Nasal polyposis is a chronic inflammatory disease of the upper airways. It has been suggested that ion transports and CFTR expression could be modified in epithelial cells from nasal polyps of non-cystic fibrosis patients. We compared human nasal epithelial cells from nasal polyps (NP) with control nasal mucosa (CM). The level of CFTR mRNA was studied by Northern blot analysis and protein expression was studied by immunoprecipitation both ex vivo and in vitro in primary cultures of human nasal epithelial cells at the air-liquid interface. Ion transports were evaluated by short-circuit measurements in vitro. CFTR gene and protein expressions were significantly decreased in NP native tissues and in culture on day 4, when a global defect of ion transports was observed in NP cultures, but not in CM. We evaluated the effect of transforming growth factor (TGF)-β1 on CFTR expression and function in NP cultures on day 14 and showed, for the first time, that TGF-β1 was able to significantly downregulate the level of CFTR mRNA and cAMP-dependent current in NP cultures. Finally, we showed that the effects of TGF-β1 on ion transports could be reversed after 48-h removal of TGF-β1 in NP cultures. In conclusion, our data strongly suggest that chronic inflammation in nasal polyposis downregulates CFTR gene and protein expression.
- transforming growth factor-β1
- respiratory epithelium
- ion transport
- primary culture
nasal polyposis is a chronic inflammatory disease of the nasal mucosa in which epithelial modifications (secretory hyperplasia or squamous metaplasia), inflammatory cells, and edema are frequently observed. Although nasal polyposis occurs in many patients, the mechanisms of development of nasal polyps (NP) still remain unclear. Nasal polyposis can be either primary or associated with another underlying disease, such as cystic fibrosis (CF). Moreover, the prevalence of nasal polyposis is ∼50% in CF (9, 31). The high frequency of nasal polyposis in CF patients has led to the hypothesis that constitutive alterations in CFTR protein expression and function may be involved in the development of nasal polyposis in CF. Conversely, it could be speculated that acquired alterations in CFTR expression and function may be involved in the pathophysiology of nasal polyposis in non-CF patients. Moreover, non-CF patients exhibit in vitro abnormalities of ion transports in epithelial cells of NP (2). The very low expression and the exclusive cytoplasmic localization of CFTR protein in NP epithelial cells from non-CF patients also constitute arguments in favor of this hypothesis (4). Such alterations of CFTR expression and function in non-CF polyps could be related to local production of cytokines by inflammatory cells. Recent studies indicate that various inflammatory cytokines may affect CFTR expression and/or function (5, 11, 16), but the effect on CFTR of TGF-β1, which is abundantly expressed in nasal polyposis by inflammatory cells (10, 27), has not yet been investigated. The aim of the present study was therefore, first, to evaluate and compare CFTR gene and protein expression and function in non-CF NP compared with control nasal mucosa (CM) both ex vivo and in vitro and, second, to investigate the in vitro effects of TGF-β1 on CFTR expression and function.
MATERIALS AND METHODS
NP were obtained from 38 non-CF patients requiring surgery for their nasal polyposis. The diagnosis of nasal polyposis was established on the basis of clinical history, endoscopic findings, and computed tomography results. Patients were requested to stop all treatments (i.e., glucocorticoids, antibiotics, and antiallergic drugs) at least 1 mo before surgery. CM was obtained from the inferior turbinate in 22 patients during turbinectomy for snoring. This protocol was approved by the Institutional Review Board and ethics committee of our institution (CCPPRB, Hôpital Henri Mondor), and informed consent was obtained from all patients. Human nasal epithelial cells (HNEC) from NP and CM were studied in either native tissues (NP or CM tissues) or primary cultures (NP or CM cultures).
Ham's F-12 (F-12) nutrient medium and Dulbecco's modified Eagle's nutrient mixture (DMEM), penicillin, streptomycin, amphotericin B, fetal calf serum (FCS), trypsin, EDTA, and Ultroser G were purchased from Invitrogen (Cergy-Pontoise, France). Dithiothreitol, pronase, gentamicin, collagen IV, Igepal, amiloride, forskolin, IBMX, bumetanide, and transforming growth factor (TGF)-β1 from human platelets were obtained from Sigma (Saint Quentin Fallavier, France). Monoclonal antibody M3A7 was purchased from Chemicon (Euromedex, Souffelweyersheim, France). Protein A/G agarose was obtained from Tebu (Le Perray en Yvelines, France). RNA plus was obtained from QBiogen (Illkirch, France). cAMP-dependent protein kinase was purchased from Promega (Charbonnières, France).
Primary cultures of HNEC.
Primary cultures of HNEC were grown from 31 NP and 12 CM samples. Samples were immediately placed in DMEM/F-12 supplemented with antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 μg/ml amphotericin B, and 100 mg/ml gentamicin) and transported to the laboratory for culture procedures. The NP or CM primary cultures were adapted from a culture model originally described with human tracheobronchial cells (18, 28). Briefly, NP or CM samples were rinsed in PBS with dithiothreitol (5 nM) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 μg/ml amphotericin B, and 100 mg/ml gentamicin) and then placed overnight at 4°C in a PBS-antibiotics solution containing 0.1% pronase. The samples were incubated in DMEM/F-12 with 5% FCS before centrifugation (300 g, 7 min). Cell pellets were then suspended in 0.25% trypsin-EDTA solution for 3 min and incubated in DMEM/F-12-antibiotics with 10% FCS before centrifugation and resuspension. Finally, HNEC were plated on permeable supports (Costar, Cambridge, MA), Transwell or Snapwell (1 × 106 cells/insert), for short-circuit measurements. All inserts had a diameter of 12 mm with polycarbonate microporous membranes coated with type IV collagen. HNEC were incubated at 37°C in 5% CO2. For the first 24 h, HNEC were incubated with 1 ml of DMEM/F-12-antibiotics with 2% Ultroser G outside the insert and DMEM/F-12-antibiotics with 10% FCS inside the insert. After 24 h, media (DMEM/F-12-antibiotics with 2% Ultroser G) were changed outside the inserts and removed inside the inserts to place the cells at an air-liquid interface, and media outside the inserts were then changed daily. HNEC reached the most differentiated state during the second and third weeks of culture with the presence of ciliated and secretory cells (28). Transepithelial resistance (Rte) and transepithelial potential difference were measured every 3 days with a microvoltmeter (World Precision Instruments, Astonbury, UK).
RNA preparation and Northern blot hybridization.
We isolated total RNA from native tissues (7 NP, 7 CM) or primary cultures (3 NP, 3 CM) on days 4 and 14 (7) by lysing the cells with the RNA plus reagent according to the manufacturer's instructions. Epithelial cells were removed from native tissues by scraping surface tissues with a surgical knife. RNA purity was checked by spectrophotometry [optical density at 260 nm (OD260)/OD280 ≥1.7] and electrophoresis and stored at −80°C until use. Ten micrograms of total RNA from each sample were separated on a 1% agarose gel with 8% formaldehyde and ethidium bromide in 10% MOPS buffer under denaturing conditions. After transfer onto Hybond nylon membrane (Amersham, Freiburg, Germany), the RNA was cross-linked (UV Stratalinker, Stratagene, Amsterdam, Netherlands). A 1.5-kb CFTR cDNA probe was generated from EcoRI-EcoRI enzyme restriction and then labeled with 32P by random priming (Prime a gene labeling system; Promega, Charbonnières, France). Blots were hybridized with Ultrahyb (Ambion, Huntingdon, UK) at 42°C overnight. mRNA from the HT29 cell line (no. HTB38; ATCC, Manassas, VA), an established cell line derived from a human colon adenocarcinoma, which expresses CFTR mRNA and protein at a high level (25, 35), was used as positive control. All amounts were normalized by hybridization to 18S probe as internal standard. The relative amount of human CFTR mRNA in HNEC was quantified by radioanalytic scanning and compared with CFTR mRNA from HT29 using a Molecular Dynamics PhosphoImager (Amersham, Saclay, France).
Cells from native tissues (3 NP, 3 CM) and primary cultures (7 NP, 3 CM) on days 4 and 14 were detached and lysed in a buffer (pH 7) containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% Igepal, and a cocktail of protease inhibitors 7× (Roche, Meylan, France). Equal amounts of protein lysates (600 μg) were immunoprecipitated (overnight incubation at 4°C) with 0.8 μg of monoclonal antibody M3A7 raised against the COOH-terminal end of the second CFTR NBF from amino acid 1,370–1,380 (20). The cell lysate was then precipitated with protein A/G agarose. Immunoprecipitated proteins were then phosphorylated in vitro for 1 h at 30°C with 10 μCi of [γ-33P]ATP (Amersham, Fribourg, Germany) with 5 units of the catalytic subunit of cAMP-dependent protein kinase and separated by 5% SDS polyacrylamide gel electrophoresis (8). The HT29 cell line was used as positive control. Labeled proteins were detected by PhosphoImager.
Ussing chamber experiments.
Measurements of short-circuit current (Isc) were performed on days 4 and 14 in primary cultures (6 NP, 6 CM). Snapwell inserts were mounted in vertical diffusion chambers and were bathed with Ringer solution (pH 7.4) continuously bubbled with 5% CO2-95% air at 37°C. The apical and basolateral chambers were filled with (in mM): 136.9 NaCl, 5.6 KCl, 1.9 CaCl2, 1.2 MgCl2, 5.9 CH3COONa, 1.3 NaH2PO4, 10 HEPES, and 10 glucose. The transepithelial potential difference was short circuited to 0 mV with a voltage clamp (World Precision Instruments, Astonbury, UK) connected to the apical and basolateral chambers via Ag-AgCl electrodes and agar bridges. The Rte33). Amiloride-treated HNEC were stimulated with forskolin (10−5 M, basolateral side) and IBMX (10−4 M, basolateral side) to reveal cAMP-dependent Cl− secretion (Ifors+IBMX) (34). Finally, we investigated the response to bumetanide (20 μM, basolateral side) (32), a blocker of the basolateral Na+-K+-2Cl− cotransporter. Changes in Isc were calculated as the difference between the initial value and the peak or sustained phase obtained in response to drug addition.Isc and Rte were allowed to stabilize, and drugs were added. Amiloride (10−4 M) was applied to the apical solution to measure the fraction of the basal Isc due to epithelial sodium channel (ENaC) activity (Iamil) (
Stimulation of HNEC cultures by TGF-β1.
We evaluated the effect of TGF-β1 in 12 NP cultures and in 3 CM cultures on day 14. Transwell or Snapwell inserts were exposed basolaterally to TGF-β1 (0.5 or 5 ng/ml) or to standard medium (control) for 24 h. CFTR mRNA and the response to amiloride, forskolin + IBMX, and bumetanide were studied after 24 h. All measurements were compared with NP or CM control cultures. Because of the absence of effect of TGF-β1 at 0.5 ng/ml (data not shown), only the results of 5 ng/ml of TGF-β1 are shown.
Reversible effect of TGF-β1 on ion transports in NP cultures.
Finally, we investigated whether the effect of TGF-β1 on ion transports could be reversed. At the end of the second week, Snapwell inserts of 3 NP cultures were exposed basolaterally to TGF-β1 (5 ng/ml) for 24 h followed by 48 h without TGF-β1. Responses to amiloride, forskolin + IBMX, and bumetanide were evaluated at these two times.
Data from Northern blot analysis and short-circuit measurements are presented as means ± SE. Data from short-circuit measurements were compared between NP and CM cultures with a Mann-Whitney nonparametric test. The same test was used to compare data from short-circuit measurements in NP or CM cultures with TGF-β1 or control. A value of P < 0.05 was considered to be statistically significant.
Decreased expression of CFTR mRNA transcripts in NP tissues and NP cultures on day 4.
In native tissues, CFTR mRNA was detected in all samples from CM and quantified as 12.5 ± 7% of the level of CFTR mRNA detected in HT29. In the samples from NP, CFTR mRNA level was either very low or undetectable as shown in Fig. 1A.
To assess whether the difference of CFTR mRNA was also present in primary cultures, we performed Northern blot on day 4 when cell differentiation was poor and on day 14 when cell differentiation was well established (28). All CM cultures clearly expressed CFTR mRNA both on day 4 and day 14 (quantified as 40 ± 11 and 31 ± 3.5% of the CFTR mRNA level in HT29, respectively) (Fig. 1B). CFTR mRNA was undetectable in NP cultures on day 4 but was clearly detected on day 14 of culture (quantified as 35 ± 5.2% of the CFTR mRNA level in HT29) (Fig. 1C).
Abnormal CFTR protein expression in NP tissues and cultures on day 4.
To verify whether the difference of CFTR gene expression between CM and NP observed in native tissues and cultures on day 4 was also reflected by a difference of CFTR protein, we evaluated CFTR protein expression by immunoprecipitation. In CM tissues, a band C (Fig. 2A) was detected. This diffuse band represents mature, fully glycosylated CFTR protein that has migrated from the Golgi apparatus to the cell membrane (molecular mass of 170 kDa) (6). In contrast, no band C was detected in NP tissues (Fig. 2A). In CM cultures on day 4, two bands were detected: band C and a thin band of ∼140 kDa (band B) that represents the core-glycosylated protein located in the endoplasmic reticulum (6) (Fig. 2B). Neither band C nor band B was detected in NP cultures on day 4 (Fig. 2B), but band C was detectable in NP cultures on day 14 (Fig. 2C).
Reduced ion transports in NP cultures on day 4.
We investigated whether the abnormalities of CFTR detected on day 4 in NP cultures could interact with ion transports and more precisely on the CFTR Cl− current. CM cultures displayed a basal Isc of 32.12 ± 4.11 μA/cm2 and an Rte of 618 ± 38 Ω·cm2. NP cultures displayed a basal Isc of 20.25 ± 2.46 μA/cm2, significantly lower (P < 0.05) than that observed in CM, and an Rte of 709 ± 52 Ω·cm2 (Fig. 3, A and B). Isc was markedly inhibited by amiloride in CM cultures. The mean amplitude of Iamil in CM cultures (12.96 ± 2.93 μA/cm2) was significantly higher (P < 0.005) than in NP cultures (Iamil was 3.42 ± 0.46 μA/cm2) (Fig. 4A). In CM cultures, the peak increase of Ifors+IBMX was 5.62 ± 0.83 μA/cm2, whereas Ifors+IBMX was significantly reduced in NP cultures (P < 0.0005) with a peak increase of 1.56 ± 0.31 μA/cm2 (Fig. 4A). The subsequent addition of bumetanide completely blocked the current induced by forskolin and IBMX in both CM and NP cultures.
Normalization of ion transports in NP cultures on day 14.
Basal Isc and Rte were not significantly different in CM (Isc, 58.57 ± 8.67 μA/cm2; Rte, 916 ± 50 Ω·cm2) and in NP cultures (Isc, 43.67 ± 5.48 μA/cm2; Rte, 801 ± 40.8 Ω·cm2) (Fig. 3, C and D). Iamil and Ifors+IBMX were consistent and not significantly different in CM (Iamil, 32.3 ± 7.7 μA/cm2; Ifors+IBMX, 5.23 ± 0.71 μA/cm2) and in NP cultures (Iamil, 17.84 ± 4.47 μA/cm2, Ifors+IBMX, 4.08 ± 1.56 μA/cm2) (Fig. 4B). Addition of bumetanide completely inhibited the current induced by forskolin and IBMX both in CM and NP cultures.
TGF-β1 downregulates CFTR mRNA transcripts in NP and CM cultures on day 14.
Levels of CFTR mRNA transcripts in NP or CM cultures were markedly reduced (2.9-fold ± 0.7 and 1.9-fold ± 0.25, respectively) by addition of 5 ng/ml of TGF-β1 compared with CFTR mRNA levels in nonstimulated NP or CM cultures (Fig. 5, A and C).
TGF-β1 decreases ion transports in NP and CM cultures on day 14.
To determine whether the decrease of CFTR gene expression induced by TGF-β1 in NP and CM cultures could be correlated with ion transport abnormalities, Isc measurements were performed after addition of TGF-β1. Basal Isc was significantly lower (P < 0.05) in NP and CM cultured with TGF-β1 (26.88 ± 6.72 and 34.7 ± 12 μA/cm2, respectively) compared with controls (43.77 ± 3.2 and 60.61 ± 5.4 μA/cm2, respectively). Rte was not different in NP and CM cultures stimulated by TGF-β1 (979.8 ± 58.4 and 1,020 ± 20.5 Ω·cm2, respectively) compared with the control (833.3 ± 218.6 and 848.5 ± 305.2 Ω·cm2, respectively). TGF-β1 exposure in NP and CM cultures significantly decreased both Iamil (NP: Iamil control: 24.38 ± 6.76 μA/cm2, Iamil TGF-β1: 10.11 ± 2.53 μA/cm2; CM: Iamil control: 31.23 ± 5.26 μA/cm2, Iamil TGF-β1: 18.3 ± 3.9 μA/cm2) (P < 0.05) and Ifors+IBMX (NP: Ifors+IBMX control: 2.04 ± 0.57 μA/cm2, Ifors+IBMX TGF-β1: 0.38 ± 0.1 μA/cm2; CM: Ifors+IBMX control: 4 ± 1.79 μA/cm2, Ifors+IBMX TGF-β1: 1.11 ± 0.42 μA/cm2) (NP, P < 0.0005; CM, P < 0.005) (Fig. 5, B and D). Bumetanide inhibited Ifors+IBMX in both control and TGF-β1-treated NP and CM cultures (data not shown).
Effects of TGF-β1 on ion transports in NP cultures are reversible.
To verify if TGF-β1 effects on ion transports can be reversed, we stimulated NP cultures with TGF-β1 (basolateral side, 5 ng/ml) for 24 h. We performed short-circuit measurements 24 h after TGF-β1 stimulation and 48 h after removal TGF-β1 from medium in the same cultures.
As expected after 24-h exposition with TGF-β1, basal Isc was significantly lower (P < 0.05) in NP cultured with TGF-β1 compared with control (51.6 ± 7 and 84.5 ± 9.2 μA/cm2, respectively), whereas Rte was not different (888 ± 36.6 and 777.8 ± 75.6 Ω·cm2, respectively). TGF-β1 significantly decreased both Iamil (Iamil control, 62.1 ± 9.35 μA/cm2; Iamil TGF-β1, 16.33 ± 2.5 μA/cm2) and Ifors+IBMX (Ifors+IBMX control, 19.81 ± 2.6 μA/cm2; Ifors+IBMX TGF-β1, 4.9 ± 0.72 μA/cm2) (P < 0.005). Forty-eight hours after removal of TGF-β1, previously TGF-β1-treated cultures showed consistent Iamil and Ifors+IBMX that were not different from these observed in untreated control cultures on the same day (basal Isc control, 76.2 ± 10.5 μA/cm2; basal Isc TGF-β1, 68.1 ± 11.8 μA/cm2; Iamil control, 53.28 ± 8.12 μA/cm2; Iamil TGF-β1, 41.4 ± 10 μA/cm2; Ifors+IBMX control, 15.09 ± 2.91 μA/cm2; Ifors+IBMX TGF-β1, 15.5 ± 2 μA/cm2) (Fig. 6).
In this study, we demonstrated first of all, that CFTR was clearly expressed in terms of both mRNA and protein in native tissues from CM, but not from NP. These results are in accordance with the results of previous studies, showing a low level of CFTR mRNA transcripts in the respiratory tract and nasal polyps using RT-PCR (12, 30). To our knowledge, the present study is the first to detect CFTR mRNA by Northern blot analysis in upper airways, allowing a real quantitative evaluation of CFTR mRNA transcripts without amplification. Moreover, in the present study, immunoprecipitation revealed a mature CFTR protein (band C) only in native tissues from CM. The absence of detectable mature CFTR protein in native tissues from NP has already been suggested by immunolabeling in non-CF NP (4). Kälin et al. (19), using immunoblotting, also showed that CFTR protein expression in NP tissues from non CF-patients was very low and only detectable in some of the specimens studied. Altogether, these data show that non-CF NP are not the most appropriate tissues for studying CFTR expression, especially compared with CF tissues.
Second, we have demonstrated that the discrepancy of CFTR gene and protein expressions between CM and NP persists at least after 4 days of culture. CFTR mRNA transcripts were only detectable after 2 wk in NP cultures. In parallel, a mature glycosylated CFTR protein was rapidly detectable in CM cultures and only detected later in NP cultures. To our knowledge, this is the first study comparing CFTR gene and protein expressions at different culture times.
Third, we found that the decreased CFTR expression in nasal polyposis is associated with altered ion transport functions. NP cultures exhibited a global defect of ion transports. Iamil and Ifors+IBMX were dramatically decreased early in NP cultures, but no longer after 2 wk. In our study, in non-CF patients, ENaC and CFTR were therefore downregulated early in NP cultures, contrasting with a previous study in which cultures from NP showed increased ion transports compared with CM using short-circuit measurements (2). In that study, Bernstein and Yankaskas (2) showed that basal Isc and Iamil were enhanced in non-CF NP cultures compared with CM, and responses to selected chloride-negative channel agonists were equivalent. It is important to note that, under their immersion culture conditions, Rte and potential difference, before short-circuit measurements, were much lower than in those observed in our NP or CM cultures at air-liquid interface. Paracellular and cellular electrophysiological properties were therefore not comparable. Actually, the air-liquid interface appears to be the best model to induce a good epithelial differentiation closely resembling in vivo conditions (18) and is therefore more suitable for studying ion transports.
In normal respiratory mucosa, CFTR downregulates ENaC activity (22, 24), suggesting that, as observed in CF tissues, the lower CFTR expression and function observed early in NP cultures would be accompanied by an increased ENaC function. On the contrary, we found a decreased ENaC function suggesting that inflammation could modify the properties of ENaC and CFTR channels. In support of this hypothesis, downregulation of CFTR and ENaC activities was abolished when nasal epithelial cells were extracted from their inflammatory microenvironment after a sufficient culture time. All these findings suggest that the results of studies on CFTR expression and function in epithelial cells from polyps should be interpreted cautiously according to the culture model and culture time.
Finally, we tested the hypothesis that some cytokines overexpressed in nasal polyposis could regulate ENaC and CFTR, especially TGF-β1, which is abundantly secreted in polyps (10, 13, 27). In the present study, addition of TGF-β1 in NP and CM cultures, when CFTR and ENaC were clearly functional, downregulated CFTR expression and function and ENaC function. Our results are in accordance with a recent study showing that TGF-β1 significantly reduces ENaC function and expression in monolayers of primary rat and human alveolar type II cells (14). It has been shown that other cytokines (i.e., IFN-γ, IL-13, and IL-4) can decrease amiloride-sensitive current in airway epithelial cells in vitro (11, 16).
Additionally, we investigated whether the effects of TGF-β1 on ion transports could be reversed. We showed that NP cultures first stimulated with TGF-β1 during 24 h could recover (48 h after TGF-β1 removal) an equivalent ENaC and CFTR function compared with untreated cultures. This result strengthens the hypothesis that inflammation can modulate ion transports in cultures and that these modifications are reversible after extracting epithelial cells from their inflammatory environment.
Various inflammatory stimuli may have contrasting effects on CFTR mRNA expression and function (3, 5, 11, 26). IFN-γ decreases cAMP-dependent current in T84 and HT29 human epithelial cell lines, whereas TNF-α causes few changes in ion transports (3, 16). To our knowledge, our study is the first showing that TGF-β1 can induce modifications of CFTR expression and function and ENaC function in HNEC. Thus in nasal polyposis, inflammatory mediators, especially TGF-β1, could downregulate CFTR expression and function. Further investigations are required to determine the impact of these CFTR abnormalities in the pathophysiology of nasal polyposis. It could be interesting to evaluate other channels and transporters potentially regulated by CFTR, i.e., outward rectifying Cl− (15), Cl− and HCO3− exchanger (23), and the rat outer medullary K+ channel (17). The potential CFTR-induced alterations of intracellular pH of epithelial cells could also be investigated, as a lack of acidification results in a decrease of enzyme-mediated defense function of these cells (29). The decreased ion transport could result in mucous modifications with mucociliary clearance alterations, potentially inducing local infections increasing the inflammatory process (21).
In conclusion, our study shows decreased CFTR expression and function in NP, which can be corrected after 2 wk of culture, suggesting that inflammatory environmental factors could induce changes in CFTR expression and function. We have shown, for the first time, that TGF-β1, a key cytokine in nasal polyposis and more largely in chronic airway inflammation, is able to downregulate CFTR expression and function and ENaC activity. It could be interesting to study the potential impact of TGF-β1 gene polymorphisms in nasal polyposis, such as has already been performed in CF, in which high TGF-β1-secreting patients were shown to have more severe pulmonary disease (1). Future therapeutic strategies could there target TGF-β1 production or its effects.
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