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Clin Exp Immunol. Mar 2002; 127(3): 423–429.
PMCID: PMC1906318

The in vivo effects of tumour necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis

Abstract

Anti-TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell-driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF-RI at days 0, 2 and 4 postinduction or 10 mg/kg anti-TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3–4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT-PCR. TNF and IFN-γ protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF-RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF-RI did not attenuate AA, in contrast to an anti-TNF antibody, which suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.

Keywords: adjuvant arthritis, anti-TNF, cell mediated immunity

INTRODUCTION

Anti-TNF therapy has been shown to improve both the clinical and laboratory parameters of rheumatoid arthritis (RA) [1] and has been shown to slow radiographic disease progression [2]. The success of this new treatment for RA, as well as in psoriatic arthritis, anklyosing spondylitis and juvenile chronic arthritis, has established TNF blockade as a new class of therapy for inflammatory arthritis. However, despite the success of this therapy, the mechanisms of action are unclear, and these diseases recur once therapy stops. Understanding the mechanisms of action of TNF-blocking therapy will be essential if the full potential of this new class of therapy is to be realized. Direct inhibition of TNF-mediated mechanisms, encapsulated in the term mesenchymal activity, is most likely inhibited by TNF blockade. Other immune pathways, mediated by T cell-driven mechanisms, are also thought to play an important role in the activity of inflammatory arthritis [3]. The effects of TNF blockade on this pathway are unclear.◊In vitro and in vivo studies have suggested that TNF may inhibit T cell function, with the suggestion that blockade of TNF bioactivity would release T cells from this inhibition [4]. Investigation of the effects of TNF blockade on T cell function in vivo is now needed to clarify this question.

Adjuvant arthritis is a commonly used T cell dependent model of inflammatory arthritis, with an incidence of around 90%, making it an ideal model in which to investigate prearthritic changes. We have previously demonstrated increased cytokine expression (TNF, IFN-γ and IL-17) in the inguinal lymph nodes of rats with adjuvant arthritis 6–9 days after adjuvant injection, prior to arthritis onset [5].

To date, studies of TNF-blocking treatment of adjuvant arthritis have investigated treatment of the later arthritic phase. Anti-TNF monoclonal antibody treatment given on day 16 improved clinical scores and was paralleled by inhibition of leucocyte accumulation in the joints [6]. Treatment with PEGylated soluble TNF receptor type 1 (PEG sTNF-RI) on days 9, 11 and 13 of adjuvant arthritis significantly inhibited joint swelling, inflammation and bone resorption [7].

This study used TNF blocking treatments to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph node activation phase of early adjuvant arthritis.

MATERIALS AND METHODS

Animals

Male Dark Agouti (DA) rats 4–6 weeks old weighing approximately 150–200 g (Gilles Plains, South Australia) were housed in cages lined with cellulose bedding and shredded paper in a temperature controlled room (22 ± 1°C) with a 12- h alternating light and dark cycle. Animals were given food (rat chow, Gordon's Speciality Stockfeeds, Yandera, Australia) and water ad libitum. All experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales, Sydney, Australia.

Induction of adjuvant arthritis

Arthritis was induced with complete Freund's adjuvant (0·1 ml of 10 mg/ml heat killed and dried Mycobacterium butyricum suspension in paraffin oil and mannide monoleate, Difco Laboratories, Detroit, Michigan, USA) injected intradermally into the tail base under anaesthesia with ketamine (50 mg/kg i.p.; Parnell Laboratories, Alexandria, Australia) and xylazine (5 mg/kg i.p.; Troy Laboratories, Smithfield, Australia).

Anti-TNF compounds

The sheep antimouse TNF polyclonal antibody was a gift from Prof Gisa Tiegs, Germany and the PEGylated soluble TNF receptor 1 (PEG sTNF-RI) was a gift from Amgen Inc, CO, USA.

Experimental protocol

PEG sTNF-RI treatment in rat adjuvant arthritis – Experiment 1. Arthritis was induced in a group of 65 male DA rats. Rats received 3 mg/kg s.c. of PEG sTNF-RI, a dose previously shown to suppress established arthritis [7], on days 0, 2 and 4. Control rats received the same volume of saline. Day 0 rats did not have adjuvant arthritis induced. Rats were sacrificed on day 0 (n = 5), day 6, when our previous studies showed maximal inguinal lymph node cytokine mRNA production [5] (n =10 treated, 10 controls), day 13, time of maximal synovial T cell cytokine mRNA expression [5] (n =10 treated, 10 controls) and day 21 (n = 10 treated, 10 controls) postadjuvant. The synovium from the right ankle joint and inguinal lymph nodes were removed, placed in cryotubes and immediately snap frozen in liquid nitrogen and stored at – 70°C for subsequent RNA extraction. For day 6 rats, one inguinal lymph node was removed for RNA extraction and the other was used for cell culture. The left paw was removed for radiology and histology analysis.

PEG sTNF-RI treatment in rat adjuvant arthritis – Experiment 2. To investigate if the early treatment effect on adjuvant arthritis was dose dependent, a group of rats were treated with a higher dose of PEG sTNF-RI. Arthritis was induced in a group of 20 male DA rats. Rats received 10 mg/kg PEG sTNF-RI (n =10) or saline (n =10) s.c. on days 0, 2 and 4 post induction. Rats were sacrificed on day 21 postadjuvant and the left paw was removed for radiology and histology analysis.

Anti-TNF polyclonal antibody treatment in rat adjuvant arthritis

Arthritis was induced in a group of 20 male DA rats. Rats were given one dose of 500 μl (10 mg/kg) i.p. polyclonal sheep antimouse TNF antibody at the time of induction of arthritis. Control rats received the same concentration of normal sheep serum (heat inactivated) diluted in PBS. Rats were sacrificed on day 21 postadjuvant and the synovium from the right ankle joint and inguinal lymph nodes were removed and immediately snap frozen in liquid nitrogen and stored at – 70°C for subsequent RNA extraction. The left paw was removed for radiology and histology analysis.

Assessment of arthritic damage

The disease progression was monitored from the induction of arthritis (day 0). Rats were euthanased using pentobarbital (60 mg i.p.; Lethobarb; Virbac, Sydney, Australia). Oedema of both ankles was measured every 3–4 days by plethysmometry (Ugo Basile, Comerio, Italy). Following sacrifice, the left ankle was removed for quantitative radiography and histology. Briefly, radiographs were scored 0–3 for soft tissue swelling, joint space loss and joint damage, range 0–9. This is a simplified version of the method originally described by Ackerman and colleagues [8] and utilized by us previously [5,9]. Following radiography, paraffin sections were prepared for histological analysis as described previously [9]. Each section was evaluated for periarticular inflammation and pannus formation using a scale of 0–8 (maximum score 16)[8].

Reverse transcriptase-Polymerase chain reaction (RT-PCR)

Samples were processed simultaneously, as previously described [10], at each step of the RT-PCR process to minimize experimental variation. Frozen synovial membrane and inguinal lymph nodes tissues were crushed and homogenized in liquid nitrogen on ice and total RNA extracted using the method of Chomczynski and Sacchi [11] and cDNA was prepared as previously described [5,12]. To minimize variability due to experimental processing, cDNA for each tissue (inguinal lymph node, synovium) of all rats was made on the same day, and then stored at – 20°C.

PCR was performed using primers for IFN-γ, IL-2, IL-4, TNF, TGF-β and IL-17 as previously described [5]. Four hour concanavalin A stimulated inguinal lymph node cells were used as the positive control as they express high levels of the cytokines. Negative controls contained all components of the reaction mix except cDNA. β-Actin expression confirmed that the RNA was not degraded.

Synovial and inguinal lymph node samples were all amplified in the same PCR reaction for each tissue, at the optimal cycle number for a specific cytokine. Synovial and inguinal lymph node samples were amplified at different cycle numbers, therefore expression of a cytokine in the two tissues cannot be directly compared; however, changes in cytokine expression over time for each tissue are directly comparable.

PCR product analysis

PCR products and molecular weight markers were separated in a 2% agarose gel (ICN, Ohio, USA) using a Tris-borate buffer. Gels were stained with ethidium bromide and photographed with Polaroid 665 film (Polaroid, Hertfordshire, UK) using UV illumination. Density of bands on the negative images of the gels were determined using densitometry (GS-700 densitometer, Biorad Laboratories, California, USA) and computer analysis (Molecular Analyst Software, Biorad Laboratories, California, USA), with sample densities normalized for background. Sample PCR product values were expressed as a percentage of the density of positive control samples amplified simultaneously in the same PCR reaction.

Inguinal lymph node culture

Inguinal lymph nodes were dissected on days 0 and 6 postinduction of adjuvant arthritis in rats treated with either 3 mg/kg PEG sTNF-RI or saline. A single cell suspension was prepared and cells were cultured as previously described [5]. Cell supernatants were collected after 48 h for protein analysis by bioassay and ELISA.

WEHI 164 bioassay (MTS cytotoxicity assay)

Biologically active TNF was detected in the WEHI 164 murine fibrosarcoma cytotoxicity assay as described previously [5,13]. The WEHI cell line was obtained from Dr Robert Miller (Peptech, Sydney, Australia). The lower limit of sensitivity of the bioassay was 5 pg/ml. Both the PEG soluble human TNF-RI and sheep antimouse TNF antisera were tested in the WEHI 164 bioassay to confirm that they blocked rat TNF. Various concentrations of each compound were added to wells containing 2500 pg/ml recombinant rat TNF in the bioassay and the amount of bioactive TNF remaining was determined. Levels of lymphotoxin (LT) were determined by subtracting the levels of TNF detected when the supernatants were incubated with PEG sTNF-RI (blocks both TNF and LT) from the level detected when the supernatants were incubated with anti-TNF antisera.

IFN-γ ELISA

Secreted rat IFN-γ protein from inguinal lymph node cell cultures was measured using a commercial rat IFN-γ immunoassay kit (Endogen, MA, USA). The lower limit of sensitivity of the assay was 2 pg/ml.

Serum PEG sTNF-RI

Blood samples were collected from each of the rats on the day of sacrifice, centrifuged and serum frozen (– 20°C) and then analysed using an ELISA at Amgen Inc. The sensitivity of the assay was 0·49 ng/ml [14].

Statistical analysis

Data are presented as mean ± standard error of the mean (s.e.m.). Raw scores for both left and right paw volumes were normalized as percentage change from day 0. Differences between means were calculated using two factor repeated measures anova's. If a significant difference was found (P < 0·05), posthoc analysis was performed on planned comparisons using Fisher's LSD multiple comparison tests (Number Cruncher Statistical System, NCSS, Kaysville, Utah, USA). For the radiology, histology and T cell data, two factor anova's or unpaired T-tests were used to compare means. For the day 0 and 6 ILN cytokine mRNA and protein data, differences between means were calculated using one-way anova's. If a significant difference was found (P < 0·05), posthoc analysis was performed on planned comparisons using Fisher's LSD multiple comparison tests (Number Cruncher Statistical System, NCSS, Kaysville, Utah, USA).

RESULTS

Effect of PEG sTNF-RI treatment on rat adjuvant arthritis

In the control rats, paw volume significantly increased, compared with preimmunization paw volumes, from days 13–21 (P < 0·05) (Fig. 1). Doses of 3 mg/kg and 10 mg/kg PEG sTNF-RI did not prevent the development of adjuvant arthritis. In the 3 mg/kg treated group there was a significant increase in paw volume at days 17 and 21 compared with controls (P < 0·05; Fig. 1a). There were no significant improvements in radiology or histology scores at days 6, 13 or 21 for either dose. Day 21 data is shown on Fig. 2a,b. All arthritic animals groomed themselves well and maintained their body weight throughout the study period.

Fig. 1
Percentage change in paw volume from day 0 in rats with adjuvant arthritis. Rats were injected with either (a) 3 mg/kg PEG sTNF-RI (•) or saline (control, ○) (n =10–35 in each group), (b) 10 mg/kg PEG sTNF-RI (•) or saline ...
Fig. 2
(a) Histology scores and (b) radiology scores on day 21 in rats with adjuvant arthritis. Rats were injected with either 3 mg/kg PEG sTNF-RI, 10 mg/kg PEG sTNF-RI or 10 mg/kg polyclonal sheep antimouse TNF antibody. Control rats received either saline ...

Effect of polyclonal anti-TNF antibody treatment on rat adjuvant arthritis

In the untreated control rats, paw volume significantly increased at days 13, 17 and 21 compared with day 0 preimmunization paw volumes, P < 0·05 (Fig. 1c). Anti-TNF antisera (10 mg/kg) significantly decreased paw volume compared with control rats at days 13, 17 and 21 (P < 0·05) (Fig. 1c) and significantly decreased both radiology and histology scores compared with untreated control rats at day 21 (Fig. 2a,b). All arthritic animals groomed themselves well and maintained their body weight.

Cytokine mRNA expression in the inguinal lymph nodes of PEG sTNF-RI treated and untreated rats in early adjuvant arthritis

There were no significant differences in TNF, IFN-γ, IL-17 or IL-2 mRNA expression between day 0 and 6 of adjuvant arthritis in the untreated rats (Table 1). There were significant increases in TNF and IL-2 mRNA expression at day 6 in the PEG sTNF-RI treated rats compared with untreated rats. IFN-γ and IL-17 mRNA expression was also increased at day 6 in the treated rats; however, this increase was not significant (P > 0·05).

Table 1
Cytokine mRNA expression (as a percentage of positive control) in inguinal lymph nodes at day 0 and 6 of adjuvant arthritis

TNF protein in inguinal lymph node cell cultures from rats treated with PEG sTNF-RI

Both the PEG sTNF-RI (500 ng/ml) and the anti-TNF antisera (20μg/ml) completely blocked rat TNF in the WEHI 164 bioassay. TNF was detected in inguinal lymph node cell cultures from rats sacrificed at day 0 and day 6 in both treated and untreated rats (Table 2). In day 6 untreated rat inguinal lymph node cultures, TNF levels were significantly higher than day 0 lymph node cultures. TNF levels in day 6 lymph node cultures from rats receiving PEG sTNF-RI treatment were significantly reduced compared to the day 6 levels from untreated rats (P < 0·05). There was a trend for increased TNF production in day 6 treated rat inguinal lymph node cultures compared with day 0 rat inguinal lymph node cultures (P =0·08). All bioactivity detected was blocked by the sheep antimouse polyclonal antibody and PEG sTNF-RI, confirming that it was TNF, not LT.

Table 2
TNF and IFN-γ protein (pg/ml) measured by ELISA in inguinal lymph node cells cultured at day 0 and 6 of adjuvant arthritis

IFN-γ protein in inguinal lymph node cell cultures from PEG sTNF-RI treated and untreated rats in early adjuvant arthritis

IFN-γ was detected in inguinal lymph node cultures from rats sacrificed at day 0 and day 6 in both PEG sTNF-RI treated and untreated rats (Table 2). There were no significant differences between day 0 and day 6 cultures (P > 0·05) or between treated and untreated rats (P > 0·05).

Serum PEG sTNF-RI levels

Serum sTNF-RI levels in the 3 mg/kg PEG sTNF-RI treated rats increased to 4407 ± 337 ng/ml at day 6, fell to 2 ± 0·2 ng/ml by day 13 and were back to zero by day 21. Serum sTNF-RI levels in the 10 mg/kg PEG sTNF-RI treated rats were zero at day 21.

DISCUSSION

This study has shown that in early adjuvant arthritis, treatment with 3 mg/kg PEG sTNF-RI resulted in increased IL-2 and TNF mRNA expression in the inguinal lymph nodes, with reduced protein production by lymph node cells after 48 h in culture. PEG sTNF-RI treatment, when given over the first 4 days of adjuvant arthritis, does not prevent disease development. In contrast, an anti-TNF polyclonal antibody significantly attenuated adjuvant arthritis.

Although the main aim of this study was to investigate the effect of TNF blockade on T cell cytokine expression and to investigate the role of TNF in the inguinal lymph node, the effects of the two different therapies on disease progression requires further comment. After treatment with 3 mg/kg PEG sTNF-RI paw volume was increased. This dose has previously been shown to attenuate adjuvant arthritis when given on days 9, 11 and 13 [7]. This lack of efficacy was not just a dose related effect, as 10 mg/kg PEG sTNF-RI given in a subsequent study also had no effect on disease development. In contrast, 10 mg/kg anti-TNF polyclonal antibody significantly reduced paw volume, histology and radiology scores.

Collagen-induced arthritis in mice is ameliorated when animals are given anti-TNF antibodies [1519] or soluble TNF receptor/Fc fusion proteins [20,21] before onset of disease. In contrast to earlier studies, a recent study of collagen-induced arthritis demonstrated similar results to those found in this study. Adenovirus-mediated transfer of a modified tumour necrosis factor receptor (p55) and soluble human TNFR Ig given at the onset of collagen-induced joint swelling reduced severity of disease, but exacerbated disease after day 12. In contrast, anti-TNF antibody treatment reduced disease severity throughout the time course [22]. In addition, similar studies have found variable results [23,24].

Why is there a difference between these two treatments? We have shown that both the anti-TNF antibody and PEG sTNF-RI block rat TNF activity in a WEHI 164 bioassay. The doses of PEG sTNF-RI may not have been large enough to block all TNF produced early in disease. However, the serum levels of PEG sTNF-RI detected in our study were similar to earlier reports for the 3 mg/kg dose, which attenuated adjuvant arthritis [14], with relatively high levels of PEG sTNF-RI still detected at day 6.

We hypothesize that differences in the half-lives of the two compounds may explain our results. As the half-life of PEG sTNF-RI in rats is 20 h [7], serum levels of PEG sTNF-RI were probably below therapeutic levels (300 ng/ml) by days 8–9. PEG sTNF-RI would have been at high levels in the early induction phase of adjuvant arthritis, when cytokine expression in the inguinal lymph node is increased [5]. This suggests that PEG sTNF-RI did not have an effect on the arthritis-inducing events in the inguinal lymph node early in adjuvant arthritis. The levels of PEG sTNF-RI would have fallen below therapeutic levels at the onset of synovial inflammation. The half-life of the anti-TNF antibody is probably considerably longer than that of PEG sTNF-RI. In rats, the half-life of human monoclonal antibodies was found to range between 12 and 16 days [25,26]. If the half-life of the antibody used in this study was similar, then therapeutic levels would have been in the serum well beyond the onset of synovial inflammation. Our results cannot distinguish if the polyclonal antisera also inhibited the early phase or if it was solely preventing joint inflammation later in the disease. It is possible that cell-mediated immune reactions in the inguinal lymph node may be a TNF-independent process, relying on other mechanisms such as cell–cell contact, whereas joint inflammation may be TNF-dependent.

Alternative explanations may also contribute to the observed difference. In the study by Quattrocchi et al. mentioned previously, adTNFR was given continuously in collagen-induced arthritis, suggesting that the arthritis flared despite therapeutic levels of the adTNFR [22]. These authors suggest that mice may have developed agonistic antibodies to the p55 TNFR, which could cross-link the TNFR and hence mimic the pro-inflammatory actions of TNF. Similarly, antibodies to PEG sTNF-RI may have been produced in the rats in this study. These antibodies may then either act against endogenously produced soluble TNF receptors, which regulate TNF, resulting in increased free TNF, or could activate membrane-bound TNF receptors, resulting in increased pro-inflammatory actions. The exacerbation of arthritis with PEG sTNF-RI treatment in this study is an interesting observation that warrants further investigation.

Studies of the binding characteristics of etanercept, a p75 TNF receptor/Fc fusion protein, and infliximab, a chimeric anti-TNF monoclonal antibody to both soluble TNF and membrane-bound TNF, demonstrate that infliximab dissociated from soluble TNF and cell-surface TNF more slowly than etanercept, suggesting that infliximab may have longer-lasting inhibitory properties [27].

The inguinal lymph nodes are an important site of cell-mediated immune activation in adjuvant arthritis. Although increases in mRNA expression of TNF, IFN-γ, IL-17 and IL-2 at day 6 in the inguinal lymph nodes compared with day 0 were not significant, increases at this time are consistent with previous studies [5]. In PEG sTNF-RI treated rats, expression of TNF and IL-2 mRNA was significantly increased in the inguinal lymph nodes at day 6 compared with untreated rats, with IFN-γ and IL-17 expression also increased, but not significantly. These findings support the suggestion that TNF blockade does not suppress T cell activity and may actually increase T cell activity [4,2830]. In studies of TNF blockade in collagen-induced arthritis, gene delivery of a human p55 TNFR-IgG fusion protein did not alter T cell proliferative responses to type II collagen [22]. However, anti-TNF monoclonal antibody treatment [31,32] and gene transfer of sTNFR-Ig [23] have been shown to inhibit the T cell response to type II collagen in cultured lymph node cells. In contrast to these previous studies, the results presented here provide a direct measure of in vivo T cell function with TNF blockade.

Significant differences are difficult to demonstrate between groups with small numbers as in this study. The experimental design requiring lymph node cultures from individual rats on the day of sacrifice was the reason for the small numbers in each group. The lymph node cells were cultured to determine changes in cytokine protein production in these tissues to complement the mRNA data. These protein culture results differed from the mRNA data for TNF, with no differences in IFN-γ protein production between PEG sTNF-RI treated and untreated rats.

Production of TNF protein by the inguinal lymph node cultures was reduced in the PEG sTNF-RI treated rats, whereas the mRNA expression of TNF was increased. TNF mRNA expression may have been up-regulated as a result of blocking TNF protein. The reduced levels of TNF may have been due to excess PEG sTNF-RI in the inguinal lymph node cultures, possibly bound to cell-surface TNF, or post-translational control of TNF protein production in the PEG sTNF-RI treated rats. The autocrine effects of TNF on TNF gene expression and protein synthesis are unclear from the literature. Synovial membrane TNF protein production is reduced in patients with RA [33,34] and in collagen-induced arthritis by TNF-blocking treatment [31]. However, the in vivo effects of this treatment on TNF mRNA expression has not been reported. It must also be noted that the mRNA data was derived from inguinal lymph node tissue prior to the 48 h culture period. The culture conditions involve preparation of single cell cultures with major disruption to the in vivo cellular distribution.

In summary, TNF blockade did not suppress mRNA expression of T cell cytokines in the inguinal lymph nodes of rats in the early phase of adjuvant arthritis, suggesting ongoing T cell activity. TNF protein production by inguinal lymph node cells in culture was reduced in treated rats, although mRNA expression in the lymph nodes was increased prior to culture. PEG sTNF-RI treatment, when given over the first 4 days of adjuvant arthritis, did not attenuate disease, in contrast to an anti-TNF polyclonal antibody, which significantly suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.

Acknowledgments

This study was funded by grants from the National Health and Medical Research Council of Australia (97851 to JSW), ARC and Glaxo Wellcome (to KB).

The authors thank Jan Frazier (Amgen Inc.) for performing the serum PEG sTNF-RI assay, Angelina Enno for her help with the immunohistochemistry, Gisa Tiegs for the donation of the sheep antimouse polyclonal antibody and Amgen Inc. for the kind donation of PEG sTNF-RI.

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