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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Plasma cytokines play an important role in the pathogenesis of Sjögren's syndrome (SS) by initiating and perpetuating various cellular and humoural autoimmune processes. The aim of the present study was to describe a broad spectrum of T-cell and B-cell cytokines, growth factors, chemokines and molecules that could contribute to cell death in SS. A novel protein array system was utilized to measure simultaneously the levels of 25 plasma cytokines of patients with primary SS and healthy individuals. Furthermore, we correlated these plasma cytokine levels with potential laboratory and clinical parameters related to disease activity in SS. A subset of plasma cytokines [e.g. interleukin-1β (IL-1β), IL-6, CXCL8 (IL-8), IL-12 p40, IL-15, tumour necrosis factor-α (TNF-α), epidermal growth factor, CCL4 (MIP-1β), CCL2 (MCP-1), CCL11 (Eotaxin), CCL5 (RANTES), TNF-RI and TNF-RII] was found to significantly differ between patients and controls. Also, distinct populations of cytokines were found to differentiate between patients with normal versus elevated ESR or IgG levels and patients with the presence or absence of extra-glandular manifestations (EGMs). Our results support the assumption that the multiplex cytokine array system can be successfully utilized in the diagnosis and disease management of SS. Furthermore, it may provide a powerful tool in the design of individualized anticytokine therapies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Sjögren's syndrome (SS) is a chronic, slowly progressive, systemic autoimmune disease, which predominantly affects middle-aged women, although it can be seen in patients of all ages, including children [1]. It is characterized by lymphocytic infiltration and destruction of the exocrine glands, resulting in xerostomia and keratoconjunctivitis sicca and the presence of other exocrinopathic symptoms [1]. SS can develop alone (primary SS) or in association with other autoimmune diseases (secondary SS) such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis (PM), systemic sclerosis (sS) or thyroiditis [2].

Cytokines are small soluble peptides used by the immune system to communicate and influence cellular function. They are released by cells and their function can be autocrine or paracrine or endocrine. The key role of a few circulating cytokines in the pathogenesis of SS by triggering the perpetuation of the cellular and humoral autoimmune processes has been described [2, 3]. In previous studies, mostly serum Th1/Th2 cytokines were selectively reported in SS [3–8]. However, no extensive studies have been carried out to describe a broad variety of cytokines and their relation to clinical and/or laboratory features of the disease.

In RA and in SLE, disease activity/severity indices have been established to evaluate the patient's disease status. In SS, a similar disease activity assessment has not been fully established yet [9, 10]. Besides clinical and general immunological tests and since circulating cytokines may correspond to the activation status of immunocompetent cells, the evaluation of these cytokines might be a good indicator of disease activity. Although it is noteworthy that several aspects, including kinetics of expression, mode of induction, regulation of receptor expression and competition for occupancy and the stage of the disease (acute versus chronic), are critical to the cytokine net effect, all of these aspects need to be understood if we are to define a cytokine's whole nature [11].

The aim of this preliminary study was to simultaneously describe a broad spectrum of T-cell and B-cell cytokines, growth factors, chemokines and molecules that could contribute to cell death in SS by utilizing a multiplex cytokine array system. Furthermore, we correlated these plasma cytokine levels with potential laboratory and clinical parameters for disease activity assessment in SS.

Application of a multiplex cytokine array system might become a powerful tool to extensively describe the SS cytokine profile and to assess the role of these molecules in the pathogenesis of the disease. Furthermore, it ought to improve the design of special, individualized, cytokine-targeted therapy for these patients.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients and controls The study population consisted of nine patients with primary SS (eight female and one male; mean age 49.2 years, range 22–86) fulfilling the revised European classification criteria for primary SS [12] and nine age- and sex-matched healthy controls. Although two out of nine patients missed the lip biopsy (patient no. 1 and 6), these patients presented ocular symptoms, ocular signs and oral symptoms, together with high titres of autoantibodies against anti-SSA and anti-SSB autoantigens. Accordingly, these patients also fulfilled the clinical and laboratory features obligatory to be involved in our study as patients with SS [12]. Patients were recruited from the inpatient and outpatient clinic at the Department of Rheumatology, Haukeland University Hospital, Bergen, Norway. Treatment included artificial tear substitution and occasionally peroral NSAIDs. Before inclusion in the study, informed consent was signed by each patient.

Laboratory and histological assessments included ESR, CRP, serum immunoglobulin, antinuclear antibody (ANA), anti-SS-A, anti-SS-B and a routine histological evaluation of minor salivary gland biopsy material. Clinical, laboratory and histological characteristics of the patients included in the study are summarized in Table 1.

Table 1.  Clinical and laboratory characteristics of patients with primary Sjögren's syndrome (SS)
Sl. no.SexAge (years)ESRCRPIgGANASS-ASS-BFocus scoreEGM
  1. ANA, antinuclear antibody (normal 0.00–0.99); CRP, C-reactive protein (normal <10); EGM, extra-glandular manifestation; ESR, erythrocyte sedimentation rate (normal 0–20 mm/h); IgG, immunoglobulin G (normal 6–15.3 g/l).

1M7154514.19.57++Not available
2M23425205.84+1Polyarthritis
3M8653512.15.03++2
4F753459.80.482Polyneuropathy
5M22301713.71.2+2Thyroiditis, polyarthritis
6M3411516.29.37++Not available
7M6920511.12.92+3Thyroiditis
8M4117516.63.11
9M2277279.80.752Thyroiditis

Plasma samples Blood samples were obtained from both patients and controls after informed consent and treated anonymously throughout the analysis. Peripheral blood was diluted 1 : 1 in phosphate-buffered saline (PBS) before centrifugation over Lymphoprep (Axis-Shield PoC A/S, Oslo, Norway) for further analysis. Plasma was collected and stored at −80 °C until analysed.

Multiplex cytokine assay Cytokines measured included interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, CXCL8 (IL-8), IL-10, IL-12 (p40), IL-13, IL-15, IL-17, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) basic, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), CCL2 [monocyte chemoattractant protein-1 (MCP-1)/MCAF], CCL11 (eotaxin), CCL3 [macrophage inflammatory protein-1α (MIP-1α)], CCL4 [macrophage inflammatory protein-1β (MIP-1β)], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], TNF-RI and TNF-RII.

A sandwich immunoassay based protein array system (Biosource International, Camarillo, CA, USA), which contains dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used. Serum samples were thawed and run in duplicates. Antibody-coupled beads were incubated with the plasma sample (antigen) after which they were incubated with biotinylated detection antibody before finally being incubated with streptavidin–phycoerythrin (Fig. 1). A broad sensitivity range of standards (Biosource International, Camarillo, CA, USA) ranging from 1.95 to 32000 pg/ml were used to help enable the quantitation of a dynamic wide range of cytokine concentrations and provide the greatest sensitivity. This captured immunoassay was then read by the Bio-Plex array reader (Bio-Rad Laboratories, Hercules, CA, USA) which uses Luminex fluorescent-bead-based technology (Luminex Corporation Austin, TX, USA) with a flow-based dual laser detector with real-time digital signal processing to facilitate the analysis of up to 100 different families of colour-coded polystyrene beads and allow multiple measurements of the sample ensuing in the effective quantitation of cytokines.

image

Figure 1. Simultaneous cytokine quan- titation with a Biosource suspension array system. For analysis of cytokine levels, a multiplex biometric sandwich immunoassay was used. Diluted plasma is mixed with fluorescently addressed microbeads bound with anticytokine antibodies {e.g. interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, CXCL8 (IL-8), IL-10, IL-12 (p40), IL-13, IL-15, IL-17, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) basic, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), CCL2 [monocyte chemoattractant protein-1 (MCP-1)] (MCAF), CCL11 (Eotaxin), CCL3 [macrophage inflammatory protein-1α (MIP-1α)], CCL4 (MIP-1β), CCL5 (RANTES), TNF-RI and TNF-RII}. Biotin anticytokine secondary antibodies are then added and allowed to bind to cytokine-bead complexes, followed by phycoerythrin (PE)-conjugated streptavidin. Total surface fluorescence for each bead is measured using a BioPlex fluorescent flow-based fluorescent detection system (essentially a FACS designed for detection of fluorescent signal detection on the surface of microbeads).

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Analysis The concentrations of analytes in these assays were quantitated using a calibration or standard curve. A regression analysis was performed to derive an equation that was then used to predict the concentration of the unknown samples. Statistical differences in measured values were analysed using a Mann–Whitney U-test. P values less than 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Diverse cytokine profile of SS patients and healthy controls

The 25 investigated plasma cytokines were sorted into five functional groups, such as ‘cytotoxic cellular cytokines’ (e.g. IL-1β, IL-2, IL-12p40, IL-15, IL-17, IFN-γ and TNF-α), ‘humoural cytokines’ (e.g. IL-4, IL-5, IL-6, IL-10 and IL-13), ‘growth factors’ (e.g. EGF, VEGF, FGF basic, G-CSF and GM-CSF), ‘chemokines’[e.g. CXCL8 (IL-8), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL2 (MCP-1), CCL11 (Eotaxin) and CCL5 (RANTES)] and ‘death receptors’ (e.g. TNF-RI and TNF-RII). In each group, we compared the plasma cytokine levels between SS patients and controls.

In the ‘cytotoxic cellular cytokines’ group, IL-1β (P = 0.019) and TNF-α (P = 0.004) were significantly elevated, while IL-12p40 levels (P < 0.0001) and IL-15 levels (P = 0.0008) were significantly decreased in SS patients (Fig. 2A).

image

Figure 2. Comparison of various plasma cytokines levels between Sjögren's syndrome (SS) patients (n = 9) and controls (n = 9). Bars show the mean in pg/ml and SEM. (A) Levels of plasma cytotoxic cellular cytokines. (B) Levels of humoural cytokines. (C) Plasma levels of growth factors. (D) Plasma chemokine levels. (E) Levels of death receptors. *significantly different from control values by t-test (P < 0.05). IFN-γ, interferon-γ; IL-1β, interleukin-1β; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; TNF-α, tumour necrosis factor-α; VEGF, vascular endothelial growth factor.

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Figure 2(B) summarizes the data obtained from the comparison of the cytokines likely to drive the humoural immune responses between patients and controls. The level of IL-6 (P = 0.0008) was significantly elevated in the SS group.

The comparison of the plasma levels of ‘growth factors’ revealed a significant decrease of EGF (P = 0.0003) in the patient group (Fig. 2C).

Figure 2(D) summarizes the plasma ‘chemokine’ levels of SS patients and controls. A highly significant decrease was observed in the SS group compared to healthy controls concerning CXCL8 (IL-8) (P < 0.0001), CCL2 (MCP-1) (P < 0.0001), CCL11 (Eotaxin) (P < 0.0001), CCL5 (RANTES) (P < 0.0001) and CCL4 (MIP-1β) (P = 0.0056) levels.

The comparison of the plasma levels of two ‘death receptors’ TNF-RI (P < 0.0001) and TNF-RII (P = 0.0002) revealed a marked decrease in both cases in the SS patient group compared to controls (Fig. 2E).

As no ‘disease activity score’, or ‘disease severity index’ has been established in SS, we could not divide our patient population into ‘active’ and ‘inactive’ groups. Therefore, we subgrouped the SS patients on the basis of various laboratory (ESR, IgG and anti-SSA/SSB) and clinical [extraglandular manifestations (EGMs)] characteristics and determined how these features correlate with the aforementioned plasma cytokine levels.

Comparison of plasma cytokine levels between SS patients with normal (n = 3) and elevated (n = 6) ESR levels

In this comparison, we found significantly elevated levels of the following cytokines in SS patients with high ESR: IL-2 (P = 0.024) from the ‘cytotoxic cellular cytokines’ group, EGF (P = 0.036) of the ‘growth factors’, CCL3 (MIP-1α) (P = 0.024) and CCL5 (RANTES) (P = 0.048) of the chemokines and the death receptor, TNF-RI (P = 0.048). Figure 3 summarizes the data of 25 cytokines.

image

Figure 3. Comparison of various plasma cytokine levels between Sjögren's syndrome (SS) patients with normal ESR (n = 3) and SS patients with elevated ESR (n = 6). Bars show the mean in pg/ml and SEM. (A) Levels of plasma cytotoxic cellular cytokines. (B) Levels of humoural cytokines. (C) Plasma levels of growth factors. (D) Plasma chemokine levels. (E) Levels of death receptors. *significantly different from control values by t-test (P < 0.05). IFN-γ, interferon-γ; IL-1β, interleukin-1β; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; TNF-α, tumour necrosis factor-α; VEGF, vascular endothelial growth factor.

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Comparison of plasma cytokine levels between SS patients without (n = 4) and with (n = 5) EGMs

The relation of EGMs and peripheral cytokine levels in SS showed significantly lower levels of IL-12p40 (P = 0.032) from the ‘cytotoxic cellular cytokines’ group in patients with EGMs. Figure 4 illustrates the rest of the cytokine groups in these populations.

image

Figure 4. Comparison of various plasma cytokine levels between Sjögren's syndrome (SS) patients without (n = 4) and with (n = 5) the presence of EGMs. Bars show the mean in pg/ml and SEM. (A) Levels of plasma cytotoxic cellular cytokines. (B) Levels of humoural cytokines. (C) Plasma levels of growth factors. (D) Plasma chemokine levels. (E) Levels of death receptors. *significantly different from control values by t-test (P < 0.05). IFN-γ, interferon-γ; IL-1β, interleukin-1β; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony stimulating factor; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; TNF-α, tumour necrosis factor-α; VEGF, vascular endothelial growth factor.

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The comparison of plasma cytokine levels between SS patients without (n = 3) and with (n = 6) the presence of anti-SSA/anti-SSB autoantibodies, the comparison of patients with normal serum IgG levels (n = 6) and hypergammaglobulinaemia (n = 3) as well as SS patients with low focus score (FS) (FS = 1) (n = 2) versus patients with high FS (FS ≥ 2) (n = 5) revealed no significantly different values in any cytokine groups (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The aim of the present study was to describe subpopulations of circulating cytokines likely to drive a broad spectrum of autoimmune processes in SS. We could identify a subset of cytokines that significantly differ between controls and SS patients as well as differentiate between various subsets of patients with SS (Table 2). Cytokines were grouped into the following categories: ‘cytotoxic cellular cytokines’, ‘humoural cytokines’, ‘growth factors’, ‘chemokines’ and ‘death receptors’. We could characterize each population of patients by their cytokine profile, compare these disease groups and pinpoint special subsets of cytokines that are attributed to the particular disease subset.

Table 2.  Plasma cytokines that significantly differ between controls and patients as well as various subsets of patients with Sjögren's syndrome (SS)
Plasma cytokines that significantly differ between healthy controls and patients
Categorized groupSignificant cytokinesHealthy controls (pg/ml) (median)SS patients (pg/ml) (median)P value
  1. CCC, cytotoxic cellular cytokine; CK, chemokine; DR, death receptor; EGM, extra-glandular manifestation; GF, growth factor; HC, humoural cytokine; IL-15, interleukin-15; TNF-α, tumour necrosis factor-α.

CCCIL-1β11.2135.140.0188
 IL-12p40124.826.51<0.0001
 IL-15122.712.280.0008
 TNF-α3.5231.220.004
HCIL-64.4234.080.0008
GFEGF42.997.950.003
CKCXCL8 (IL-8)6.363.23<0.0001
 CCL4 (MIP-1β)59.2621.990.056
 CCL2 (MCP-1)343.556.57<0.0001
 CCL11 (Eotaxin)109.65.67<0.0001
 CCL5 (RANTES)7903.21304.01<0.0001
DRTNF-RI127396.74<0.0001
 TNF-RII1593470.50.0002
Plasma cytokines that significantly differ between subsets of SS patients
Normal versus elevated ESR
Categorized groupSignificant cytokinesNormal ESR (pg/ml) (median)Elevated ESR (pg/ml) (median)P value
CCCIL-23.0713.480.024
GFEGF6.288.010.036
CKCCL3 (MIP-1α)21.7240.130.024
 CCL5 (RANTES)790.115740.048
DRTNF-R115.74116.60.048
Presence versus absence of EGMs
Categorized groupSignificant cytokinesWithout EGM (pg/ml) (median)With EGM (pg/ml) (median)P value
CCCIL-12p4032.2419.480.032

The key cytokines that discriminate between SS and healthy individuals were IL-1β, IL-12p40, IL-15 and TNF-α of the ‘cytotoxic cellular cytokines’ group; IL-6 of the ‘humoral cytokines’; EGF of the ‘growth factors’; CXCL8 (IL-8), CCL4 (MIP-1β), CCL2 (MCP-1), CCL11 (Eotaxin) and CCL5 (RANTES) of the ‘chemokines’ and TNF-RI, TNF-RII of the ‘death receptors’ group. These findings support the hypothesis that a broad spectrum of impaired immune functions are involved in the pathogenesis of SS, affecting cellular, humoural immune responses, leucocyte function, cell proliferation/growth and cell death. Although a few of the cytokines related to SS have been described previously [13–17], most of these significant cytokines have not been related to SS before. With the novel, advanced multiplex cytokine array system, a distinct SS-related plasma cytokine pattern has been revealed that discriminated between patients and healthy individuals.

Additionally, we analysed the correlation between the expression of these cytokines and various laboratory and clinical parameters to characterize the disease status of SS patients (e.g. ESR, serum IgG levels, the presence of anti-SSA and/or anti-SSB autoantibodies, FC values of minor salivary gland histology and the presence of EGMs besides glandular features).

The most prominent cytokines, likely to differentiate between SS patients with normal or elevated ESR levels were IL-2, EGF, CCL3 (MIP-1α), CCL5 (RANTES) and TNF-RI. Our results indicate that behind the elevated ESR in SS lies a complex disturbance of lymphocyte and leucocyte function, characterized by inappropriate regulation of cell-proliferation/growth and cell death.

The presence or absence of EGMs, the most essential ‘clinical candidate’ of disease activity evaluation in SS, was correlated with the circulating levels of 25 cytokines simultaneously. From the ‘cytotoxic cytokines’ group, IL-12p40 was found to significantly differ between SS patients with or without EGMs.

Interestingly, the peripheral plasma cytokine levels did not correlate significantly with the presence or absence of hypergammaglobulinaemia in SS or with the disease-specific autoantibody levels. It is more likely that factors other than peripheral plasma cytokines drive the humoural autoimmune response in SS alternatively, an indirect association between these cytokines and immunoglobulin production exists.

Furthermore, the cytokine comparison in patients with low and high salivary gland FC values did not show significant differences between the two populations raising the possibility that the development of sialadenitis in SS might be driven by a local network of pro-inflammatory cytokines. Further investigations of salivary gland tissue cytokines, utilizing the multiplex cytokine assay, may be used to describe the in situ cytokine milieu in SS salivary glands.

Our results imply that a complex disorder of secreted plasma cytokine levels driving various immunocompetent cell types can be found in SS patients. Also, different patterns of circulating cytokines are specific to diverse types of the disease. We assume that the utilization of the multiplex cytokine array system in SS provides a powerful tool to subcategorize the disease and along with common clinical and laboratory parameters help to evaluate disease activity.

In this study, we focused on individual time points to describe the cytokine profile of the patients, although fluctuations in cytokine levels sometimes accompanies disease flare or remissions. However, in separate ongoing studies utilizing the multiplex cytokine assay, serial serum cytokine level measurements did not seem to fluctuate signficantly (unpublished results).

In this study, cytokine levels with known indicators of disease flare used in the clinical management of SS (e.g. ESR, serum IgG levels, the presence of anti-SSA and/or anti-SSB autoantibodies, FS values of minor salivary gland histology or the presence of EGMs besides glandular features) were assessed in order to describe the characteristic cytokine pattern accompanying a flare as a measure of ‘disease activity’. Moreover, to exclude modulations in the circulating cytokine levels, we selected patients in this study, who at the time of the analysis were not taking immunomodulatory medications, which would seriously affect the cytokine levels.

One of the interesting future applications of this technique is on patients with secondary SS. As this is a preliminary study, we wanted to describe the effective utilization of the multiplex cytokine assay in a clearly defined disease population and avoid complex overlap syndromes, or associated diseases, like secondary SS. As the multiplex cytokine assay is a recently developed, unique technique, we believe that primarily it is important to gain information from relatively well-defined diseases, and furthermore, on the basis of such knowledge apply the technique on more complex diseases. Our further goal is to apply this technology on other systemic autoimmune diseases, also to describe complex disease entities (e.g. secondary SS). A future important application of this technology will be to evaluate the circulating cytokine pattern and thereby help subcategorizing patients with SS and develop this technology to become a powerful diagnostic tool in the future. By the simultaneous monitoring of general laboratory values and serum multiplex cytokine levels, individual therapy planning could be achieved. Additionally, this assay could be used to evaluate the efficacy of an ongoing therapy. Instead of long, empirical therapies, one can optimize combination therapies by individual pro-inflammatory cytokine targeting and also plan advanced cost-benefit strategies. Although this strategy is relatively expensive (100$ per sample for reagents alone), the amount of information obtained from the multiplex cytokine assay is considerable and gives a near-complete description of the pathogenic cytokines and is therefore both powerful and represents a good cost-benefit. We assume that the utilization of the multiplex cytokine array system in the diagnosis and therapy design in SS will provide an advanced disease management in the future.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was funded by the National Institutes of Health National Center for Research Resources (grant numbers NIH 1 P20 RR15577 and NIH 1 P20 RR16478), The Fund for Arthritis and Inflammatory Research (FAIR) and the Faculty of Medicine, University of Bergen, Bergen, Norway.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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