PMC:17824 / 7204-10745 JSONTXT

{"project":"2_test","denotations":[{"id":"11178126-8630732-4389686","span":{"begin":166,"end":168},"obj":"8630732"},{"id":"11178126-10371511-4389687","span":{"begin":594,"end":596},"obj":"10371511"},{"id":"11178126-10371511-4389688","span":{"begin":1351,"end":1353},"obj":"10371511"},{"id":"11178126-1612762-4389689","span":{"begin":2667,"end":2669},"obj":"1612762"},{"id":"11178126-9536122-4389690","span":{"begin":2775,"end":2777},"obj":"9536122"}],"text":"Materials and methods\n\nMice\nC57BL/6 mice were purchased from ALAB (Stockholm, Sweden). IL-12 P40 knockout mice were kindly provided by Dr J Magram (Nutley, NJ, USA) [21]. All mice were housed in the animal facility of the Department of Rheumatology, University of Göteborg. Male mice 6–8 weeks of age were used in all the experiments.\n\nOligonucleotides and injection\nPhosphorothioate-modified oligonucleotide (CpG ODN) 1668 were synthesized by Scandinavian Gene Synthesis AB (Köping, Sweden). The sequence of oligodinucleotide (ODN) 1668 (containing the CpG motif) has been reported elsewhere [10]: 5'-TCC ATG ACG TTC CTG ATG CT-3'. CpG ODN (6 μg in a volume of 20 μl) was injected into the knee joints of the mice.\n\nTissue preparation\nThe mice were killed 0, 1, 3, 7, 14, or 21 d after inoculation. At the end of each time interval, the synovial tissues from the joints of four animals were excised under an inverted microscope. These tissues were then snap-frozen in OCTTM compound (Tissue-TeK®; Sakura Finetek Europe B.V., The Netherlands) by immersion in liquid nitrogen. Frozen tissue was stored at -70°C until use. Serial 6-μm sections were cut and thaw-mounted onto probe-on-slides (Fisher Scientific, Pittsburgh, PA, USA).\n\nHybridization in situ\nHybridization was conducted in situ to analyze cytokine mRNA expression, as previously detailed [10]. Briefly, synthetic oligonucleotide probes (Table 1) — TNF-α, IFN-γ, IL-1β, and IL-12 (the gift of Dr Tomas Olsson, Karolinska Institute, Stockholm, Sweden) — were labeled at the 3' end using terminal deoxynucleotidyl transferase (Advanced Biotechnologies, Leatherhead, UK) and [35S]ATP (Dupont Scandinavia, Stockholm, Sweden). Sections (6 μm thick) of freshly frozen synovial tissues were thaw-mounted onto slides and hybridized with 1 × 106 cpm of labeled probe per 100 μl hybridization mixture. After emulsion autoradiography, development, and fixation, the coded slides were examined by dark-field microscopy for positive cells, which were defined as those containing \u003e15 silver grains in a star-like distribution. The intracellular distribution of the grains was checked by light microscopy at higher magnification. The data were expressed as the number of cells (mean ± SEM) expressing mRNA per mm2 of the tissue section.\n\nHistopathological examination\nJoints were fixed, decalcified, and embedded in paraffin for histopathological examination. Tissue sections from knee joints were cut and stained with hematoxylin–eosin. All the slides were coded and evaluated blind. The specimens were evaluated with regard to synovial hypertrophy, pannus formation, and destruction of cartilage and sub-chondral bone [22].\n\nTNF-α levels in supernatant and serum\nSpleen mononuclear cells were prepared as described previously [20]. The cells (1×106/ml) were cultured in Iscove's complete medium (10% fetal calf serum, 5 × 10-5 m 2-mercaptoethanol, 2 mm L-glutamine, and 50 μg/ml gentamicin) and stimulated with 1 μg/ml lipopolysaccharides or 1 μm CpG ODN. The cultures were maintained in 24-well plates (Nunc; Roskilde, Denmark) at 37°C in 5% CO2 and 95% humidity. The supernatants were collected after 18 h for determination of TNF-α. TNF-α levels in supernatant and serum taken from IL-12 knockout and wild-type mice at day 3 after intra-articular inoculation with 6 μg CpG ODN were analyzed using a TNF-α ELISA kit (Genzyme, Cambridge, MA, USA).\n\nStatistical analysis\nThe differences between mean values were tested for significance with the Fisher's Exact test and the Mann–Whitney U test."}

{"project":"Colil","denotations":[{"id":"T20","span":{"begin":166,"end":168},"obj":"8630732"},{"id":"T21","span":{"begin":594,"end":596},"obj":"10371511"},{"id":"T22","span":{"begin":1351,"end":1353},"obj":"10371511"},{"id":"T23","span":{"begin":2667,"end":2669},"obj":"1612762"},{"id":"T24","span":{"begin":2775,"end":2777},"obj":"9536122"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Materials and methods\n\nMice\nC57BL/6 mice were purchased from ALAB (Stockholm, Sweden). IL-12 P40 knockout mice were kindly provided by Dr J Magram (Nutley, NJ, USA) [21]. All mice were housed in the animal facility of the Department of Rheumatology, University of Göteborg. Male mice 6–8 weeks of age were used in all the experiments.\n\nOligonucleotides and injection\nPhosphorothioate-modified oligonucleotide (CpG ODN) 1668 were synthesized by Scandinavian Gene Synthesis AB (Köping, Sweden). The sequence of oligodinucleotide (ODN) 1668 (containing the CpG motif) has been reported elsewhere [10]: 5'-TCC ATG ACG TTC CTG ATG CT-3'. CpG ODN (6 μg in a volume of 20 μl) was injected into the knee joints of the mice.\n\nTissue preparation\nThe mice were killed 0, 1, 3, 7, 14, or 21 d after inoculation. At the end of each time interval, the synovial tissues from the joints of four animals were excised under an inverted microscope. These tissues were then snap-frozen in OCTTM compound (Tissue-TeK®; Sakura Finetek Europe B.V., The Netherlands) by immersion in liquid nitrogen. Frozen tissue was stored at -70°C until use. Serial 6-μm sections were cut and thaw-mounted onto probe-on-slides (Fisher Scientific, Pittsburgh, PA, USA).\n\nHybridization in situ\nHybridization was conducted in situ to analyze cytokine mRNA expression, as previously detailed [10]. Briefly, synthetic oligonucleotide probes (Table 1) — TNF-α, IFN-γ, IL-1β, and IL-12 (the gift of Dr Tomas Olsson, Karolinska Institute, Stockholm, Sweden) — were labeled at the 3' end using terminal deoxynucleotidyl transferase (Advanced Biotechnologies, Leatherhead, UK) and [35S]ATP (Dupont Scandinavia, Stockholm, Sweden). Sections (6 μm thick) of freshly frozen synovial tissues were thaw-mounted onto slides and hybridized with 1 × 106 cpm of labeled probe per 100 μl hybridization mixture. After emulsion autoradiography, development, and fixation, the coded slides were examined by dark-field microscopy for positive cells, which were defined as those containing \u003e15 silver grains in a star-like distribution. The intracellular distribution of the grains was checked by light microscopy at higher magnification. The data were expressed as the number of cells (mean ± SEM) expressing mRNA per mm2 of the tissue section.\n\nHistopathological examination\nJoints were fixed, decalcified, and embedded in paraffin for histopathological examination. Tissue sections from knee joints were cut and stained with hematoxylin–eosin. All the slides were coded and evaluated blind. The specimens were evaluated with regard to synovial hypertrophy, pannus formation, and destruction of cartilage and sub-chondral bone [22].\n\nTNF-α levels in supernatant and serum\nSpleen mononuclear cells were prepared as described previously [20]. The cells (1×106/ml) were cultured in Iscove's complete medium (10% fetal calf serum, 5 × 10-5 m 2-mercaptoethanol, 2 mm L-glutamine, and 50 μg/ml gentamicin) and stimulated with 1 μg/ml lipopolysaccharides or 1 μm CpG ODN. The cultures were maintained in 24-well plates (Nunc; Roskilde, Denmark) at 37°C in 5% CO2 and 95% humidity. The supernatants were collected after 18 h for determination of TNF-α. TNF-α levels in supernatant and serum taken from IL-12 knockout and wild-type mice at day 3 after intra-articular inoculation with 6 μg CpG ODN were analyzed using a TNF-α ELISA kit (Genzyme, Cambridge, MA, USA).\n\nStatistical analysis\nThe differences between mean values were tested for significance with the Fisher's Exact test and the Mann–Whitney U test."}