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Centre for Infectious Diseases and International Health, Royal Free and University College Medical School
Departments of Thoracic and HIV Medicine and Radiology, Royal Free Hospital NHS Trust, London, United Kingdom
ABSTRACT
Rationale: Tuberculosis progresses despite potent Th1 responses. A putative explanation is the simultaneous presence of a subversive Th2 response. However, interpretation is confounded by interleukin 42 (IL-42), a splice variant and inhibitor of IL-4. Objective: To study levels of mRNA encoding IL-4 and IL-42, and their relationship to treatment and clinical parameters, in cells from lung lavage and blood from patients with pulmonary tuberculosis. Methods: IL-42, IFN-, IL-4, and soluble CD30 (sCD30) levels were measured by polymerase chain reaction and relevant immunoassays in 29 patients and matched control subjects lacking responses to tuberculosis-specific antigens. Results: mRNA levels for IL-4 and IL-42 were elevated in unstimulated cells from blood and lung lavage of patients versus control subjects (p < 0.005). In control subjects, there were low basal levels of IL-4 and IL-42 mRNA expressed mainly by noneCT cells (p < 0.05). However, in patients, there were greater levels of mRNA for both cytokines in both T- and noneCT-cell populations (p < 0.05 compared with control subjects). Radiologic disease correlated with the IL-4/IFN- ratio and sCD30 (p < 0.005). After chemotherapy, IL-4 mRNA levels remained unchanged, whereas IL-42 increased in parallel with IFN- (p < 0.05). Sonicates of Mycobacterium tuberculosis upregulated expression of IL-4 relative to IL-42 in mononuclear cell cultures from patients (p < 0.05). Conclusions: A Th2-like response, prominent in T cells and driven by tuberculosis antigen, is present in tuberculosis and modulated by treatment, suggesting a role for IL-4 and IL-42 in the pathogenesis of tuberculosis and their ratio as a possible marker of disease activity. The specific antigens inducing the IL-4 response require identification to facilitate future vaccine development strategies.
Key Words: cytokines; human; interleukin 4; Th1/Th2 cells; tuberculosis
Immunity to human mycobacterial disease is known to require tumor necrosis factor and a Th1 response because genetic defects in signaling by IFN- or interleukin 12 (IL-12) lead to susceptibility (1, 2), and neutralization of tumor necrosis factor reactivates latent infection (3). Nevertheless, it is becoming evident that this is not the complete answer. First, most individuals with tuberculosis (TB) have strong antigen-specific Th1 responses (4, 5), particularly at the site of disease (6). Second, bacille Calmette-Gueerin, a predominantly Th1-inducing vaccine, has poor protective efficacy (7), and recombinant bacille Calmette-Gueerin strains or adjuvants that evoke still stronger Th1 responses do not increase vaccine efficacy (8eC10). Third, data relating to genetically modified strains of Mycobacterium tuberculosis (11) suggest that relatively few Th1 cells are sufficient to control proliferation, and that the remaining 90% may be more concerned with immunopathology. Indeed, immunopathology is essential to the pathogenesis of M. tuberculosis because cavities opening into bronchi allow the dissemination of bacilli by coughing.
These and other considerations have led several authors to postulate that something else is involved in addition to the production of IL-12, IFN-, and tumor necrosis factor . There are two logical possibilities: either immunity requires some additional Th1-associated activity, such as an unidentified macrophage function with variable interindividual efficacy (12), or immunity mediated by the Th1 response is only effective in the absence of another corrupting influence. Many authors have suggested that this corrupting influence might be a form of Th2-like response (reviewed in Reference 13), because IL-4 downregulates inducible nitric oxide synthase (14), drives an inappropriate alternative form of macrophage activation (15), and, in animal models (16, 17), can contribute to tissue damage and fibrosis. Moreover, recent human data suggest that those with preexisting lymphocyte IL-4 responses have a high rate of progression to active TB (18).
However, our tenuous understanding of the role of IL-4 production in human TB is confounded by the fact that earlier studies did not distinguish between the agonist IL-4 and its splice variant (19) and antagonist (20), IL-42, resulting in data that are difficult to interpret. The two forms can easily be distinguished by reverse transcriptaseeCpolymerase chain reaction (RT-PCR). Using this method, we previously established that, whereas in asthma there is a selective increase in expression of IL-4, indicating a "classical" Th2 response (21), in TB there is increased expression of both IL-4 and IL-42 in peripheral blood mononuclear cells (PBMCs) (22). Because IL-42 is an antagonist, it was also interesting to discover that individuals with latent TB, which does not progress to active disease, have increased levels of expression of IL-42 (but not of IL-4) (23, 24).
In this study, we ask fundamental questions about expression of IL-4 and IL-42 by cells in peripheral blood and bronchoalveolar lavage (BAL) from patients with pulmonary TB (PTB). To clarify further the causeeCeffect relationship of IL-4, we studied antigen-driven cultures and the effect of anti-TB treatment. This report adds to the available murine data (16, 17, 25), which suggest that effective vaccines against TB might need to concentrate on elimination of the IL-4 response, rather than on boosting the Th1 response, which develops spontaneously as a consequence of infection. Future work will need to elucidate which specific antigenic components of M. tuberculosis are responsible for driving the IL-4eCsecreting T cells. These components could be combined with adjuvants capable of converting the response to Th1 or of driving a regulatory T-cell response. Some of the work presented here has previously been reported in the form of an abstract (26).
METHODS
Patients and Samples
Twenty-nine patients with culture-positive, HIV-negative (three ELISAs on two samples) PTB, 15 of whom underwent BAL, were recruited in London, United Kingdom. All patients with PTB had pan-sensitive isolates, received standard short-course chemotherapy (6eC9 months), and responded clinically and radiologically to anti-TB treatment. Control donors (n = 21) were healthy volunteers matched to the first 21 sequentially recruited patients with TB for age (within 4 years), sex, and ethnicity. They were asymptomatic, had no risk factors for HIV infection (but were not formally tested), had normal chest radiographs, and were selected for high risk of exposure to M. tuberculosis (health care workers and household contacts of patients with PTB). Six of these volunteers underwent BAL. Antigen-specific IFN- responses were used to exclude latent infection in the control subjects (27, 28).
Whole blood (2.5eC20 ml) was taken, after informed consent, within the first 2 weeks of anti-TB treatment (baseline), and 2.5 ml was immediately transferred from the patient into PAXgene-RNA tubes (Qiagen Ltd., Crawley, West Sussex, UK) to fix the mRNA profile (29). The remaining blood was used for further experiments. Ten donors with PTB provided blood again after 3 months or within 4 weeks of stopping chemotherapy. More detailed methods and manufacturers' details for experimental reagents used are provided in an online supplement. Approval was obtained from the relevant hospital ethics committees.
BAL and Radiographic Scoring
BAL fluid, obtained from a radiologically affected lung segment, was concentrated approximately 10-fold before analysis, and cell pellets were fixed in RNA stabilization buffer. In control donors, the right middle lobe was lavaged. To determine the extent of pretreatment radiologic disease, two radiologists blinded to patient details scored chest radiographs for airspace shadowing, reticular opacities, and cavitation.
Cell Separation/Flow Cytometry
For cell subpopulation studies, PBMCs from 10 patients and matched control subjects were enriched for CD3+, CD3+CD4+, and CD3+CD8+ T cells, and depleted of CD3+ T cells (for noneCT-cell fraction), after checking cell viability with trypan blue, by using enrichment reagents according to the manufacturer's directions (StemCell Technologies, Vancouver, BC, Canada). Purity of cell subpopulations in whole blood and lymphocyte counts in BAL were confirmed by flow cytometry (104 gated events) after staining approximately 7.5 x 105 cells with anti-CD4 fluorescein isothiocyanate, anti-CD8 phycoerythrin and anti-CD3 peridinin chlorophyll protein antibodies (BD Tritest; BD Biosciences, Oxford, UK) and fixation in 1% formaldehyde. Mean cell purities of the relevant whole blood fractions were greater than 95, 99, 93, and 90% for T, total non-T, CD4+, and CD8+ fractions, respectively.
Enzyme-linked Immunospot and ELISA Assays
T-cell IFN- enzyme-linked immunospot (ELISPOT) assay responses to early small antigenic target 6 and culture filtrate protein 10 peptide pools were determined to exclude latent TB infection (T SPOT TB; Oxford Immunotec, Oxford, UK). IL-4 production was detected by ELISPOT (significant if > 2 SD above the mean of unstimulated wells) on stimulated and unstimulated PBMCs, noneCT cells, T cells, and T-cell subfractions. Soluble CD30, IL-4, and IFN- were measured by ELISA in plasma and BAL fluid. The sensitivity of the assays was 0.33 U/ml, 0.5 pg/ml, and 5 pg/ml, respectively.
PBMC Culture
PBMCs from eight patients with PTB were cultured over 6 days and nonadherent cells harvested every 2 days; proliferation assays were performed on Day 5. Cells were treated with whole cell sonicates of H37RV M. tuberculosis, phytohemagglutinin, an environmental mycobacterium (Mycobacterium vaccae National Collection of Type Cultures 11659), or medium alone.
RT- and Real-Time PCR
RNA was isolated from whole blood and from lavage cell pellets or cells using the PAXgene and RNeasy kit, respectively. RT- and real-time PCR for IL-4, IL-42, and IFN- were performed on samples, as previously described, after quality control of RNA templates (30). The mRNA values were normalized to a validated housekeeping gene, human acidic ribosomal protein (HuPO; Table 1) (30).
Data Analysis
Data were analyzed using the Mann-Whitney U test, Wilcoxon matched pairs test, Spearman rank sum correlation, and linear regression on logged data. Cytokine ratios were expressed as log differences between respective cytokine pairs.
RESULTS
Clinical and Baseline Parameters
Demographic details of matched patients and control subjects (n = 21) and BAL cell counts are shown in Table 2. When all patients with TB (n = 29) were compared with control subjects, there were no differences in baseline characteristics. None of the study subjects had elevated eosinophil counts and all control patients had normal chest radiographs and were not latently infected with M. tuberculosis by antigen-specific IFN- assay.
Th2 Markers in Whole Blood and BAL at Baseline
Both IL-4 and IL-42 mRNA levels (mRNA copies per 106 copies HuPO) were significantly elevated in whole blood and lung lavage of patients with PTB versus control subjects (p < 0.005 for IL-4 and IL-42; Figures 1A and 1B); all median and 25theC75th percentiles are shown in Table 3. Levels of IL-4 and IL-42 were similar when whole blood and BAL were compared in paired samples. However, compared with control subjects, IFN- was massively upregulated in BAL (p = 0.0007) but not in whole blood (Figure 1C). Furthermore, sCD30 was elevated in the plasma (p = 0.0025) and BAL fluid (p = 0.01; Figure 2) of patients with PTB versus control subjects.
In whole blood, there was a significant correlation between the PTB total radiographic score and the IL-4/IFN- ratio (p = 0.0034; Figure 3A) and a negative correlation with IFN- (p = 0.005). In BAL, there was a significant correlation between the PTB total radiographic score and sCD30 levels (p = 0.0001; Figure 3B).
Cell Subpopulations in Whole Blood Making mRNA Encoding IL-4 and IL-42
The IL-4 and IL-42 mRNA levels in CD3+ T-cell fractions were significantly higher in patients with PTB than in control subjects (p = 0.01 for IL-4 and p = 0.02 for IL-42; Figures 4A and 4B, respectively; median 25theC75th percentiles shown in Table 3 and flow cytometric dot plots for T-cell and noneCT-cell purities are shown in Figures 4C and 4D, respectively). Patients with PTB had higher absolute levels of IL-4 and IL-42 mRNA in both T cells (IL-4 25-fold and IL-42 100-fold greater) and noneCT cells (IL-4 6-fold and IL-42 100-fold greater). For a given production by noneCT cells, the T cells of patients with TB produced significantly more IL-4 than those in control subjects (p = 0.02; data not shown); T cells contained 25% of the IL-4 mRNA in TB.
By contrast, in healthy, exposed volunteers, both IL-4 and IL-42 were produced mainly by noneCT cells compared with T cells (p < 0.05; Figures 4A and 4B); 93% of IL-4 mRNA in control patients was made by noneCT cells. Furthermore, because the T-cell fraction was approximately 95% pure, it is possible that the expression of IL-4 by T cells of control subjects is even lower.
The increase in IL-4 mRNA in T cells from patients with PTB was present both in CD4+ T cells (IL-4 mRNA 7-fold greater in TB) and CD8+ T cells (IL-4 mRNA 10-fold greater in TB). However, the increase in IL-42 mRNA in T cells from patients with TB was confined to CD4 T cells ( 500-fold more than in control subjects). There was no difference in IL-42 expression in the CD8+ T cells from patients with PTB and control subjects.
IL-4 and IL-42 in Response to Anti-TB Treatment
After the intensive phase of short-course chemotherapy (Month 3, n = 5), there was no significant change in IL-4 or IL-42 mRNA levels (data not shown). However, 6 months or more after initiation of short-course chemotherapy (n = 10), there was a nonsignificant decrease in IL-4 mRNA expression, which was still higher than in matched control subjects (p = 0.03; Figure 5A). By contrast, there was a significant increase in IL-42 (p = 0.02; Figure 5B) and IFN- mRNA levels (p < 0.0001) after treatment. Furthermore, the median IL-4/IL-42 ratio and the IL-4/IFN- ratio (expressed as log differences between cytokine pairs) significantly decreased 6 months or more after initiation of chemotherapy (1 vs. 0.4 for the former, p = 0.04, and 2.3 vs. 0.3 for the latter, p < 0.0002).
ELISPOT Assays
PBMCs from control donors cultured with ESAT-6 and CFP-10 yielded median (range) IFN- spot counts (per million PBMCs) that did not differ significantly from negative control wells (0, 0eC52, vs. 0, 0eC65 spots), suggesting that they were not latently infected.
ELISPOT assays were performed to confirm the production of IL-4 and/or IL-42 protein. It is not known whether the ELISPOT assay detects one or both of these cytokines. Significant numbers of IL-4/IL-42 spots were detected in 90% of PBMCs, 60% of noneCT cells, 92% of T cells, 92% of CD4+ T cells, and 92% of CD8+ T cells where either cytokine was detectable by PCR. In the PBMC fraction of one patient with TB, IL-4 was detected by ELISPOT but not by RT-PCR. However, all other cell fractions of this patient had IL-4/IL-42 detectable by both methods. There was no correlation between the magnitudes of IL-4/IL-42 responses between the two methods, but these findings confirm that IL-4 mRNA correlates with production of active protein.
ELISA for IL-4 and IFN-
IL-4 was elevated in the BAL of patients with PTB versus control subjects (median, 12.2 vs. 6.9 pg/ml; p = 0.5) and detectable in 11 of 15 patients with PTB and 4 of 6 control subjects. In plasma, IL-4 levels were not different in patients with PTB versus control subjects, but in most cases, levels were around the detection limit of the assay. IFN- protein was largely undetectable in BAL or plasma (2 of 15 TB BAL samples and 3 of 13 TB plasma samples).
IL-4 and IL-42 Expression in PBMC Culture
Sonicated TB antigen induced the expression of IL-4 but not of IL-42 when PBMCs from eight patients with TB were stimulated over 8 days (Figures 6A and 6B). When cells stimulated with TB sonicate were harvested on Days 1 or 2, expression of IL-4 was increased compared with baseline (p < 0.05), with unstimulated wells (p < 0.005), and with wells stimulated with M. vaccae sonicate (p < 0.05). When harvested on Days 3 or 4, expression of IL-4 was increased compared with unstimulated wells (p < 0.005). A similar pattern was observed using cells from four healthy volunteers, though the median IL-4 mRNA levels were fivefold lower than in the cells of patients with TB (data not shown).
DISCUSSION
This study examines the expression of mRNA encoding the novel and little studied cytokine IL-42, which is an antagonist to IL-4 (19, 20) in human PTB. Findings in whole blood and BAL indicate that patients with PTB have significantly greater expression of mRNA encoding both IL-4 and IL-42, and the pretreatment radiographic scores correlated with the IL-4/IFN- ratio. IL-4 and IL-42 were prominently expressed in CD4 and CD8 T cells. Interestingly, antigen from M. tuberculosis caused cells from patients with PTB to express increased levels of IL-4, but not of IL-42, in vitro. After short-course chemotherapy, IL-4 mRNA levels did not decline significantly, whereas IL-42 mRNA significantly increased.
Collectively, our results are compatible with the hypothesis that a small but significant IL-4 component may subvert the protective Th1 response, whereas IL-42 might be protective. IL-42 is raised in contacts of patients with latent TB infection who remain healthy (23, 24), suggesting that this represents a protective response that deters progression to active disease. We have reviewed the arguments for and against this view in detail elsewhere (13, 30), and discuss some of them below in relation to the findings presented here. Briefly, the hypothesis can explain why TB develops and progresses despite the prominence of a protective Th1 response (6, 12), why bacille Calmette-Gueerin vaccine fails in countries close to the equator, and why current vaccine candidates are rarely more protective than bacille Calmette-Gueerin, even when they drive larger Th1 responses (8, 13, 31). If this hypothesis is correct, then radically new approaches to vaccine design are needed, especially because the global burden of TB continues to increase despite the availability of effective chemotherapy. Furthermore, these considerations may need to be extended into the fields of HIV and certain parasitic diseases.
Whether the IL-4 represents cause or effect has remained a contentious issue (22, 32eC34). Moreover, proof that TB antigen drives IL-4 in patients with TB is sparse. However, studies in knock-out (16) and preimmunized mice indicate that a preexisting Th2 component, even to a single 16eCamino acid epitope (17), can undermine the immune response to subsequent challenge, exacerbate immunopathology and fibrosis, and increase bacterial load. We provide evidence that in humans, too, the Th2 response is unlikely to be an inflammatory epiphenomenon. First, all patients improved clinically and radiologically during anti-TB therapy, but the IL-4 response did not decline substantially. This argues against the IL-4 being a nonspecific, inflammation-driven, bystander response. Rather, if IL-4 antagonizes mycobactericidal macrophage activity, it may explain why, even after several months of treatment, when bacterial load is very low, the immune response still cannot contain the disease, and treatment must continue. Second, the dominant T-celleCmediated production of IL-4 and IL-42, which in patients with TB was at least 25-fold higher than in control subjects, suggests that this is a true antigen-driven response. Previously described phenomena, such as TB-specific IgE (35) and antigen-driven T-cell clones producing IL-4, support this contention (36). Third, the observation that in vitro M. tuberculosis, but not M. vaccae, markedly increased expression of IL-4 relative to IL-42 suggests that M. tuberculosis differentially upregulates the agonist cytokine.
Although T cells produce significant amounts of IL-4 and IL-42 in PTB, it is unclear whether these are from effector or Th2-derived regulatory T cells or both. Although not conclusive, the increasing IL-4 response seen with more extensive pulmonary involvement in this study and others (22, 33) argues against regulatory T cells being the primary source of IL-4. Although IL-4 responses have been shown in CD4+ and CD8+ T cells (33), the production of IL-42 has not been taken into account. Moreover, the antibodies used for flow cytometric studies do not distinguish between the agonist and the antagonist. It is possible, but unproven, that the balance between IL-4 and IL-42 determines the net Th2 response. Although it was not logistically possible, it would have been instructive to repeat the cell subpopulation experiments at the end of TB treatment.
IL-42, which is an inhibitor of IL-4 (19, 20), rises in parallel with clinical improvement. These findings are relevant to the study of surrogate immunologic and serologic markers denoting successful treatment and cure of TB (37); the latter are a priority for the evaluation of new therapeutic vaccines and anti-TB drugs. IL-42 and the ratio of IL-42/IL-4 deserve further study in this respect. There were two patients in whom IL-42 levels did not increase at the time of assay; both had disseminated disease, and it is possible that the rise in IL-42 was delayed in those patients. Further studies are required to clarify the role of IL-42 in TB infection and longitudinal changes during active disease. Indeed, IL-42 illustrates the underlying complexity of the Th2 response in TB and this may explain the difficulty in categorizing TB into the classical Th1/Th2 paradigm. This complexity is exemplified by the exceedingly different ratio of IL-4 and IL-42 in human bronchial asthma where IL-4 is raised 1,000-fold more than the IL-42 (21).
There are few human data comparing in vivo cytokine findings in both whole blood and the lungs. If we had studied whole blood alone, our data would be consistent with other reports (38, 39) showing an insufficient Th1 response in the context of active TB and recovery of this response with treatment. However, our data demonstrate that, at the site of disease, the lungs, there are very high IFN- mRNA levels. Nevertheless, we have also shown a significant IL-4 and IL-42 response, which is similar in the blood and lungs, indicating different cytokine balance in the different body compartments. This cytokine- and compartment-specific disparity has to be taken into account for meaningful interpretation of TB-specific immunologic data. Accordingly, we also studied sCD30 in TB BAL, a finding that has not previously been reported. Elevated levels were not significantly different from plasma despite the high IFN- in the lungs and correlated with disease extent. Although expression of sCD30 is not confined to Th2 cells (40), there are quantitative differences in cellular expression, such that sCD30 correlates well with Th2-based markers in asthma, seasonal allergy, and atopic dermatitis (reviewed in Reference 40). The functional relationship between sCD30 and membrane CD30 is uncertain. A putative mechanism by which IL-4 mediated expression of CD30 modulates immune responses is by promoting tumor necrosis factor eCmediated lymphocyte apoptosis, which may facilitate immunopathology (41). Recent work has suggested that CD30 is expressed on CD25+ regulatory T cells that can suppress the activity of CD8+ T cells by causing them to undergo apoptosis (42). A regulatory role that decreases T-cell function is compatible with our data.
This is the first study using a specific probe real-time PCR assay to measure IL-4 and IL-42 mRNA levels in TB. Earlier studies that failed to find IL-4 (32, 34) used conventional PCR assays, which are limited by imprecise quantification (43), poor reproducibility (44), low dynamic range (45), the use of nonvalidated methods to normalize mRNA levels (46), and the failure to assess the quality of the initial RNA template (47). To avoid these pitfalls, we used real-time PCR with a validated housekeeping gene for normalization (30) and only used quality-controlled RNA templates. Probe-specific assays have been shown to give better resolution when low copy number genes of interest are studied (48). Because IL-4 is expressed at low levels that approach the detection limit of the assay, it is essential that the above considerations are adhered to if meaningful results are to be obtained. As seen in this study, real-time PCR is useful to study profiles in BAL fluid, where detection of cytokines is hampered by substantial dilution and lack of a satisfactory normalization strategy (49).
The lack of correlation between the magnitude of IL-4 detected by ELISPOT and real-time PCR is consistent with other reports (50), and may reflect the different sensitivities of the assays. This is related to the fact that the ELISPOT may have measured both IL-4 and IL-42 simultaneously, dichotomy between RNA and protein levels, or that the ELISPOT detects the number of cells secreting the cytokine, not the total quantity of cytokine produced. Indeed, the difficulty in detecting IL-4 by ELISA, even when blocking antibodies are used (50), is not surprising because IL-4 is produced at concentrations 1,000-fold lower than IFN- (51, 52), and is quickly taken up by widely distributed cellular receptors (53). Other methods, such as flow cytometry coupled with intracellular cytokine staining, require potent and unphysiologic cell stimulation to detect IL-4 (54). This study is unique because we measured mRNA levels in unstimulated cells with "immediate bedside" stabilization of the mRNA profiles. This is important because the mRNA encoding IL-42 has a very short half-life (unpublished observations). Delayed processing of specimens and cell stimulation protocols often used to detect IL-4 reflect an unphysiologic in vitro "snapshot" (54). Consequently, our strategy may represent a valuable alternative.
In conclusion, our data add to the accumulating evidence that a small but significant Th2-like response is indeed present in TB. However, we demonstrate that the IL-4 response is a complex one encompassing both IL-4 and IL-42, and we show for the first time that antigens from M. tuberculosis selectively drive expression of IL-4 itself in PBMCs from patients, without increasing expression of the antagonist IL-42. The usefulness of IL-42 or of the IL-42/IL-4 ratio as longitudinal markers of disease activity deserves investigation. Similarly, the identification of IL-4eCinducing TB antigen fractions might lead to the design of novel anti-TB immunotherapies and vaccines that concentrate on suppression of the detrimental Th2 component (13, 31).
Acknowledgments
The authors thank Drs. M. C. Lipman, I. A. Cropley, B. Bannister, M. Beckles, H. Booth, R. F. Miller, and J. Scott for assisting with patient recruitment, Dr. Luciene Lopes and Mr. Ian Gerrard for their technical advice, Dr. C. Hartigen for reading the x-rays, and Dr. F. C. Lampe for her statistical input.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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