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ORIGINAL ARTICLE
Year : 2015  |  Volume : 28  |  Issue : 3  |  Page : 757-764

Effect of the antirheumatic drug leflunomide (Avara) on the lungs of adult male albino rats and the possible protective effect of N-acetylcysteine: histological and immunohistochemical study


Department of Histology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission28-Jan-2015
Date of Acceptance15-Mar-2015
Date of Web Publication22-Oct-2015

Correspondence Address:
Shaimaa M Abdel-Fattah Hasn
MSc, El-Harm, Giza 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.167898

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  Abstract 

Objective
The present study was conducted to throw light on the histological effects of the antirheumatic drug leflunomide (Avara) on the lungs of adult male albino rats, and to clarify the possible protective effect of N-acetylcysteine.
Background
Leflunomide (Avara) is an immunomodulating agent and disease-modifying antirheumatic drug with anti-inflammatory and immunosuppressive activity. N-acetylcysteine belongs to a class of drugs called antioxidants which may reduce the amount of damage caused by inflammation or active damage of the lungs.
Materials and methods
A total of 60 adult male albino rats were used in the present study. They were divided into four groups. Group I: The control group composed of 20 rats. Group II: The N-acetylcysteine-treated group composed of 10 rats received N-acetylcysteine in a daily oral dose of 200 mg/kg body weight. Group III: The leflunomide-treated group composed of 20 rats receiving leflunomide on a daily oral dose of 10 mg/kg body weight. Then, half the animals were killed 4 weeks after the treatment (subgroup IIIa), and the other half were left without treatment for another 2 weeks and served as the recovery group (subgroup IIIb). Group IV: The protected group composed of 10 rats receiving combined treatment of both leflunomide and N-acetylcysteine in the same previous doses and route of administration for 4 weeks. At the end of the study, animals were killed and specimens from the lungs were processed for both L/M, E/M, and immunohistochemical study (using caspase-3 immunostaining). Morphometric study for the number of pneumocyte type II was counted, and the thicknesses of the interalveolar septa were done and statically analyzed.
Results
The results of this study revealed that leflunomide treatment caused considerable histological, immunohistochemical, and ultrastructural changes in the lungs. Concomitant administration of N-acetylcysteine with leflunomide resulted in remarkable improvement, whereas arrest of treatment for 2 weeks revealed mild improvement.
Conclusion
N -acetylcysteine is proven to have a protective effect on the lungs against hazardous effects induced by leflunomide treatment. Therefore, strict follow-up and coadministration of N-acetylcysteine are highly recommended for those patients who use leflunomide.

Keywords: Leflunomide, lung, N-acetylcysteine


How to cite this article:
Soliman ME, Atteya SE, El Saify GH, Ghoneim NS, Yasein RI, Abdel-Fattah Hasn SM. Effect of the antirheumatic drug leflunomide (Avara) on the lungs of adult male albino rats and the possible protective effect of N-acetylcysteine: histological and immunohistochemical study. Menoufia Med J 2015;28:757-64

How to cite this URL:
Soliman ME, Atteya SE, El Saify GH, Ghoneim NS, Yasein RI, Abdel-Fattah Hasn SM. Effect of the antirheumatic drug leflunomide (Avara) on the lungs of adult male albino rats and the possible protective effect of N-acetylcysteine: histological and immunohistochemical study. Menoufia Med J [serial online] 2015 [cited 2020 Feb 24];28:757-64. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/757/167898


  Introduction Top


Leflunomide (Avara) is an immunomodulating agent and disease-modifying antirheumatic drug with anti-inflammatory and immunosuppressive activity. It is used for the management of signs and symptoms of rheumatoid arthritis [1]. Previous reports suggest that leflunomide is the most likely cause of pneumonitis. However, there are no reports showing that pneumonitis occurs after the administration of leflunomide by itself, because it may be used with other drugs. In addition, rheumatoid arthritis itself can also affect the lung, usually leading to an interstitial pneumonitis [2].The controversy about the associated risk of respiratory infection in patients treated with leflunomide depended exclusively on the clinical studies. Hence, this is the main drive for this present work to demonstrate the histological and immunohistochemical effects of leflunomide on the lung tissue and the possibility of recovery after its withdrawal.

N-acetylcysteine belongs to a class of drugs called antioxidants which may reduce the amount of damage caused by inflammation or active damage of the lungs. Inflammation can lead to fibrosis (scarring) in the lungs. By reducing the damage caused by inflammation, it is hoped that N-acetylcysteine will limit the progression of lung fibrosis and allow the inflamed lung to return to normal [3]. Hence, the aim of this work was to study the effect of leflunomide on the lungs and to evaluate the protective effect of N-acetylcysteine.


  Materials and methods Top


A total of 60 adult male albino rats weighing nearly 100-120 g were used in the present study. They were divided into four main groups:

Group I (control group)

It was composed of 20 rats that were further subdivided into two equal subgroups, each comprising 10 rats.

The first: kept without any treatment and served as control for all experimental groups.

The second: given 1 ml saline (vehicle of leflunomide)/day orally for 4 weeks and served as control for leflunomide-treated group.

Group II (N-acetylcysteine-treated group)

A total of 10 rats that were treated with N-acetylcysteine at a dose of 200 mg/kg body weight/orally daily for 4 weeks [4] in distilled water by modified plastic syringe. N-acetylcysteine was dissolved in 1 ml distilled water to produce the desired concentration.

Group III (leflunomide-treated group)

A total of 20 rats were treated with leflunomide in the form of leflunomide tablet at dose of 10 mg/kg [5]. Each tablet containing 10 mg leflunomide was dissolved in 1 ml saline and administered orally daily by modified plastic syringe for 4 weeks. Half of the animals were killed after 4 weeks in subgroup IIIa, and the other half were left without treatment for further 2 weeks in subgroup IIIb for recovery and then killed.

So group III includes two subgroups:

Subgroup IIIa: Rats were killed 4 weeks after treatment with leflunomide.

Subgroup IIIb: Rats were killed 2 weeks after arrest of leflunomide treatment.

Group IV (leflunomide- and N-acetylcysteine-treated group)

A total of 10 rats were treated with both leflunomide and N-acetylcysteine at dose similar to that of leflunomide-treated and N-acetylcysteine-treated groups, respectively. At the end of the experiment (4 weeks), animals were killed and their lungs were extracted and prepared for the following studies:

Histological studies: using hematoxylin and eosin stain and Mallory trichrome stain [6].

Immunohistochemical study: using caspase-3 immunostaining [7].

Electron microscopic study [8].

Morphometric study and statistical analysis: From H&E sections of different experimental groups (control and treated groups), the number of pneumocyte type II was counted and the thickness of the interalveolar septa was measured. Then, statistical analysis for these data was performed in the form of mean and SD using t-test and P-value [9].


  Results Top


Macroscopic results

The lungs obtained from control rats and N-acetylcysteine-treated rats showed the normal homogeneous glistening bright red appearance [Figure 1]a. On the other hand, the lungs obtained from leflunomide-treated rats for 4 weeks appeared shrunken (collapsed) and showed dark opaque red appearance (patches of hemorrhage) [Figure 1]b; however, 2 weeks after the arrest of the treatment, the lungs obtained showed little hemorrhage [Figure 1]c. Leflunomide- and N-acetylcysteine-treated rats for 4 weeks showed bright red appearance and small areas of hemorrhage [Figure 1]d.
Figure 1: A naked eye photomicrograph of the rat lung of (a) group I showing homogeneous glistening bright red appearance. (b) Group IIIa appearing shrunken (collapsed) with dark opaque red appearance (patches of hemorrhage). (c) Group IIIb showing little hemorrhage. (d) Group IV showing bright red appearance and small areas of hemorrhage.

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Histological results

Hematoxylin and eosin

Group I: They showed normal lung architecture with the thin interalveolar septa. There were numerous alveoli with the thin alveolar walls. An alveolus may open into alveolar sacs [Figure 2]a. An alveolus was lined by pneumocyte type I which was flattened with the thin membrane-like cytoplasm and flattened nucleus and pneumocyte type II with large central rounded nucleus and dome-shaped surface facing the alveolar lumen. There were alveolar macrophages in the alveolar lumen [Figure 3]a1.
Figure 2: A photomicrograph of the rat lung of (a) group I, (b) group II, (c1, c2) subgroup IIIa, (d) subgroup IIIb and (e) group IV.

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Figure 3: A photomicrograph of the rat lung of (a) group I, (b) group II, (c) subgroup IIIa, (d) subgroup IIIb and (e) group IV.

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Group II: They showed normal lung architecture detected by the patent alveoli, normal bronchioles, and normal blood vessels [Figure 2]b. The patent alveoli were lined by flattened pneumocyte type I and rounded pneumocyte type II [Figure 3]b.

Group III: Subgroup IIIa: They showed multiple areas of diffuse alveolar damage in the form of thickened pulmonary interstitium by the inflammatory cells and inflammatory exudates with extensive interstitial hemorrhage. There were collapse of some alveoli and thickened interalveolar septa and overexpansion of the other alveoli with destroyed alveolar walls. Interstitial and alveolar hemorrhage were the prominent feature in this group and represented by numerous red blood cells (RBCs) filling the pulmonary interstitium and alveolar lumen. Pulmonary vascular congestion was detected in the form of dilated congested blood vessels. In addition, there was partial shedding of the bronchiolar mucosa and epithelial proliferation in some areas of the mucosa [Figure 2]c1, c2. Moreover, pneumocyte type II showed apparent increase in the number, whereas pneumocyte type I appeared unchanged. Haemosiderin granules were seen as dark brown dots in some sections [Figure 3]c.

Subgroup IIIb: They showed some improvement. There were dilated, congested pulmonary vessels, moderate inflammatory cellular infiltrate, and some hemorrhage [Figure 2]d. Pneumocyte type II showed apparent increase in the number, whereas pneumocyte type I appeared unchanged [Figure 3]d.

Group IV: As a result of simultaneous administration of N-acetylcysteine to leflunomide-treated rats, lung sections showed minimal inflammatory cell infiltrate and minimal interstitial hemorrhage. The mucosal lining of the bronchiole appeared quite normal without any epithelial changes [Figure 2]eThe alveoli appeared normal and lined by flattened pneumocyte type I and rounded pneumocyte type II [Figure 3]e

Mallory trichrome stain

Group I: They showed minimal amount of collagen fibers (tinge of blue color) in the pulmonary interstitium and surrounding the blood vessels. In addition, the adventitia of the bronchioles showed collagen fibers (blue color) [Figure 4]a.
Figure 4: A photomicrograph of the rat lung of (a) group I, (b) group II, (c) subgroup IIIa, (d) subgroup IIIb and (e) group IV.

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Group II: They showed normal minimal amount of collagen fibers in the pulmonary interstitium [Figure 4]b.

Group III: Subgroup IIIa: They showed excess deposition of collagen fibers at the peribronchiolar and perivascular areas [Figure 4]c.

Subgroup IIIb: They showed excess collagen in the pulmonary interstitium and the peribronchiolar and perivascular areas [Figure 4]d.

Group IV: They showed mild amount of collagen fibers in the peribronchiolar and perivascular areas and the pulmonary interstitium [Figure 4]e.

Immunohistochemical study: caspase-3 immunostaining

Group I: They showed negative immune reaction to caspase-3 protein in the interalveolar septa and weak reaction in the alveolar epithelial cells. It appeared as dark brown punctuate granules filling the cytoplasm [Figure 5]a.
Figure 5: A photomicrograph of the rat lung of (a) group I, (b) group II, (c) subgroup IIIa, (d) subgroup IIIb, and (e) group IV.

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Group II: They showed negative immune reaction to caspase-3 protein in the interalveolar septa and weak reaction in the alveolar epithelial cells [Figure 5]b.

Group III:

Subgroup IIIa: They showed intense positive immune reaction to caspase-3 protein in the thickened interalveolar septa and infiltrating cells. In addition, there was intense positive immune reaction in the wall of the alveoli, alveolar sacs, and alveolar epithelial cells [Figure 5]c.

Subgroup IIIb: They showed moderate positive immune reaction to caspase-3 protein in the thickened interalveolar septa, infiltrating cells, and alveolar epithelial cells [Figure 5]d.

Group IV: They showed mild positive immune reaction to caspase-3 protein in the interalveolar septa, infiltrating cells, and alveolar epithelial cells [Figure 5]e.

Electron microscopic results

Group I: Examination of ultrathin lung sections showed pneumocyte type I containing large oval nucleus with the peripheral condensed chromatin and thin attenuated cytoplasm [Figure 6]a1. Pneumocyte type II was rounded with large rounded central vesicular nucleus with prominent nucleolus. Its cytoplasm showed mitochondria and lamellar bodies with concentric lamellae of secretory material (surfactant). Its surface showed short apical microvilli [Figure 6]a2. Alveolar macrophage appeared with the kidney-shaped indented nucleus. Its cytoplasm demonstrated multiple lysosomes. It had multiple pseudopodia on its surface [Figure 7]a3.
Figure 6: Electromicrograph of ultrathin section of a1: group I (TEM magnification 3000), a2: group I (TEM magnification 2000), b: group II (TEM magnifi cation 2000), c1: subgroup IIIa (TEM magnification 2000), c2: subgroup IIIa (TEM magnifi cation 1000), d1: subgroup IIIb (TEM magnification 2000), e1: group IV (TEM magnification 2000).

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Figure 7: Electromicrograph of ultrathin section of a3: group I (TEM magnification 3000), c3: subgroup IIIa (TEM magnification 2000), e2: group IV (TEM magnification 2000).

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Group II: Examination of ultrathin lung sections showed the alveoli similar to that of the control group. The alveoli was lined by normal pneumocyte type I and pneumocyte type II. Pneumocyte type I appeared with large oval nucleus and thin cytoplasm. Pneumocyte type II had large rounded nucleus and short apical microvilli. The cytoplasm of pneumocyte type II contained numerous mitochondria, cisternae of rough endoplasmic reticulum, and multiple lamellar bodies [Figure 6]b.

Group III

Subgroup IIIa: Examination of ultrathin lung sections showed degenerated pneumocytes type I with pyknotic nuclei and surrounded by collagen fibers [Figure 6]c1. Pneumocytes type II appeared degenerated with pyknotic nuclei and vacuolated cytoplasm. There were hyperplasia, loss of characteristic lamellar pattern of lamellar bodies leaving empty vacuoles and swollen ballooned mitochondria. There was monocellular infiltration-included eosinophils with their characteristic elongated granules and neutrophils in interalveolar space [Figure 6]c2. Alveolar macrophages were also demonstrated. They showed electron dense bodies and numerous degenerated mitochondria in their cytoplasm [Figure 7]c3.

Subgroup IIIb: Examination of ultrathin lung sections showed no restoration of the normal histological structure of the alveoli. Pneumocyte type I appeared flattened and surrounded by collagen fibers deposited by fibroblasts. Pneumocytes type II showed irregular lamellar bodies and disturbed microvillous border. The alveolar space showed alveolar macrophage with its characteristic pseudopodia, congested blood capillaries (which lined by normal endothelial cell, and others with an abnormal one), and extravasted RBCs [Figure 7]d1.

Group IV: Examination of ultrathin lung sections showed a great reduction in pathological changes that were demonstrated in leflunomide-treated rats. The patent alveoli was lined by normal flattened pneumocyte type I and pneumocyte type II. Pneumocytes type II had short apical microvilli; their cytoplasm demonstrated numerous mitochondria with intact cristae and lamellar bodies with regular lamellar pattern [Figure 6]e1.

The blood capillaries were lined by regular flattened endothelial cell and contained RBCs. Alveolar macrophage appeared with its characteristic pseudopodia in alveolar space [Figure 7]e2.

Morphometric and statistical results

The number of pneumocyte type II

The mean number of pneumocyte type II of group IIIa showed highly significant increase (P < 0.001) when compared with the control group (group I). However, other groups including groups II, IIIb, and IV showed a nonsignificant change (P > 0.05) when compared with the control group (group I) ([Table 1] and Histogram 1 [Additional file 1]).
Table 1: The mean number of pneumocyte type II in the control group and different groups of rats

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The thickness of interalveolar septa

The mean thickness of the interalveolar septa of group IIIa showed highly significant increase (P<0.001) when compared with the control group (group I). However, other groups including groups II, IIIb, and IV showed a nonsignificant change (P > 0.05) when compared with the control group (group I) ([Table 2] and Histogram 2 [Additional file 2]).
Table 2: The mean thickness of interalveolar septa in the control group and different groups of rats

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  Discussion Top


Interstitial lung disease (ILD), also known as diffuse parenchymal lung disease, refers to a group of lung diseases affecting the interstitium (the tissue and space around the air sacs of the lungs) [10]. It concerns alveolar epithelium, pulmonary capillary endothelium, basement membrane, perivascular, and perilymphatic tissues [11]. ILD has been reported during treatment with leflunomide and has been associated with fatal outcome. The risk of its occurrence is increased in patients with a history of ILD. ILD is a potentially fatal disorder that may occur acutely at any time during therapy and has a variable clinical presentation [12].

The present study was designed to clarify the role of antioxidant N-acetylcysteine with regard to the histological, immunohistochemical, and the ultrastructural changes in the lungs of leflunomide-treated rats.

The pathogenesis of cytotoxic lung injury could be due to direct injury to pneumocytes or the alveolar capillary endothelium, with subsequent release of cytokines and influx of the inflammatory cells as had been explained by Matsuno [13].

The stagnant blood in the dilated capillaries would cause hypoxia and hypoxia leads to the upregulation of the expression of neurokinin-1 receptors in alveolar macrophages and in epithelial cells. Activation of these receptors leads to inflammatory responses mediated by cytokines interleukin-1, interleukin-6, and tumor necrosis factor-a. Furthermore, alveolar macrophages have been implicated in the synergistic effects of hypoxia on pathogen-induced lung inflammation [14].

Interstitial and alveolar hemorrhage noticed in leflunomide-treated rats of this study was in harmony with Zhang et al. [15] who attributed these changes to increased vascular endothelial permeability. In addition, hypoxia leads to increased lung vascular endothelial and alveolar epithelial permeability. The pulmonary vascular congestion noticed in leflunomide-treated rats may be attributed to vasodilator substances released by the blood vessels into the blood stream [16]. In addition, hypoxia of the lung tissue results in more congestion [17].

The dark brown haemosiderin granules in the areas of extravasted blood in the pulmonary intersititium and inside the alveoli could be explained by breakdown of RBCs followed by phagocytosis of the released iron pigment by the pulmonary alveolar macrophages as had been explained by Cythia et al. [18]. The present study showed collapse of some alveoli and thickened interalveolar septa and subsequent compensatory overexpansion of neighboring alveoli with the destroyed alveolar walls. This is compatible with emphysematous changes as leflunomide induces recurrent pulmonary inflammation which damages and eventually destroys the alveolar walls, creating large air spaces (emphysematous changes). The alveolar septa were initially destroyed, eliminating a portion of the capillary bed and increasing air volume in the alveoli as explained by Eckman and Bartelmo [19].

Moreover, Grommes and Soehnlein [20] explained that destruction of the alveolar septa to be due to proteolytic destruction of the lung parenchyma by the proteolytic enzymes released from the inflammatory cells. Pneumocyte type II hyperplasia might be due to their proliferation in an attempt to regenerate the damaged alveolar cells as pneumocyte type II are the progenitors of pneumocyte type I. This was in harmony with Vallbracht et al. [21]. On the other hand, simultaneous administration of N-acetylcysteine to leflunomide-treated rats resulted in the improvement of general condition of the treated rats. This could be explained by the ability of N-acetylcysteine to act as a free radical scavenger and aprecursor of the antioxidant-reduced glutathione as mentioned by Blesa et al. [22]. Oxidative stress has been suggested as a potential mechanism in the pathogenesis of lung inflammation [23]. N-acetylcysteine has the capacity to inhibit various inflammatory elements related to oxidative stress such as nuclear factor, tumor necrosis factor, inducible nitric oxide synthetase, and cell adhesion molecules [24]. In addition, oxidative stress stimulates mucin synthesis in airways, a process also inhibited by N-acetylcysteine. Deposition of collagen fibers mainly in the pulmonary interstitium, perivascular and peribrochiolar areas was observed in leflunomide-treated rats. The same was reported by Mosquero et al. [25] who explained this by fibroblast activity brought to the irritated area and starts to lay down collagen fibers replacing severely damaged epithelial cells.

Caspase-3 immunostaining of leflunomide-treated rats showed positive immune reaction to caspase-3 protein in thickened interalveolar septa, infiltrating cells, and alveolar epithelial cells, whereas other groups showed negative or mild positive immune reaction in the cytoplasm of alveolar epithelial cells. This agreed with Vassilev et al. [26] and Colic et al. [27] who proved in their studies that leflunomide induced activation of caspase-3 enzymes in human t-lineage leukemia cells and rat thymocytes in vitro.

On an ultrastructural level, the findings demonstrated by electron microscopic study confirming the same were found on light microscopic study of the same group. Injury to pneumoctye type II reduces their ability to synthesize, secrete, and recycle surfactant. Therefore, degenerative changes of lamellar bodies were observed in the form of empty lamellar bodies, leaving empty vacuoles and loss of regular lamellar pattern. These changes might result in decrease in the total pulmonary surfactant and more decrease in the free pulmonary surfactant. This was in harmony with Muller et al. [28] who demonstrated impaired recycling of surfactant-like liposomes in type II pneumocytes from injured lungs. Kumar et al. [29] explained the changes of mitochondria observed in leflunomide-treated rats to be due to inhibition of dihydroorotate dehydrogenase by leflunomide. Moreover, this mitochondrial damage results in cellular degeneration and necrosis with appearance of the nuclear changes as indentation, lysis and pyknosis. Many alveolar macrophages were detected in leflunomide-treated rats. The hypertrophied macrophages filled with large pleomorphic residual bodies containing lamellar-like bodies (dense bodies). This suggests their role in phagocytosis of degenerated pneumocyte type II with their deformed secretory material [30]. In the present work, morphometric and statistical studies showed that there was an increase in the number of pneumocyte type II in leflunomide-treated rats. This increase in number could be considered as an early response of the alveolar wall to injury [31],[32]. It was a regenerative trial to replace the damaged alveolar cells as pneumocyte type II are progenitors of pneumocyte type I [30].

In addition, morphometric and statistical studies showed that there was an increase in the thickness of interalveolar septa in leflunomide-treated rats. This increase was due to excess cellular infiltrate (inflammatory cells), inflammatory exudates, congested capillaries, increased interstitial connective tissue, excess collagen deposition, and the associated alveolar collapse as mentioned before.


  Conclusion Top


It was demonstrated that leflunomide resulted in considerable histological and ultrastructural changes in the lung. N-acetylcysteine minimizes this hazardous effect of leflunomide on the lung and provides a good protective role. Therefore, coadministration of N-acetylcysteine in the patients under leflunomide treatment is highly recommended. To date, there is very little research on the effects of leflunomide on the lung. Therefore, it is essential to encourage other studies. Further studies are recommended to study the effects of leflunomide on other parts of the body to assess its hazardous effects. Moreover, other alternative safe drugs could be used instead of leflunomide.


  Acknowledgements Top


Conflicts of interest

None declared.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

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