PMC:3537549 / 26392-53132 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3537549","sourcedb":"PMC","sourceid":"3537549","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3537549","text":"Results\n\nPrototype CpG-A, ODN 2216, induce proliferation of primary feline immune cells and enhance their expression of costimulatory surface molecules\nProliferation of PBMCs in response to treatment with CpG ODN gives strong indications about the biological activity of the stimulatory molecule and has been used to screen ODN in many species[25]. In relation to this, the potential of CpG-A to induce proliferation of feline PBMCs were assessed by measurement of 3 H-thymidine incorporation after stimulation. Although considerable variability was observed between individuals, a 2-fold increase in proliferation was observed after stimulation with ODN 2216 when compared to stimulation with inactive control ODN 2243 (p = 0.0281, Figure1A). The cells of three cats (c08, c09, c12) could be stimulated to particularly high proliferative rates by ODN 2216 (values above the mean of eight cats illustrated in Figure1A) in the following order: c08 \u003e c12 \u003e c09.\nAnother characteristic feature of stimulatory ODN is their ability to enhance interactions between various immune cell populations by upregulation of cell surface costimulatory molecules. With the objective to test whether ODN 2216 could exert such properties in feline immune cells, the expression of B7.1 and MHCII was measured by flow cytometry in stimulated PBMCs of the same eight cats as above. The expression of both co-stimulatory molecules was evaluated in gates defined to contain a PBMC population, a lymphocyte population and a non-lymphocyte population of cells. The observed effects varied considerably between the cells of individual cats, ranging from no alterations to an increase of 400% stained cells in some cellular subpopulations after ODN 2216 stimulation. Also, in some animals, stimulation with the control ODN 2243 indicated similar staining patterns as PBS, whereas in other cats the induction of effects comparable to those of ODN 2216 could be observed. The response of cells originating from a particular cat to stimulation with ODN 2243 did not however correlate with the influence of ODN 2216 on the expression of either surface molecule on these same cells. Overall, an increased expression of both B7.1 and MHCII was measured in gated PBMCs upon a 24 h stimulation with ODN 2216 when overlaid with the expression of these molecules after ODN 2243 or PBS stimulation (Figure1B and C). Considering all eight cats, lymphocytic cells expressed significantly higher levels of B7.1 and MHCII when exposed to ODN 2216 than after treatment with PBS (p = 0.0195 and p = 0.039 respectively, Figure1D and F). Feline MHCII expression on lymphocytes also seemed to be affected by ODN 2216 and ODN 2243 in a similar manner (Figure1F). For non-lymphocytic cells, ODN 2216 more specifically affected the expression of B7.1 molecules, inducing significant upregulation of surface levels of this molecule when compared to stimulation with ODN 2243 (p = 0.0391) and PBS (p = 0.0039) for 24 h (Figure1E). In contrast, the expression of MHCII was not significantly altered in cells gated as non-lymphocytes at this time point after stimulation (Figure1G). Finally, the cells of the three cats (c08, c09 and c12) that exhibited the highest proliferation rates in response to ODN 2216 (Figure1A) also indicated the strongest increase in expression of both cell surface molecules in all cellular subpopulations tested.\n\nODN 2216 influence type I IFN and proinflammatory gene expression in primary feline immune cells\nThrough interaction with the TLR9, CpG-A typically induce expression of both type I IFN and proinflammatory cytokines in stimulated cells[12]. In order to understand whether ODN 2216 exert similar effects in the cat, treated cultures of feline PBMCs, FEA, CrFK and fcwf-4 cells were systematically screened for increased mRNA expression of IFNα and IL-6 following stimulation. Although all tested immortalized feline cell lines expressed TLR9 mRNA (data not shown), they failed to respond to stimulation with ODN 2216 (Figure3A and B and data not shown). However, this molecule exhibited potent immunomodulatory properties in feline PBMCs: when measured 24 h post stimulation, a concentration of only 1 μg/mL ODN 2216 was sufficient to enhance transcription of IFNα by 40-fold, and a maximum induction of this gene was observed when 4 μg/mL ODN were used (Figure3C). Although affected in a similar pattern, the mRNA expression of the proinflammatory cytokine IL-6 remained comparatively low at all concentrations tested (Figure3D). In experiments foreseen to determine gene expression kinetics in feline cells after stimulation with the ODN 2216 molecule, an influence on IFNα transcription could be measured as early as 3 h after treatment of PBMCs, whereas increased levels of IL-6 mRNA were observed only as of 6 h post stimulation (Figure3E and F). Notably, the highest induction of both genes was measured 24 h after addition of ODN 2216 to the cultures, with transcription increasing by 9000-fold and 39-fold at this time point for IFNα and IL-6 respectively (Figure3E and F). The observed effects were specifically conferred by the CpG motifs comprised in the 2216 ODN, since specific control ODN 2243 only induced slight elevations in the expression of the genes tested (Figure3C-F). Importantly, trypan blue exclusion experiments indicated no evidence of cellular toxicity after treatment neither with ODN 2216 nor with ODN 2243 at all concentrations tested (data not shown).\nFigure 3 ODN 2216 influence IFNα and IL-6 expression in primary feline immune cells. mRNA expression factors of IFNα and IL-6 were measured in (A, B) fcwf-4 cells stimulated with ODN 2216 and (C-F) feline PBMCs from one cat belonging to group 2 stimulated with either ODN 2216 or control ODN 2243. The transcription of both genes was assessed either 24 h after treatment of the cells with increasing concentrations of ODN (A, C, D), or over time after a single stimulation with 4 μg/mL ODN (B, E, F). Experiments C-F were repeated for 4 adult cats and representative results are shown.\n\nODN 2216 broadly influence the gene expression profile in primary feline immune cells of adult cats\nIn an effort to assess both the breadth of the effects conferred by treatment with a CpG-A and possible variability in the responses obtained in individual cats, the mRNA expression of ten genes relevant to early immune responses was measured in ODN 2216 stimulated PBMCs of fourteen cats divided in four different age groups (group 1: 10 weeks (n = 4), group 2: 1.5 years (n = 4), group 3: 7 years (n = 4), group 4: 14 years (n = 2)). Only slight individual variability in gene induction was observed after 24 h stimulation of immune cells from adult cats ranging between 1.5 and 14 years of age (Figure2B-D). Overall, the mRNA expression profile measured in stimulated PBMCs of these cats corroborated their induction of strong antiviral immune responses. The expression of type I IFN mRNA, including IFNα, IFNβ and IFNω was substantially increased in the immune cells subjected to ODN 2216 stimulation from every adult cat, with minimal inductions of 490, 60 and 1600-fold respectively observed in the older animals of group 4 (Figure2D). Moreover, all individual IFNα subtypes tested were induced at similar levels in the cells of four adult cats from group 2 (Figure2E). Increased levels of proinflammatory cytokine mRNA were also measured in most individuals of groups 2–4, with IL-6 more systematically increased than TNFα. The cells from these cats indicated a typical Th1 orientation after stimulation, with enhanced transcription of IL-12 in 9/10 and IFNγ in 6/10 animals, together with absent or only low induction of IL-4. Stimulation of PBMCs with ODN 2216 also created an optimal environment for NK cell activity, as indicated by the increases in mRNA expression of the NK cell stimulator IL-15 by up to 20-fold and of the NK cell effector Granzyme B by up to 7-fold. The cells of those cats (c08, c09, c12) that had most effectively proliferated (Figure1A) and/or exhibited the strongest expression of co-stimulatory molecules (Figure1B-E) following ODN 2216 stimulation also consistently expressed the highest mRNA levels of IFNα, IFNω, IL-6, Il-12, IL-15 and Granzyme B. PBMCs of cat c08 (group 2, Figure1B) were also by far most responsive to stimulation with ODN 2216. When cells from kittens of group 1 were stimulated, higher individual variability was observed than in adult animals (Figure2A). The PBMCs of only 2 out of 4 cats from this group could be stimulated with ODN 2216 to increase mRNA expression of the tested genes. In both individuals, not only the mRNA expression of type I IFN genes was enhanced at much lower levels than observed in adult cats but the overall immune response favored a Th2 direction, characterized by upregulated IL-4 and downregulated IL-12 mRNA levels (Figure2A).\nIn order to determine whether a discrepancy in expression of TLR9 between the PBMCs of adult animals and kittens could play a role in these observations, mRNA levels of this gene were measured in immune cells of each cat. Although basal TLR9 expression was similar in the PBMCs of the cats of all age groups (Figure2F), ODN 2216 stimulation increased TLR9 transcription in the cells of kittens, but decreased transcription of this gene in the cells of adult cats so that differences in the mRNA levels of this receptor were significantly higher in young cats (group 1) than in adult cats of groups 2 and 3 (p = 0.0286) (Figure2G).\n\nODN 2216 induce the production of soluble molecules that activate intracellular antiviral mechanisms in feline target cells\nProtective properties of type I IFN against viruses originate from their ability to trigger the production of potent antiviral proteins in nearby cells. The expression of one of these antiviral proteins, the Mx GTPase, is known to be directly stimulated by type I IFN and can be used as marker for the induction of intracellular antiviral mechanisms by these cytokines in cats as well as in other species[19, 64]. Thus, as detection of feline type I IFN on a protein level is rendered difficult by the unavailability of antibodies specifically recognizing these proteins, mRNA levels of Mx were measured in feline PBMCs after stimulation with ODN 2216, as indication for production of type I IFN. Transcription of Mx was significantly enhanced and remained proportional to mRNA expression of type I IFN in stimulated PBMCs of individual cats (Figure4A). Moreover, peak levels of Mx mRNA were measured 24 h after stimulation of the cells, indicating the presence of optimal effects of the type I IFN present in the cell culture medium at this time point (Figure4B). In order to further determine the potential of the type I IFN liberated by ODN 2216 stimulated PBMCs to induce intracellular antiviral mechanisms in non-immune target cells, cell-free supernatants of PBMCs derived from the blood of individual adult cats and stimulated in vitro for 24 h with ODN 2216, ODN 2243 or endotoxin-free PBS (Sup 2216, Sup 2243 and Sup Neg respectively), were incubated with CrFK and fcwf-4 cells. Mx gene transcription was significantly enhanced in cells treated with Sup 2216 compared to Sup 2243 (p = 0.0078) and Sup Neg (p = 0.0078), at levels comparable to those achieved by stimulation of the cells with 100U recombinant feline IFNα (rfeIFNα) (Figure4C and data not shown). The Sup 2216 derived from cells of individual animals (c08, c09, c12) that had shown particularly strong responses to ODN 2216 in previous experiments also induced the highest Mx mRNA expression in both cell lines tested. Although treatment with Sup 2243 systematically increased Mx transcription in target cells, protein levels remained low (Figure4D and data not shown). Furthermore, peak induction of Mx transcription was observed in both cell lines already within 6 hours of incubation with Sup 2216 (Figure4E and data not shown), while protein levels achieved maximum levels within 24 h post stimulation and remained stable thereafter for at least another 24–48 h (Figure4F and data not shown).\nFigure 4 ODN 2216 induces an antiviral state both in stimulated PBMCs directly and in target cells incubated with supernatants from stimulated PBMCs. (A) mRNA expression factors of the indicated genes were measured in PBMCs of the individual cats (c01-c14) from four different age groups after stimulation with ODN 2216 for 24 h. (B) Mx mRNA expression factors were assessed at the indicated time points in PBMCs of one cat after a single stimulation with either ODN 2216 or ODN 2243. (C) Mx mRNA expression factors were measured in fcwf-4 cells incubated for 24 h with supernatants (Sup 2216, Sup 2243, Sup Neg) derived from PBMCs of eight adult cats (groups 2 and 3) or 100U recombinant feline IFNα (rfeIFNα). (D) Mx protein was detected by Western blot in fcwf-4 cells incubated with the indicated supernatants derived from PBMCs of one cat belonging to group 2 or with 100U recombinant feline IFNα (rfeIFNα) for 24 h. (E) Mx mRNA expression factors were measured in fcwf-4 cells at the indicated time points after a single stimulation with Sup 2216, Sup 2243 and Sup Neg respectively. (F) Mx protein was detected by Western blot in fcwf-4 cells at indicated time points after stimulation with Sup 2216 derived from PBMCs of the same cat (right panel). **p \u003c 0.01. rfeIFNα = recombinant feline IFNα.\n\nODN 2216 inhibit replication of common feline viruses in vitro\nFelids are frequently affected by four viruses of different families: the feline herpesvirus (FHV), calicivirus (FCV), parvovirus (FPV) and coronavirus (FCoV). Although these viruses cannot productively infect purified PBMCs in vitro, they share the ability to induce cytopathic effects (CPE) in CrFK and fcwf-4 cells. These feline cell lines, however, do not alter their mRNA levels of genes selected as markers for innate immunity upon direct treatment with ODN 2216 (Figure3A and B and data not shown), preventing from assessing the potential of this molecule to inhibit viral replication directly in these cells. Thus, CrFK and fcwf-4 cells were incubated, prior to their inoculation, with the cell-free supernatants of PBMCs mentioned above: Sup 2216, Sup 2243 and Sup Neg. To this aim, supernatants derived from PBMCs treated for 24 h were selected, as Sup 2216 produced by the cells of several adult cats were estimated to contain optimal type I IFN amounts at this time point, according to the induction of Mx transcription measured directly in these immune cells (Figure4A and4B). CrFK and fcwf-4 target cells were then incubated with the supernatants for 24 h before inoculation, as this time span had indicated highest induction of antiviral mechanisms (Figure4F). The antiviral effects of the supernatants were initially tested on vesicular stomatitis virus (VSV) as control, as this virus is widely recognized for both its potential to induce CPE in cell lines of multiple species and for its particularly high sensitivity to the effects of type I IFN[65, 66]. When the Sup 2216 derived from the PBMCs of eight adult cats that had broadly responded to in vitro ODN 2216 stimulation (Figure2B and C, groups 2 and 3) were incubated with fcwf-4 cells prior to their inoculation, significant inhibition of VSV replication was observed (p = 0.0039) (Figure5A). The replication of this virus was also to some degree repressed by Sup 2243 (p = 0.0078), an observation reminiscent of the slight induction of Mx in target cells incubated with these supernatants (Figure4C and D). In turn, the propagation of FCV, FCoV, FHV and FPV on fcwf-4 cells was also significantly suppressed by the Sup 2216 when compared to Sup 2243 (p = 0.0039, p = 0.0039, p = 0.0078 and p = 0.0039 respectively) and Sup Neg (p = 0.0039), however with expected lower sensitivity than VSV (Figure5B-E and Table1). Both Sup 2243 and Sup Neg failed to inhibit replication of this heterogeneous group of feline viruses, underlining the essential role of the 2216 molecule in conferring the observed effects. Importantly, cells stimulated with ODN 2216 directly did not indicate any resistance to viral replication, in concordance to their impaired response to this molecule already measured on a genetic level (Figure3A and B and data not shown). With respect to the younger cats, the Sup 2216 of those 2 kittens whose cells responded to ODN 2216 stimulation (Figure2A, group 1) could also inhibit both VSV and FCV on fcwf-4 cells, while the supernatants derived from the PBMCs of the other 2 kittens indicated no inhibition potential on these viruses (Figure5K and L). Altogether, the viral suppression potential of Sup 2216 from all the cats could be compared to that conferred by treatment of the cells with 10 to 100U rfeIFNα, a quantity determined in titration experiments of rfeIFNα conducted together with the viral inhibition assays (data not shown).\nFigure 5 Supernatants derived from ODN 2216 stimulated PBMCs inhibit viral replication in target cells. (A-E) fcwf-4 cells were incubated for 24 h with the indicated supernatants derived from PBMCs of eight adult cats (groups 2 and 3) or medium only as control before inoculation with the indicated viruses. Each dot represents mean optical density (OD) values from spectrophotometric readings of viral inhibition assays conducted on duplicate wells treated with supernatants from an individual cat. (F-J) Correlation of individual inhibition ratios of each virus with Mx mRNA expression induced in fcwf-4 cells incubated with supernatants of ODN 2216 stimulated PBMCs from the eight cats of groups 2 and 3. Note the different scale on the x-axis for each graph indicating the differences in the inhibitory effects of these supernatants on the different viruses. (K, L) fcwf-4 cells were incubated for 24 h with the indicated supernatants derived from PBMCs of 4 kittens (group 1) or medium only as control before inoculation with VSV or FCV. Each dot represents mean OD values from spectrophotometric readings of viral inhibition assays conducted on duplicate wells treated with supernatants from one cat. **p \u003c 0.01. VSV = vesicular stomatitis virus, FCV = feline calicivirus, FPV = feline parvovirus, FCoV = feline coronavirus, FHV = feline herpes virus.\nTable 1 Means of viral inhibition rates measured in fcwf-4 cells after treatment with supernatants derived from stimulated PBMCs of 8 adult cats Depicted are mean viral inhibition rates for 8 cats. Viral inhibition rates for each cat were calculated with the following formula:\nMean optical densityODvalues of duplicate wells treated with SupernatantMean OD values of quadruplicate wells treated with medium alone.\nAbbreviations: VSV Vesicular Stomatitis Virus, FCV Feline Calicivirus, FCoV Feline Coronavirus, FHV Feline Herpesvirus, FPV Feline Parvovirus. The differential inhibition of the viruses tested by the same supernatants is reflected by the distinct viral inhibition ratios observed (Figure5B-E and Table1). Sensitivity of each virus to rfeIFNα correlated with sensitivity to the Sup 2216 and induction of Mx in target cells by the supernatants highly correlated with the inhibition of all viruses (Figure5F-I). Finally, Sup 2216 derived from PBMCs of cats c08, whose cells had indicated strong responsiveness to stimulation with ODN 2216 in previous experiments, most efficiently inhibited the replication of all viruses.\nSimilar results were notably obtained when the supernatants of PBMCs derived from all cats were incubated with CrFK cells prior to their inoculation with all the above-mentioned viruses (data not shown). This cell line has previously indicated less sensitivity to the antiviral effects of type I IFN[66] and generally 10-fold higher amounts of rfeIFNα were found in titration experiments to be required for the inhibition of all five viruses. Concordantly, average fold viral inhibition in CrFK cells by the Sup 2216 was approximately half that observed in fcwf-4 cells.\n\nODN 2216 inhibits replication of a retrovirus in vitro\nThe life cycle of retroviruses is characterized by the reverse transcription of their genomic RNA into DNA and subsequent integration of this viral DNA as provirus into the genome of the host, causing permanent infection most often accompanied by persistent virus production by infected cells. The feline leukemia virus (FeLV), a gammaretrovirus that can be propagated on FEA cells in vitro, affects domestic cats worldwide. As viral replication in chronically infected cats can be lowered by treatment with IFNα[67], the potential of Sup 2216 produced by the PBMCs of five adult cats (c06 and c08 from group 2; c09, c10, c12 from group 3) to inhibit productive infection of FEA cells was analyzed. In initial experiments, this cell line exhibited similar responses as fcwf-4 and CrFK cells to both direct treatment with ODN 2216 and incubation with the different supernatants (Figure3A,4C and D and data not shown). Compared to incubation with medium alone, treatment of FEA cells with Sup 2216 for 24 h prior to inoculation with FeLV followed by repetitive treatments of the cells with this supernatant every 2 days thereafter significantly reduced viral RNA (p \u003c 0.05) and DNA (p \u003c 0.05) measured in the cell culture supernatants and cells respectively as of 4 days post inoculation (Figure6A and B). The antiviral potential of the supernatants from the individual cats was very similar; however the best results were conferred by the Sup 2216 derived from cells of c08 (depicted in Figure6A and B).The kinetic curve of viral RNA loads in cultures of cells treated with Sup 2216 of this cat was similar to those obtained in cells treated with 50U rfeIFNα (data not shown). Also, treatment of the cells with ODN 2216 directly affected neither viral RNA nor viral DNA loads measured in the supernatants and the cells respectively (data not shown). Mx mRNA expression was 80-fold higher in the Sup 2216 treated cells than in all controls 8 days post inoculation, indicating the ability of these supernatants to sustain antiviral mechanisms when applied to the cells repeatedly (Figure6C). Furthermore at this time point, the Sup 2216 treated cells exhibited significantly lower viral DNA loads (p = 0.0313) and produced significantly less virus (p = 0.0313) than cells treated with Sup 2243, Sup Neg or medium alone (Figure6D and E). The extent of Mx transcription conferred by the Sup 2216 of the cats strongly correlated with lower provirus (p = 0.0053) and virus (p = 0.0012) loads measured in the FEA cells and supernatants respectively on day 8 post inoculation (p = 0.0053). Also, the highest Mx mRNA levels in target cells were conferred by Sup 2216 of cat c08 and reflected by the lowest viral and proviral loads measured at this time point in our experiments.\nFigure 6 Supernatants derived from ODN 2216-stimulated PBMCs decrease retroviral DNA and RNA loads in target cells. (A) FEA cells were incubated for 24 h with the respective supernatants or medium alone, before inoculation with the feline leukaemia virus (FeLV), as well as every 2 days thereafter. Viral RNA loads were measured at the indicated time points by real time RT-PCR and 45-Ct values are depicted. (B) FeLV DNA loads in the cells were measured at the indicated time points and Ct values were normalized to detection of a housekeeping gene (GAPDH). Mean values from duplicate experiments carried out simultaneously with the supernatants derived from PBMCs of a selected cat (belonging to group 2) and with medium alone are shown as an example. Results for (A) and (B) are indicative of those obtained with supernatants from PBMCs of two additional adult cats (from group 3). Stars represent statistical differences in area under the curve (AUC) measurements between the curves of all three cats obtained in cells incubated with Sup 2216 and each of the other treatments. Mx mRNA expression factors (C), viral loads (45-Ct values depicted) (D) and proviral loads (E) were measured in the FEA cells of five cats (two from group 2 and three from group 3) on day 8 post inoculation. Each dot represents the mean of duplicate measurements for an individual cat. *p \u003c 0.05, **p \u003c 0.01.\n\nODN 2216 induces an antiviral state in the domestic cat in vivo\nIn a final step, the induction of antiviral responses by ODN 2216 was measured in vivo and characterized using ex vivo assays. The molecule was administered once subcutaneously to two cats (c07 and c08), while two other cats (c05 and c06) received endotoxin-free PBS as a negative control. The injections were well tolerated by all cats: no rise in body temperature, no local reactions at the injection site and no other undesired side effects were noted. Absolute monocyte counts increased 1.5 to 2-fold in the first 12 h in cats that had received ODN 2216 (data not shown). No other treatment-specific alterations were noted in hematological values throughout the experiment. Mx expression was measured in blood at early time points after injection as marker for the induction of antiviral immune processes. mRNA levels of Mx increased by 8.8 and 3.8-fold within 24 h in both treated cats and decreased gradually thereafter until 192 h post treatment (Figure7A). In accordance with observations from in vitro experiments, cat c08 exhibited a stronger response to ODN 2216 than cat c07. No increase in Mx expression was observed in the blood of the control cats (c05 and c06). In a further experiment, plasma was collected from all four cats at regular intervals post injection and was utilized in an in vitro inhibition assay. The plasma obtained from both treated cats 24 and 48 h after administration of the molecule was able to inhibit replication of FCV in vitro (Figure7B). In contrast, plasma from untreated cats seemed to slightly enhance the susceptibility of target cells to FCV inoculation.\nFigure 7 Subcutaneous injection of ODN 2216 induces a systemic antiviral state in vivo. (A) The cats received an injection of either 200 μg/kg ODN 2216 (cats 1 and 2) or endotoxin-free PBS (cats 3 and 4). Mx mRNA expression was measured by qPCR in whole blood. Depicted values represent the ratio of mRNA levels for a given cat (c05-c08) at the indicated time point to mRNA levels for the same cat at time point 0 h. (B) fcwf-4 cells were incubated for 24 h in duplicates with plasma collected from the cats 1 to 4 at the indicated time points post injection, and inoculated with the FCV. Indicated are viral inhibition factors calculated as described in the material and methods section. (C) Mx mRNA expression factors measured in the blood of all four cats at time points 8 h-192 h were correlated with the inhibition of FCV induced in vitro by the corresponding plasma samples.\n","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":3388}},{"label":"Title","span":{"begin":9,"end":151}},{"label":"Section","span":{"begin":3390,"end":6057}},{"label":"Title","span":{"begin":3390,"end":3486}},{"label":"Figure caption","span":{"begin":5472,"end":6057}},{"label":"Section","span":{"begin":6059,"end":9510}},{"label":"Title","span":{"begin":6059,"end":6158}},{"label":"Section","span":{"begin":9512,"end":13408}},{"label":"Title","span":{"begin":9512,"end":9635}},{"label":"Figure caption","span":{"begin":12106,"end":13408}},{"label":"Section","span":{"begin":13410,"end":19975}},{"label":"Title","span":{"begin":13410,"end":13472}},{"label":"Figure caption","span":{"begin":16907,"end":18264}},{"label":"Table caption","span":{"begin":18265,"end":18827}},{"label":"Section","span":{"begin":19977,"end":24190}},{"label":"Title","span":{"begin":19977,"end":20031}},{"label":"Figure caption","span":{"begin":22801,"end":24190}},{"label":"Section","span":{"begin":24192,"end":26739}},{"label":"Title","span":{"begin":24192,"end":24255}},{"label":"Figure caption","span":{"begin":25859,"end":26739}}],"tracks":[{"project":"2_test","denotations":[{"id":"22906110-11763350-143530979","span":{"begin":344,"end":346},"obj":"11763350"},{"id":"22906110-11449369-143530980","span":{"begin":3625,"end":3627},"obj":"11449369"},{"id":"22906110-17683972-143530981","span":{"begin":10041,"end":10043},"obj":"17683972"},{"id":"22906110-16740097-143530982","span":{"begin":10045,"end":10047},"obj":"16740097"},{"id":"22906110-4308914-143530983","span":{"begin":15038,"end":15040},"obj":"4308914"},{"id":"22906110-7515537-143530984","span":{"begin":15042,"end":15044},"obj":"7515537"},{"id":"22906110-7515537-143530985","span":{"begin":19705,"end":19707},"obj":"7515537"},{"id":"22906110-17507184-143530986","span":{"begin":20545,"end":20547},"obj":"17507184"}],"attributes":[{"subj":"22906110-11763350-143530979","pred":"source","obj":"2_test"},{"subj":"22906110-11449369-143530980","pred":"source","obj":"2_test"},{"subj":"22906110-17683972-143530981","pred":"source","obj":"2_test"},{"subj":"22906110-16740097-143530982","pred":"source","obj":"2_test"},{"subj":"22906110-4308914-143530983","pred":"source","obj":"2_test"},{"subj":"22906110-7515537-143530984","pred":"source","obj":"2_test"},{"subj":"22906110-7515537-143530985","pred":"source","obj":"2_test"},{"subj":"22906110-17507184-143530986","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecc793","default":true}]}]}}