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(Journal of Leukocyte Biology. 2000;68:131-136.)
© 2000 by Society for Leukocyte Biology

Proteasome-mediated regulation of interleukin-1ß turnover and export in human monocytes

Marlena A. Moors and Steven B. Mizel

Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, North Carolina

Correspondence: Steven Mizel, Dept. of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, NC 27157. E-mail: smizel{at}wfubmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-1ß is a secreted protein that accumulates in the cytosol as an inactive precursor (pIL-1ß) before processing and release of biologically active protein. To understand the impact of this property on IL-1ß production, we examined the intracellular stability of pIL-1ß in lipopolysaccharide (LPS)-stimulated human monocytes. Precursor IL-1ß was degraded with a relatively short half-life of 2.5 h in the promonocytic cell line, THP-1, and in primary monocytes. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) stabilized pIL-1ß levels in THP-1 cells, suggesting that degradation was proteasome-mediated, but this inhibitor was toxic for primary monocytes, causing release of pIL-1ß as well as the cytoplasmic enzyme, lactate dehydrogenase (LDH) into supernatants. In contrast, clasto-lactacystin ß-lactone, a specific inhibitor of the proteasome, caused a dose-dependent stabilization of intracellular pIL-1ß, and this led to a corresponding increase in mIL-1ß and pIL-1ß but not LDH release into culture supernatants. Therefore, by regulating intracellular levels of precursor IL-1ß, the proteasome plays an important and previously unrecognized role in controlling the amount of biologically active IL-1ß that is exported by activated monocytes.

Key Words: THP-1 • MG132 • lactacystin • precursor interleukin-1ß • degradation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-1ß (IL-1ß) is produced predominantly by monocytes in response to inflammatory stimuli such as lipopolysaccharide (LPS) and other bacterial products. It has a broad range of effects on immune and nonimmune cells, but its major roles are in lymphocyte activation and the induction of inflammatory responses [1 ]. Injection of nanogram quantities of recombinant IL-1ß into humans rapidly induces fever, hypotension, and the production of other proinflammatory mediators [2 , 3 ], effects that are similar to those induced by LPS. In addition, IL-1ß has been implicated in the etiology of certain inflammatory and autoimmune diseases [reviewed in ref. 1 ]. Given these biological effects, it is important to understand the regulatory factors that control IL-1ß production and release by inflammatory monocytes.

IL-1ß is synthesized as a 33-kDa precursor protein (pIL-1ß) that is cleaved intracellularly by IL-1ß converting enzyme (ICE) to yield the 17-kDa mature form (mIL-1ß) [4 , 5 ]. Monocytes have the capacity to secrete both pIL-1ß and mIL-1ß, however, mIL-1ß is the biologically active form [6 , 7 ]. Precursor but not mIL-1ß is found in cellular lysates, therefore it is likely that processing and secretion are intimately coordinated events [8 ].

A feature of IL-1ß regulation that is of particular interest is its capacity to be exported from the cell in the absence of a classic, hydrophobic signal sequence. Export of IL-1ß proceeds via a novel pathway that bypasses the endoplasmic reticulum and Golgi apparatus [9 , 10 ]. Although little is known about this export mechanism, it has been demonstrated that, compared to proteins secreted by the conventional, signal sequence-directed pathway, release of IL-1ß is a relatively slow process. The precursor protein accumulates in the cytosol before release [9 , 10 ] and significant quantities of IL-1ß are not detectable in monocyte supernatants until approximately 2 h after synthesis [8 ]. In contrast, proteins secreted by the conventional pathway appear in supernatants within minutes after synthesis [11 ].

Proteins that reside in the cytosol for any length of time after synthesis are potential targets for proteolysis. The proteasome, an ATP-dependent multicatalytic protease complex, is responsible for degrading the majority of normal and damaged cytosolic proteins, as well as foreign proteins for immune recognition [12 ]. However, under certain circumstances other proteases, including the calcium-activated calpains and lysosomal proteases, can also degrade cytosolic proteins [13 , 14 ]. Given the unusual features of IL-1ß export, an important issue in the regulation of IL-1ß production is the intracellular stability of the precursor protein. We demonstrate here that cytosolic pIL-1ß is degraded fairly rapidly by the proteasome and that blocking the proteolytic activity of the proteasome enhances release of IL-1ß by activated monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). PD-150606 and clasto-lactacystin ß-lactone were obtained from Calbiochem (San Diego, CA). E-64d was obtained from Sigma Chemical (St. Louis, MO). Inhibitors were prepared as stock solutions in dimethyl sulfoxide (DMSO). The preparation and use of polyclonal anti-IL-1ß antibody has been described previously [15 ].

Cell culture
The human monocytic cell line, THP-1, obtained from the American Type Culture Collection, was maintained at a density of 2.5 x 105/mL to 1 x 106/mL in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 50 µg/mL gentamicin sulfate (complete RPMI).

Isolation of human peripheral blood monocytes
Peripheral blood mononuclear cells from healthy donors were purified by density gradient centrifugation and cultured as described previously [16 ]. Briefly, 5 x 106 to 1 x 107/well mononuclear cells per well were cultured in 24-well dishes for 2 h in serum-free RPMI 1640, after which nonadherent cells were removed by washing with phosphate-buffered saline (PBS). Adherent cells were cultured for 16–18 h in complete RPMI before assay.

Pulse/chase analysis, THP-1 cells
For assays, 5 x 106 log-phase THP-1 cells per sample were centrifuged and pellets were resuspended in 1.5 mL of methionine- and cysteine-free RPMI 1640 (ICN Pharmaceuticals, Irvine, CA) containing 5% dialyzed FBS (labeling medium). Cells were cultured in six-well dishes for 1 h in the presence of 100 pg/mL Escherichia coli LPS (Sigma Chemical) after which 250 µCi/mL Tran35S-Label (ICN) was added for an additional hour. Labeled cells were centrifuged and cell pellets were immediately lysed with 0.5 mL ice-cold immunoprecipitation buffer (150 mM NaCl, 0.4% Nonidet P-40, 50 mM Tris, pH 8.0, 10 mM EDTA) containing Calbiochem Protease Inhibitor Cocktail Set 1, or were chased with RPMI 1640 containing 5% FBS and 5 mM methionine (chase medium). After the indicated chase periods, cells were centrifuged at 4°C, supernatants were collected and concentrated to approximately 0.5 mL using Centricon-10 microconcentrators (Amicon, Danvers, MA), and cell pellets were lysed as described above. Lysates were cleared of particulate material by centrifugation at 4°C before immunoprecipitation.

Pulse/chase analysis, primary human monocytes
For assays, adherent cells were washed once with PBS and 0.25 mL/well labeling medium containing 100 pg/mL LPS was added. Cells were cultured for 1 h and labeled for an additional hour as described for THP-1 cells. After the labeling period, cells were washed once with PBS and lysed with ice-cold immunoprecipitation buffer or chased with 0.25 mL/well chase medium. After the indicated chase periods, supernatants were collected and gently cleared of detached cells by centrifugation at 260 g, and monolayers were lysed and cleared of particulate material as described above.

Immunoprecipitation
IL-1ß was immunoprecipitated from THP-1 or monocyte lysates and supernatants using polyclonal anti-IL-1ß antibody as described previously [17 ]. Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gels were processed for autoradiography as described previously [17 ]. In addition, dried gels were scanned and pIL-1ß bands were quantitated using an Ambis radioanalytic imaging system. Results are expressed as the % IL-1ß remaining after the chase period, relative to the time 0 (no chase) control. Experiments were performed at least three times, and in each case, a representative gel is shown.

Assay for IL-1ß release by primary human monocytes
Monocytes were prepared and cultured as described for immunoprecipitations but were activated to synthesize IL-1ß for 3 h with 1 µg/mL LPS. This was followed by an additional incubation in medium containing LPS with or without clasto-lactacystin-ß lactone. Culture supernatants were harvested after 5 h and gently cleared of detached cells by centrifugation. The amount of pIL-1ß and mIL-1ß in the samples was determined by enzyme-linked immunosorbent assay (ELISA; R & D Systems, Minneapolis, MN). As determined by the manufacturer, the pIL-1ß ELISA is specific for pIL-1ß and does not cross-react with mIL-1ß. The mIL-1ß ELISA cross-reacts at a level of approximately 13% with pIL-1ß. ELISAs were run in parallel on each sample, and values reported for mIL-1ß have been corrected by subtracting 13% of the total pIL-1ß value detected for each sample.

Assay for lactate dehydrogenase (LDH) activity
Supernatants were collected after the indicated chase periods from monocyte cultures that were treated exactly as described for pulse/chase analysis except the 35S-methionine label was omitted. LDH activity released into supernatants was measured using the Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Madison, WI) according to the manufacturer’s instructions. Percent cytotoxicity was calculated as follows: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Half-life of pIL-1ß in THP-1 cells
To address the issue of protein half-life in the regulation of IL-1 production, we examined the stability of pIL-1ß in the human monocytic cell line, THP-1. To measure the rate of protein turnover due to intracellular degradation, we stimulated the cells for 2 h with 100 pg/mL LPS. As shown by pulse/chase and immunoprecipitation analysis, THP-1 cells stimulated with a low concentration of LPS synthesize pIL-1ß but do not process or secrete the protein (Fig. 1B ). As shown in Figure 1A and 1B , the 33-kDa pIL-1ß protein is degraded with a half-life of 2.5–3 h in THP-1 cells.



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  		Figure 1. Pulse/chase analysis of pIL-1ß in THP-1 cells. (A) IL-1ß was immunoprecipitated from 35S-methionine-labeled THP-1 cell lysates immediately after the labeling period (time 0) or after chasing with cold methionine for 1, 2, 3, or 5 h. pIL-1ß bands were quantitated using an Ambis radioanalytic imaging system and results are expressed as the percent pIL-1ß remaining after the chase, relative to the time 0 control (% control). (B) IL-1ß was immunoprecipitated from 35S-methionine-labeled THP-1 lysates and supernatants immediately after the labeling period (time 0) or after chasing for 2.5 or 5 h. Percent control values are indicated as in panel A. Molecular weight markers are shown in the first lane. The arrow indicates the 33-kDa pIL-1ß band.

 
MG132 stabilizes pIL-1ß in THP-1 cells
Degradation of most cytosolic proteins is mediated by the proteasome [12 ]. To determine whether the proteasome degrades pIL-1ß, THP-1 cells were pulse-labeled and chased for 2.5 and 5 h in the presence and absence of MG132, one of a group of tripeptide aldehydes that block activity of the proteasome [12 , 18 ]. As shown in Figure 2 , treatment with 50 µM MG132 significantly stabilized pIL-1ß levels. Approximately 90% of the total pIL-1ß synthesized was found in cell lysates after a 2.5-h chase in the presence of inhibitor, compared with 60% in the absence of inhibitor. Similarly, after a 5-h chase, 79% of the total pIL-1ß remained compared with 27% in the absence of inhibitor. MG132 stabilized pIL-1ß levels almost completely at 10–50 µM, the effective concentration range of tripeptide aldehydes for inhibition of proteasome function in intact cells (data not shown) [12 ]. In no case was IL-1ß found in significant amounts in THP-1 cell supernatants (Figs. 1 and 2) . These results suggest that degradation of pIL-1ß in THP-1 cells is proteasome-mediated.



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  		Figure 2. Stabilization of pIL-1ß in THP-1 cells by MG132. Pulse/chase analysis was performed as described in Figure 1 , except that in some samples MG132 was included during the chase period at a final concentration of 50 µM. IL-1ß was immunoprecipitated from THP-1 cell lysates and supernatants at the indicated time points.

 
Degradation of pIL-1ß in primary human monocytes
Having obtained evidence that the proteasome degrades pIL-ß in THP-1 cells, it was important to analyze the regulation of pIL-1ß turnover in primary human monocytes. As for THP-1 cells, pIL-1ß was degraded with a half-life of approximately 2.5 h (Fig. 3 ), although some donor-to-donor variability was observed (mean half-life = 2.3 ± 0.6 h; range 1–3 h; n = 16). Again, little to no IL-1ß was detected in monocyte supernatants, indicating that turnover of cytosolic pIL-1ß was due to degradation and not to processing and secretion. In contrast to THP-1 cells, MG132 failed to stabilize pIL-1ß in primary monocytes (Fig. 4 ). There was no change in the degradation of pIL-1ß in the presence and absence of inhibitor as measured over 2.5- and 5-h chase periods. However, significant quantities of pIL-1ß were found in supernatants of cells treated with 50 µM MG132. To determine whether release of pIL-1ß in the presence of MG132 was specific for IL-1ß or was a result of general cytotoxicity, we measured levels of the cytoplasmic enzyme lactate dehydrogenase (LDH) in parallel. LDH was detectable in supernatants of cells treated with MG132 but not in culture supernatants from control cells (Fig. 4) . In a similar experiment, the appearance of pIL-1ß and LDH in supernatants after a 2.5-h chase in the presence of MG132 correlated with an increase in the percentage of trypan blue-positive (TB+) cells (5 and 27.5% TB+ cells in the absence and presence of MG132, respectively). These results indicate that release of pIL-1ß in MG132-treated cells was due to cytotoxicity. In addition, we were unable to demonstrate stabilization of pIL-1ß at MG132 concentrations that were not toxic for monocytes; treatment with 3.5-fold and 10-fold lower concentrations of inhibitor did not stabilize pIL-1ß or induce its release (data not shown).



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  		Figure 3. Pulse/chase analysis of pIL-1ß in primary human monocytes. IL-1ß was immunoprecipitated from 35S-methionine-labeled monocyte lysates (A) and supernatants (B) immediately after the labeling period (time 0) or after chasing with cold methionine for 2, 3, or 5 h. pIL-1ß bands were quantitated, and results are expressed as described in Figure 1 .

 


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  		Figure 4. MG132 induces release of pIL-1ß and LDH from primary human monocytes. Pulse/chase analysis was performed as described in Figure 3 , except that in some samples, the tripeptide aldehyde MG132 was included during the chase period at a final concentration of 50 µM. pIL-1ß was immunoprecipitated from monocyte lysates and supernatants at the indicated time points. In parallel, LDH activity was measured in supernatants using the Promega Cytotox96 kit, and results are expressed as percent cytotoxity, calculated as described in Materials and Methods.

 
Because MG132 stabilizes pIL-1ß in THP-1 cells but is toxic for primary monocytes we evaluated the effects of three additional protease inhibitors on pIL-1ß degradation in monocytes. Unlike the peptide aldehyde class of inhibitors [12 ] clasto-lactacystin ß-lactone is a specific inhibitor of the proteasome [19 , 20 ]. PD-150606 is a specific inhibitor of the calcium-activated µ- and m-calpains, which are found in the cytosol [21 ]. E-64d inhibits the activity of cysteine proteases including cathepsins, which are present in lysosomes, as well as the calpains [22 ]. As shown in Figure 5 , treatment of monocytes with clasto-lactacystin ß-lactone stabilized pIL-1ß in a dose-dependent manner. In similar experiments, 50 µM PD-150606 and E-64d had no effect on degradation of pIL-1ß. For PD-150606, 47% of the pIL-1ß synthesized was detected after a 2.5-h chase in the absence of inhibitor, versus 40% in the presence of inhibitor. Similarly, 57% of pIL-1ß remained after a 2.5-h chase in the absence of E64-d compared with 58% in the presence of E64-d. No IL-1ß was detected in supernatants of cells treated with PD-150606 or E-64d (data not shown). In three of four monocyte donors treated with clasto-lactacystin ß-lactone, pIL-1ß was stabilized in a dose-dependent manner and no IL-1ß was not detected in culture supernatants by immunoprecipitation (data not shown). In the fourth donor, shown in Figure 5 , despite stabilization of intracellular pIL-1ß, some protein was detected in supernatants, and the amount increased with increasing doses of inhibitor. This is in contrast to the effect of MG132 on monocytes, in which stabilization of intracellular pIL-1ß could not be demonstrated, and large quantities of pIL-1ß were immunoprecipitated from supernatants.



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  		Figure 5. The proteasome degrades pIL-1ß in primary human monocytes. Pulse/chase analysis was performed as described in Figure 3 , except that in some samples, clasto-lactacystin ß-lactone was included at the indicated concentrations during the 2.5-h chase period. IL-1ß was immunoprecipitated from monocyte lysates and supernatants at the indicated time points.

 
Clasto-lactacystin B-lactone augments release of IL-1ß from activated human monocytes
We next determined the effect of stabilizing intracellular pIL-1ß on levels of IL-1ß released into supernatants. For these experiments, we used an ELISA rather than radiolabeling and immunoprecipitation to achieve greater sensitivity of detection. As shown in Table 1 , both pIL-1ß and mIL-1ß levels in supernatants of LPS-activated monocytes increased approximately twofold in the presence of 15 µM lactacystin relative to controls without lactacystin. These results are consistent with the results of pulse/chase experiments that demonstrated an approximately twofold stabilization of intracellular pIL-1ß levels in the presence of this concentration of lactacystin (Fig. 5 , compare chase without lactacystin to chase with 15 µM lactacystin). Unlike MG132 treatment, lactacystin treatment did not induce release of significant quantities of LDH into culture supernatants, indicating that toxicity did not account for the increase in IL-1ß release. These results demonstrate that proteasome-mediated degradation of intracellular pIL-1ß serves to restrict the amount of IL-1ß released by activated monocytes.


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  		Table 1. IL-1ß Release in Clasto-Lactacystin ß-Lactone-Treated Monocytes

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IL-1ß is an exported protein that accumulates in an inactive precursor form before processing and release from activated monocytes. This quality distinguishes IL-1ß from other proinflammatory cytokines such as tumor necrosis factor {alpha} and IL-6 that have an amino-terminal signal sequence, and are exported via the conventional secretory pathway within minutes after synthesis [8 , 26 ]. Using pulse/chase analysis and a series of cell-permeable protease inhibitors, we have shown that one consequence of this property is that the IL-1ß precursor protein is subject to degradation by the proteasome, the major cytosolic protease, with an intracellular half-life of approximately 2.5 h. That the proteasome is responsible for the degradation of intracellular pIL-1ß is consistent with immunostaining studies that have localized pIL-1ß to the cytoplasm of activated monocytes [9 , 10 ].

A previous study designed to address the kinetics of IL-1 release reported a similar turnover rate for pIL-1ß in monocytes [8 ]. However, in that study, monocytes were activated with a 100-fold higher concentration of LPS than was used in this study and the intracellular half-life of pIL-1ß was a function of processing and release as well as degradation. To focus on the impact of protein stability on the regulation of IL-1ß production, we have used a low concentration of LPS to promote synthesis of pIL-1ß, but not processing and secretion [27 ]. This approach allowed us to accurately determine the rate as well as the mechanism of degradation of precursor protein restricted to the cytosol. Increasing the activating dose of LPS from 100 pg/mL to 1 µg/mL did not change the intracellular half-life of pIL-1ß as measured by pulse/chase analysis over 2.5 h (data not shown), but it did induce processing of pIL-1ß and release of both forms of IL-1ß into supernatants (Table 1) [27 ]. Clasto-lactacystin ß-lactone, a specific inhibitor of the proteasome, stabilized intracellular pIL-1ß levels approximately twofold and enhanced IL-1ß release approximately twofold without a concomitant increase in LDH release. Therefore, enhancement of IL-1ß release by lactacystin is, unlike MG132, not related to a cytotoxic effect. These data indicate that susceptibility of pIL-1ß to proteasome-mediated degradation is functionally relevant.

The susceptibility of a given cytosolic protein to degradation is a function of multiple factors, including primary sequence, posttranslational modification and folded structure of the protein, and subcellular location [28 ]. Reported half-lives of normal cytoplasmic proteins range from less than 30 min, as exemplified by ornithine decarboxylase, to greater than 200 h, as exemplified by phosphoglycerate kinase [29 , 30 ]. In this context, the 2.5-h intracellular half-life of pIL-1ß is relatively short. The relative instability of cytosolic pIL-1ß has important implications for IL-1ß regulation, especially in light of a study which demonstrated that significant quantities of IL-1ß are not released from LPS-activated monocytes until approximately 2 h after synthesis of the precursor protein [8 ]. Our results expand on that observation by showing that the relatively slow IL-1ß release mechanism exposes pIL-1ß to proteasome-mediated digestion in the cytosol. The rate of pIL-1ß degradation is therefore a critical factor that dictates availability of pIL-1ß for processing and release by activated monocytes. Because the half-life of pIL-1ß and the delay in release are similar, the data collectively suggest that a delicate balance of two pathways, degradation versus processing and release, controls the amount of IL-1ß released by activated monocytes.

Proteasome inhibitors have been reported to exert cytotoxic effects on various cell types [23 24 25 ]. We have found that for monocytes, the severity of this effect is donor-dependent as well as inhibitor-dependent. MG132 induced release of IL-1ß as well as LDH by primary monocytes, whereas lactacystin induced release of IL-1ß but not LDH. Unlike primary monocytes, we did not observe MG132-induced cytotoxicity with THP-1 cells, at least by the criteria of pIL-1ß and LDH release. This variable toxicity might be explained simply by concentration effects and/or may be due to different levels of in vivo priming among donors; that is, more than one signal may be required to sensitize monocytes to proteasome inhibitor-mediated toxicity. Variability among monocyte donors was clearly reflected in the small but significant differences observed for pIL-1ß half-life (see Results). Because we were able to demonstrate effects of MG132 in THP-1 cells and lactacystin in primary monocytes in the absence of cytotoxicity, these observations were not central to our conclusions, and we did not investigate them further.

IL-1ß is a potentially toxic cytokine, and as such its production and activity are regulated at many levels, including transcription, message stability, translation, and the production of a receptor antagonist [reviewed in 1, 31]. In particular, transcription of the IL-1ß gene in response to LPS is rapid and vigorous, but begins to decline within several hours of stimulation [31 ]. The opportunity to synthesize high levels of IL-1ß is therefore relatively transient. The results of this study indicate another limiting step in IL-1ß production, imposed by proteasome-mediated degradation of the inactive IL-1ß precursor. This represents an additional and previously unrecognized regulatory mechanism that may serve two purposes: first to restrict intracellular levels of pIL-1ß until the monocyte receives an appropriate activation signal, and second, to limit the amount of biologically active IL-1ß secreted upon monocyte activation.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Ciblex Corporation and by the Signal Transduction Mechanisms and Cell Function training program, Grant CA-09422, from the National Institutes of Health.

Received October 22, 1999; revised February 28, 2000; accepted February 29, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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