24 h from collection. At least six cultures (two for each analysis performed) were assessed from each subject. 0.3 ml of blood were added to 4.7 ml of RPMI-1640 culture medium (Invitrogen, Milano, Italy) supplemented with 20% FBS, 1% antibiotics and antimycotics and 1.5% phytohemagglutinin (Invitrogen, Milano, Italy). Cultures were kept at 37° for a variable time depending on the specific analysis requirements. For γH2AX analysis, cultures were immediately harvested in order to avoid lymphocytes stimulation, while for GSTO1 immunostaining they were harvested after 24 h proliferation. For assessment of MN induction in mononucleated cells, cultures where harvested after 72 h of incubation as described in the appropriate subsection (see below). In all the analyses performed, lymphocytes harvesting was performed using the same protocol described elsewhere [31 (link)]. Briefly, cells were initially precipitated by a 5 min centrifugation at 2100 rpm. After replacing the medium with 5 ml of hypotonic solution (KCl, 0.075 M), cells were pre-fixed in 0.4 ml of 5:3 acetic acid:methanol, re-centrifuged and the obtained pellet was resuspended in pure methanol for sample storage at −18°C.

Peripheral blood samples were obtained from parents with healthy children, from parents with aneuploid children and from healthy or aneuploid children. Whole blood was cultured in RPMI medium (Invitrogen, California, USA), supplemented with 1% of phytohemagglutinin (Invitrogen, USA), and incubated for 72 h at 37 °C. Colchicine (0.08 g/mL) (Sigma, San Luis Missouri, USA) was added for the last two hours of culture to obtain metaphase spreads. Harvesting was made using hypotonic solution of 0.075M KCl (Sigma, USA), and cold Carnoy solution (Methanol: Acetic acid, 3:1), then cells were dropped on cold slides and immediately placed on a thermal plate at 60 °C; GTG banding was performed and 20 metaphases per individual were analyzed, at a resolution of 450–550 bands per haploid set. In cases where the gain or loss of a chromosome was observed in one cell, the analysis was extended to 50–100 cells to rule out or confirm mosaicism, according to the criteria of the International Cytogenetic Nomenclature System [50 ]. Slides were blind coded by a person not involved in the study.

For the analysis of antigen-specific T-cell responses, briefly, BD ELISPOT 96-well plates were coated with anti-mouse IFN-γ or IL-4 capture antibodies in 100 μl of PBS/well and incubated at 4°C overnight. The plates were blocked with complete RPMI 1640 medium containing 10% fetal bovine serum (Gibco, USA) and incubated in RT for 2 h. Freshly isolated splenocytes were added at 1 × 10⁶ cells/well in media containing the 1 μg/well of sM2HA2 protein or M2 or HA2 peptide (Table 3) or 1 μg/well of inactivated H9N2 or only medium (negative control), or 0.5 μg/well phytohemagglutinin (positive control, Invitrogen, Carlsbad, CA, USA). Plates were incubated for 24 h for IFN-γ and 48 h for IL-4 at 37°C in 5% CO₂. After discarding the cells, the plates were treated sequentially with biotinylated anti-mouse IFN-γ and IL-4 antibodies, streptavidin-HRP, and substrate solution. Finally, the plates were washed with deionized water and dried for at least 2 h in the dark. Spots were counted automatically using an Immuno Scan Entry analyzer (Cellular Technology Ltd., Shaker Heights, OH, USA).

The development and counting of cytokines were performed by ELISPOTs, as described previously [31] (link), [32] (link). Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-γ and IL-4 capture antibodies (5 μg/ml) in PBS and incubated at 4°C overnight. The plates were washed with PBS, and 200 μl/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added (Invitrogen, Carlsbad, CA, USA) and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5×10⁴ cells/well in media containing sM2 protein, M2 peptide (SLLTEVETPTRNGWECKCSD) (1 μg/well), only medium (negative control), or 5 μg/ml phytohemagglutinin (positive control, Invitrogen, Carlsbad, CA, USA). After adding cells and stimulators, the plates were incubated for 24 hours at 37°C with 5% CO₂. The plates were sequentially treated with biotinylated anti-mouse IFN-γ and IL-4 antibodies, streptavidin-horseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer (Cellular Technology, Shaker Heights, USA).