EL MARCO JURÍDICO DE LA LEY 678 DE 2001 NO ESTÁ DOTADA DE LA EFECTIVIDAD Y EL ALCANCE SUFICIENTES PARA QUE LA ACCIÓN DE
2.1.7 Las medidas cautelares
Mice
C57BL/6 (WT), Rag1–/– and Rag2–/–γc–/– mice were purchased from Taconic Farms. All mice were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care accredited animal facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by the NIAID Animal Care and Use Committee. All mice were used between 6 and 13 weeks of age.
Diet Studies
Vitamin A-deficient (TD.10991) and -sufficient (20,000 IU vitamin A/kg, TD.10992) diets were purchased from Harlan Teklad Diets. At day 14.5 of gestation, pregnant females were administered either vitamin A-deficient or -sufficient diet and maintained on diet until weaning of litter. Weanlings were maintained on special diet throughout study.
Cell isolation from the lamina propria and Flow Cytometry
Cells from small and large intestinal lamina propria (Lp) were prepared as previously
described41. Single-cell suspensions were stained with CD16/32 (eBioscience) and with
fluorochrome-conjugated antibodies against any combination of the following surface antigens: CD4, CD8α, CD11b, CD11c, DX5, CD49b, TCR-β, TCR-γδ, CD19, Ter119, NK1.1, Gr-1 and Thy1.2. Prior to fixation Live/Dead Fixable Blue Cell Stain Kit (Invitrogen) was used to exclude dead cells. For examination of transcription factors and
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cellular proliferation, cells were subsequently treated with the FOXP3 staining kit (eBioscience) in accordance with the manufacturer's instructions and stained for 20 min at room temperature with fluorochrome-conjugated antibodies against the following:
RORγt, GATA3 and Ki67.
Intracellular cytokine staining
Isolated cells from lamina propria were stimulated for 2 h with phorbol 12- myristate 13- acetate (PMA) (50ng/ml) and ionomycin (2.5µg/ml) in the presence of Brefeldin A (1µg/ml) (GolgiPlug, BD Biosciences).
Cell isolation from the lung and skin
Tissues were diced followed by digestion with 0.25mg/ml liberase TL (Roche) at 37˚C for 45 min (lung) or liberase and 1mg/ml DNase at 37˚C for 1h 15min (skin). Isolated lung cells were further purified using a 37.5% Percoll gradient followed by lysis of red blood cells with ACK.
Fluorescence-activated cell sorting and in vitro culture of ILC and ILC2P
ILC3 were sorted by flow cytometry from small intestine Lp cells from WT and VAI mice based on the absence of lineage markers (CD4, CD8α, CD11b, CD11c, DX5, CD49b, TCR-β, TCR-γδ, CD19, Ter119, NK1.1 and Gr-1) but expression of Thy1.2 and IL-7R.
ILC2 were sorted as lineage negative, Thy1.2+, KLRG1+. ILC2 progenitors (ILC2P) were
sorted from the bone marrow of Rag1–/– mice by the absence of lineage makers and c- Kit, but expression of IL-7R, CD25 and high levels of Sca-159, 62, 143. RPMI 1640 supplemented with 10% FBS, penicillin, streptomycin, HEPES, glutamine, nonessential
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amino acids, and 50 mM of β-mercaptoethanol (Complete Media) was used for in vitro
culture. ILC3 or ILC2P were cultured in the presence of IL-7 (10ng/ml) and SCF (10ng/ml) in the presence or absence of 1 µM all-trans RA (Sigma-Aldrich) or 1.8 µM RA inhibitor BMS 493 (Tocris, UK) for 7 days. IL-13 cytokine expression in the cell free supernatant was assessed using a bead based cytokine detection assay (FlowCytomix, eBioscience) and was adjusted to the plated density of 5x103 cells in 50 µl culture volume.
Real time PCR
Tissues were homogenized using metal beads (Precellys). RNA was extracted using RNAeasy mini kit (Qiagen) and reverse transcribed with Omniscript (Qiagen) according to the manufacturer’s intructions. The cDNA served as template for the amplification of genes of interest and the housekeeping gene (b-actin) by real-time PCR using SYBR green. Target gene expression was calculated using the comparative method for relative quantification upon normalisation to b-actin gene expression. Primer sequences: (actb: 5’- AAGTGTGACGTTGACATCCGTAA-3’ and 5’-TGCCTGGGTACATGGTGGTA-3’;
retnlb: 5’-ATGGGTGTCACTGGATGTGCTT-3’ and 5’-AGCACTGGCAGTGGCAAGTA- 3’)
In Vivo RA Reconstitution
A total of 250 μg of all-trans-RA (Sigma Aldrich) in 30 μl of biotechnology performance certified DMSO (Sigma Aldrich) was administered intraperitoneally to vitamin A- insufficient mice every other day for 12 days. Mice not receiving RA received DMSO vehicle.
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In Vivo retinoic acid inhibitor (BMS493) treatment
A total of 220μg of the pan retinoic acid receptor inverse agonist BMS 493 (R&D)
resuspended in 30 μl of biotechnology performance certified DMSO (Sigma Aldrich) was
administered intraperitoneally to Rag1–/– mice every day for 8 days as previously described98. Control mice received DMSO only. In some experiments Rag1–/– mice were treated with RAi for 5 days followed by infection with C.rodentium. Injection of RAi or DMSO continued until the mice were sacrificed but not longer than 14 days of total treatment.
Vaccine Protocol
For vaccination, mice were orally inoculated with an isotonic bicarbonate buffer. Ten minutes later, mice were gavaged with a mixture of 1 mg of OVA and 20 μg of the
doublemutant form of E. coli LT (R192G/L211A) prepared in the same buffer.
LT(R129G/L211A) was kindly provided by J. Clements. For experiments, mice were vaccinated once per week and immune responses were assessed 1 week after challenge on Day 14.
Oral antigen administration/Treg conversion Assay
T lymphocytes were extracted from the secondary LNs (excluding the spleen) of RAG1−/− OT-II Tg mice (Ly5.1) and adoptively transferred into recipient mice. Each
mouse received 106 cells. Recipient mice were received a 1.5% OVA solution dissolved
in drinking water that was replaced every 48 h (grade V; Sigma-Aldrich) for 6 consecutive days. On day 7 cells were isolated from the hosts and Foxp3 expression was assessed in transferred cells.
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Aldefluor Assay
Cell suspensions were surface stained (1×106 cells/ml) and incubated for 30 minutes at 37°C in ALDEFLUOR assay buffer containing activated ALDEFLUOR per manufactures recommendation (Stem Cell Technologies). Cells were analyzed via flow cytometry. Background ALDEFLOUR staining was less than 0.5% in the presence of the RALDH inhibitor diethylaminobenzaldehyde (DEAB).
Infection withCitrobacter rodentium
For Citrobacter rodentium infections (formerly Citrobacter freundii, biotype 4280) strain DBS100 (provided by David Artis, University of Pennsylvania, Philadelphia, Pennsylvania, USA) was prepared by selecting a single colony and culturing in LB broth for 8 hours. Mice pre-treated for 5 days with vehicle control or RAi were inoculated with
approximately 1 × 1010 CFU in 200 µL of PBS via oral gavage. In some case VAI mice
were treated with 400ng of recombinant mouse IL-22 (rmIL-22, Biolegend) every day for 4 days starting 2 days before infection. Treatment with rmIL-22 continued every other day from day 2 after infection until mice were sacrificed.
Infection withTrichuris muris
Mice were infected with 25-30 embryonated Trichuris muris eggs (a gift from Joseph Urban, USDA, Beltsville, MD, USA) on day 0 by oral gavage. 12-13 days later infected mice were sacrificed and their worm burden was assessed in the cecal epithelial cell layer. In some case C57Bl/6 WT mice were simultaneous treated with vehicle or RAi every day starting upon infections with T. muris.
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Goblet cells scoring in the small intestine
Sections of the small intestine of control mice or VAI mice were stained with periodic acid-schiff (PAS). 5-10 villi per slide were assessed for PAS positive goblet cells. A total of 2 slides distributed over 3 randomly selected areas were counted. In some cases VAI mice were treated with 500ng of anti-IL-13 antibody163 (a gift from Centocor Inc.) every 5
days for up to two weeks.
Statistical analysis.
A two-tailed Student’s t-test was used for all statistical analysis, * p≤ 0.05, **p ≤ 0.005, *** p≤0.0005.
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