Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability

Zoltán Prohászka1,2, Jenõ Duba3, Gabriella Lakos4, Emese Kiss4, Lilian Varga5, Lívia Jánoskuti1, Albert Császár1,2, István Karádi1,2, Kálmán Nagy6, M. Singh7, László Romics1,2 and George Füst1,2

1 Third Department of Medicine, Semmelweis University of Medicine, Budapest, Hungary
2 Research Group of Metabolism, Genetics and Immunology, Hungarian Academy of Sciences, Budapest, Hungary
3 National Institute of Cardiology, Budapest, Hungary
4 Third Department of Medicine, University Medical School of Debrecen, Debrecen, Hungary
5 National Institute of Haematology and Immunology, Budapest, Hungary
6 Child Health Center, County Hospital Miskolc, Miskolc, Hungary
7 German National Research Center for Biotechnology, 38124 Braunschweig, Germany

Correspondence to: : Z. Prohászka, Kútvölgyi út 4, Budapest 1125, Hungary


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Although complement activation appears to have an important role both in the early and late phases of atherosclerosis, the exact mechanism of the initiation of this activation is still unknown. Since injuries of the endothelial cells are known to result in increased stress-protein expression we tested the complement-activating ability of recombinant human 60 kDa heat-shock protein (hsp60). Human hsp60 was found to activate the complement system in normal human serum in a dose-dependent manner. Activation took place through the classical pathway. The lack of complement activation in agammaglobulinemic serum indicates that the classical pathway is triggered by anti-hsp60 antibodies. Hsp60 activated complement in the sera of 74 patients with coronary heart disease as well, and a strong positive correlation (r = 0.459, P < 0.0001) was found between the extent of complement activation and the level of anti-hsp60 IgG antibodies but there was no correlation to the level of anti-hsp65 IgG antibodies. Further distinction between anti-hsp60 and anti-hsp65 antibodies was obtained from competitive ELISA experiments: binding of anti-hsp60 antibodies to hsp60-coated plates was inhibited only by recombinant hsp60 and vice versa. Our present findings indicate that anti-hsp60 and anti-hsp65 antibodies are distinct, showing only partial cross-reactivity. Since complement activation plays an important role in the development of atherosclerosis and the levels of complement-activating anti-hsp60 antibodies are elevated in atherosclerosis-related diseases, our present findings may have important pathological implications.

Keywords: anti-hsp60 antibodies, anti-hsp65 antibodies, atherosclerosis, classical pathway, complement activation, human heat-shock protein 60, mycobacterial heat-shock protein 65


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
According to the recent experimental and clinical data, complement activation has an important role in the development of atherosclerosis (reviewed in 1). Complement activation may contribute to the increase of vascular permeability and accumulation of a special cholesterol-rich lipid fraction, called lesion complement activator, that may further amplify activation of the complement within the arterial wall (2). Complement activation seems to have an important role in the later events of atherogenesis, too, such as in recruitment of monocytes to the site of initial lesion (3), the foam cell formation from blood monocytes (4) and the activation of the smooth muscle cells that express receptors for the most potent chemoattractant complement fragment, C5a (5).

One of the initial steps of atherogenesis is the injury of the endothelial cells by different stimuli, like mechanical stress, smoking, immune reactions, toxins, cytokines, infectious agents and oxidized low-density lipoprotein (6). Injury of the endothelial cells may induce increased expression of some cellular proteins such as different families (60 and 70 kDa) of heat-shock proteins (hsp) or adhesion molecules (ICAM-1 and VCAM-1) (7). Although there are data indicating that complement may be activated by antibodies against different epitopes on endothelial cells (8), these epitopes have not been definitely characterized yet.

One of the most frequently used model of the physical injury which may lead to endothelial cell activation and dysfunction is ischemia followed by reperfusion. Reperfusion injury of ischemic myocardium and skeletal muscle is mediated by complement activation (9,10). Recently, Collard et al. (11) demonstrated that, by an unknown mechanism, hypoxia renders human endothelial cells to be able to activate complement in human serum through the classical pathway and this activation is markedly augmented by reoxygenation.

Considering the well-established increased expression of hsp60 in activated endothelial cells (7) as well as the presence of increased amounts of IgG-type anti-mycobacterial hsp65 antibodies in sera of patients with atherosclerotic diseases (12), we assumed that the hsp60 proteins may possess complement-activating ability, possibly through anti-hsp60 antibodies. Since, to the best of our knowledge, no data on the complement-activating ability of any type of hsp have been reported yet, we investigated complement activation by solid-phase recombinant hsp60. In order to characterize the activation, we also assayed the ability of anti-hsp60 antibodies to activate complement. For this purpose sera of patients with coronary heart disease (CHD) were used for the study since they were reported to contain anti-mycobacterial hsp65 antibodies in elevated concentration as compared to healthy individuals (12). We observed a similar difference in anti-hsp65 titers like that of Hopplicher et al. (12) in a large number (357) of patients with severe CHD (Prohászka et al., manuscript in preparation) which allowed us to select serum samples stored at –70°C for the present investigation. Since not only antibodies against Mycobacterium bovis hsp65, but anti-human hsp60 were found to be elevated in this samples, a possible cross-reaction between anti-hsp60 and anti-hsp65 antibodies was also investigated.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hsp
Recombinant human hsp60 (SPP-740; batch 712403) antigen was obtained from StressGen Biotechnologies (Victoria, Canada); the recombinant hsp65 (M. bovis BCG 65K; batch MA14) antigen was produced in GBF (Braunschweig, Germany), financially supported by the UNDP/World Bank/WHO Special programme for Research and Training in Tropical Diseases (TDR).

Patients
Seventy-four patients (60 males and 14 females, median age 60 years) with CHD were enrolled in this study. The basic data of the patients are summarized in Table 1Go. All patients underwent coronary angiography and by-pass operation. After informed consent serum samples were taken 6 months after the operation to rule out the influence of acute disease and stress. Sera were aliquoted and kept at –70°C until use. The 74 patients were randomly selected from 357 patients according to their anti-hsp60 antibody levels [37 patients with `high' (higher than median) and 37 with `low' (lower than median) levels.


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Table 1. Basic data of the patients with known levels of anti-hsp60 antibodies
 
Complement-activation ELISA
ELISA for the determination of the complement-activating ability of solid-phase human hsp60 or mycobacterial hsp65 was performed as described before (13,14). In brief, ELISA plates were coated with different amounts of hsp60 or with 0.1 µg/well M. bovis hsp65. After washing wells were incubated either with 50 µl of normal human serum (NHS; pooled serum from 10 young, healthy individuals) or with heat-inactivated (56°C, 30 min) human serum (HIHS), prediluted 1:1 with veronal-buffered saline containing Ca2+ and Mg2+ or with Mg2+-EGTA-chelated serum or with a serum of a homozygous C2-deficient systemic lupus erythematosus patient for 30 min at 37°C. In an other set of experiments individual patient's sera were used prediluted 1:1 with veronal-buffered saline containing Ca2+ and Mg2+. The amounts of complement proteins fixed to the plate were determined with specific goat anti-C4b and goat anti-C3b antibodies (Atlantic Antibodies, Stillwater, MN).

Agammaglobulinemic sera
Two serum samples with very low levels of antibodies were used in the complement-activation enzyme-immune assays. Agammaglobulinemic serum AGS-1 was obtained from a 4-year-old boy. The levels of IgG-, IgA- and IgM-type antibodies were 0.375, <0.01 and 0.037 g/l respectively; all complement parameters tested (CH50, C3 and C4 levels, and hemolytic activity) were in the normal range. The agammaglobulinemic serum AGS-2 was donated by a 33-year-old woman. IgG, IgA and IgM levels were 0.380, <0.063 and <0.042 g/l respectively; all complement parameters tested (C3 and C4 levels, and haemolytic activity) were in the normal range. The sera were obtained and used after informed consent.

Determination of anti-hsp60/65 antibodies
The amounts of IgG-type antibodies reacting with chaperonin 60 family (recombinant human hsp60 and recombinant M. bovis hsp65) proteins were assessed by ELISA as described previously (15). Briefly, plates were coated with 0.1 µg/well human hsp60 or M. bovis hsp65. After washing and blocking (PBS, 0.5 % gelatine) wells were incubated with 100 µl of serum samples diluted 1:500 in PBS containing 0.5% gelatine and 0.05% Tween 20. Binding of anti-hsp antibodies was determined using {gamma}-chain-specific anti-human IgG peroxidase-labeled antibodies (Sigma, St Louis, MO) and the o-phenylenediamine (Sigma) detection system. The optical density was measured at 490 nm (reference at 620 nm) and means of duplicate wells were calculated. A serial dilution of a control anti-hsp60 rabbit polyclonal antiserum (StressGen SPA-804) was used as standard. Data obtained as optical density values were calculated to U/ml values related to this standard.

Competition ELISA
One step of the above described ELISA test was modified in order to assess the antibody-competing effect of recombinant hsp. The sera to be tested were diluted in PBS containing 0.5% gelatine and 0.05% Tween 20, and an additional 5 µg/ml hsp60 or hsp65. PBS was added in control samples. The assay was performed as described above.

Statistical analysis
Correlation coefficients were calculated by the non-parametric Spearman method. Group comparisons were computed either with Student's t-test or with the non-parametric Mann–Whitney test using GraphPad Prism 2.0 (San Diego, CA; www.graphpad.com).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Complement activation by human hsp60
In order to test whether solid-phase human hsp60 can induce the activation of the complement cascade, a previously established (13,14) solid-phase ELISA test system was applied. Hsp60-coated ELISA plates were incubated with NHS [containing medium-level (114.8 ± 15.7 U/ml, mean ± SD) anti-hsp60 antibodies], and C4b and C3b binding was measured (Table 2Go). Markedly higher binding of both complement proteins to hsp60-coated plates than uncoated ones indicates that human hsp60 can trigger the complement cascade. Since heat inactivation of the NHS reduced the binding of C4b and C3b to the background level, the activation of the complement system by human hsp60 seems to be specific. This activation takes place through the classical pathway since neither C4b nor C3b binding was seen in Mg2+-EGTA-containing serum. The chelation of the Ca2+ ions is known to inhibit the classical pathway. The classical pathway activation by human hsp60 was further evidenced by showing the lack of C3b binding from a serum obtained from a homozygous C2-deficient systemic lupus erythematosus patient, despite the fact that the initiation of the classical pathway took place, since significant C4b binding was seen in this serum (Table 2Go). The specificity of the classical pathway activation by human hsp60 was proved by the demonstration of its dose dependence (Fig. 1Go).


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Table 2. Complement activation by solid-phase human hsp60
 


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Fig. 1. The complement activation by solid-phase hsp60 is dose dependent. Human hsp60 was incubated either with NHS ({blacksquare}) or with HIHS ({blacklozenge}), the amount of C4b fixed to the plate was detected with specific anti-C4b antibody. As control, an uncoated plate was incubated with NHS ({circ}) or with HIHS ({triangledown}). The figure shows mean OD values ± SD of four parallel measurements. One representative experiment out of three similar ones. Significantly increased C4b binding was seen in the indicated (**) case as compared to the uncoated plate (Student's t-test, P < 0.01).

 
Hsp60 triggers the classical pathway through anti-hsp60 antibodies
The initiation of the classical pathway of the complement cascade can proceed through two ways: by immune complexes, or by direct binding of the first component (C1q) by certain molecules, viruses or bacterial products. In the next set of experiments the initiation of the classical pathway activation by hsp60 was tested. To tackle this question solid-phase hsp60 was incubated with agammaglobulinemic serum samples. As shown in Fig. 2Go, no significant classical pathway activation was observed in either agammaglobulinemic serum. The reconstitution of the AGS-1 serum with antibodies (HIHS) resulted in a significant classical pathway activation, indicating that hsp60 triggers the classical pathway only in the presence of antibodies.



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Fig. 2. Lack of classical pathway activation by human hsp60 in agammaglobulinemic serum. Human hsp60 (0.1 µg/well) was incubated either with NHS or with two different agammaglobulinemic serum, the amount of C4b fixed to the plate was detected with specific anti-C4b antibody. Uncoated wells were applied as control. AGS + IgG means reconstitution of agammaglobulinemic serum with antibodies (5% HIHS). One representative experiment out of two identical ones. Significant C4b binding was seen in the indicated (***P < 0.0001, *P < 0.05) case as compared to the uncoated plate (Student's t-test).

 
Correlation between hsp60-induced complement activation and the level of anti-hsp60 antibodies
In order to assess the influence of specific anti-hsp60 antibodies (known to be present in different amounts in sera of patients with atherosclerotic lesions) on the classical pathway activation by human hsp60, we tested the extent of hsp60-induced classical pathway activation (binding of C4b to hsp60-coated plates) in individual serum samples with known amounts of anti-human hsp60 and anti-M. bovis hsp65 antibodies. These samples were taken 6 months after by-pass surgery of 74 CHD patients. A highly significant positive correlation (r = 0.459, P < 0.0001) between the extent of hsp60-induced classical pathway activation (measured by C4b binding) and the amount of anti-human hsp60 antibodies was seen (Fig. 3AGo). By contrast, the extent of classical pathway activation did not correlate with the level of anti-M. bovis hsp65 (r = –0.09, P = 0.451) antibodies despite the fact that the amount of anti-hsp65 antibodies correlated very strongly with the amount of anti-human hsp60 antibodies (r = 0.425, P = 0.0005). In a part of the same serum samples the classical pathway activation by M. bovis hsp65 was analyzed (Fig. 3BGo). The classical pathway activation in individual sera by hsp65 was much weaker as compared to hsp60 and seemingly not influenced by the amount of anti-hsp65 antibodies (Spearman correlation coefficient r = 0.106, P = 0.554). Next we analyzed if there was a difference in the extent of hsp60-induced classical pathway activation in serum specimens with a high or low level of anti-human hsp60 antibodies. The classical pathway activation by hsp60 in sera with higher-than-median level of anti-hsp60 antibodies was significantly higher than in sera with lower-than-median level anti-hsp60 antibodies (P = 0.0001, Mann–Whitney test). The same difference was not seen in the case of mycobacterial hsp65-induced classical pathway activation and anti-hsp65 antibodies (P = 0.787).



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Fig. 3. (A) Correlation of anti-human hsp60 antibodies to the amount of hsp60-fixed C4b. A highly significant (r = 0.459, P < 0.0001) positive correlation between the level of anti-hsp60 antibodies and hsp60-fixed C4b was found. (B) Lack of correlation of anti-M. bovis hsp65 antibodies to the amount of hsp65-fixed C4b (r = 0.1068, P = 0.554). Normalization of the results obtained on different ELISA plates was done by using the results of eight individual sera tested on each plate as standard.

 
Differentiation between anti-hsp60 and anti-hsp65 antibodies
The data on the correlation of hsp60-triggered classical pathway activation with anti-hsp60 antibodies but lack of correlation with anti-hsp65 antibodies presented above prompted us to test by the means of a competitive ELISA whether the anti-hsp60/65 antibodies are `identical' (the same antibodies cross-reacting with different antigens) or clearly distinct antibodies. ELISA plates were coated either with human hsp60 or with M. bovis hsp65 proteins and incubated with serial dilution of the test sera. The incubation mixture contained 5 µg/ml hsp60 or hsp65 as competitive antigen in each case (or buffer as control). A total of 10 sera samples from our study group were tested in this assay. Figure 4Go shows the results obtained in the sera of three patients. The serum of patient 4101 contained significant amounts of anti-hsp60 antibodies but not anti-hsp65 antibodies. Incubation of this serum with hsp60 markedly decreased the binding activity of antibodies to solid-phase hsp60, whereas incubation with hsp65 did not influence antibody fixation. Serum of patient KM exhibited a strong activity against hsp65 but not against hsp60. Binding of antibodies from this serum sample to solid-phase hsp65 was inhibited by the homologous hsp but not by the hsp60 preparation. The serum of patient 4395 contained antibodies reacting with both hsp60 and hsp65. In this sample incubation with the homologous hsp preparations almost fully abolished antibody binding while hsp60 only slightly inhibited anti-hsp65 fixation and vice versa. Similar results were obtained in the other seven serum samples not shown in the Fig. 4Go.



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Fig. 4. Distinction between anti-human hsp60 and anti-M. bovis hsp65 antibodies by competition ELISA. ELISA plates were coated either with human hsp60 (left panels) or with M. bovis hsp65 (right panels) and incubated with serial dilution of patients sera. The binding of IgG-type antibodies to the plate was detected by specific antisera conjugated with peroxidase. The binding of antibodies was blocked by 5 µg/ml hsp60 ({circ}) or hsp65 ({square}) added to the incubation mixture (buffer was added in control cases, {blacksquare}). One representative experiment out of three identical ones. Values represent mean ± SEM OD values of three parallel measurements.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Two important conclusions can be drawn from our novel findings. First, hsp60 alone does not activate the complement system but complexing of antibodies to solid-phase hsp60 leads to a marked complement activation which is dependent on the amount of specific antibodies. Second, the antibodies against human hsp60 and M. bovis hsp65 differ from each other in their antigen specificity and complement-activating ability.

Furthermore, solid-phase recombinant human hsp60 is able to activate the complement system in pooled NHS as well as in the sera of CHD patients. Since inhibition of the first complement component by Mg2+-EGTA blocked C4 activation and binding, and no C3b binding to hsp60 occurred in the sera of a patient with homozygous C2 deficiency, hsp60 activated complement in human sera via the classical pathway. The classical pathway can be activated by different substances either directly or with the contribution of specific antibodies, i.e. by immune complex formation. In the case of human hsp60 the initiation of the classical pathway seems to occur by immune complex formation since no complement activation was observed in agammaglobulinemic serum samples. Our present findings indicate that specific antibodies directed to human hsp60 trigger and dose-dependently enhance complement activation by hsp60. In addition, anti-IgG antibodies (rheumatoid factors) may also influence this activation.

Furthermore, mycobacterial hsp65 was found to activate the classical pathway, too, but to our surprise the extent of this activation did not correlate either to anti-hsp60 or anti-hsp65 antibody levels. Since anti-hsp65 and anti-Helicobacter pylori antibodies correlate to each other (16) we tested whether the amounts of the later one does correlate to the extent of hsp65-induced complement activation. We found a very strong positive correlation between the amount of C4b fixed to hsp65 and levels of anti-H. pylori antibody in the sera of CHD patients (Prohászka et al., unpublished observations). Another possible explanation for the lack of correlation between hsp65-induced classical pathway activation and anti-hsp65 antibody levels might be a different (complement non-activating) subtype of anti-hsp65 antibodies.

This novel finding suggests that the complement system may be efficiently activated when it is exposed to complexes of hsp60 and specific antibodies. The ability of anti-hsp60 antibodies to activate complement is in accordance with the previous observations of Schett et al. (17,18) on the lysis of endothelial cells, heat-stressed U937 cells and peripheral blood-derived macrophages by human anti-hsp65/60 antibodies in the presence of guinea pig complement. Since endothelial cells are in primary contact with the blood such activation of complement by hsp60 on the surface of this cell type may occur in vivo in the blood of patients who developed anti-hsp60 antibodies. Different pathological conditions and stress situations may markedly increase hsp60 expression in endothelial cells (6,19). For instance, non-toxic ischemia reperfusion was found to render human umbilical vein endothelial cells capable of activating the complement system in human serum (11). The mechanism of this process is not fully understood. We suggest that overexpression of hsp60 on the surface of endothelial cells could be one of the factors that increases complement-activating ability of the cultured endothelial cells especially when high titers of anti-hsp65 antibodies are present. Since complement activation has an important role both in the early and late phase of atherogenesis (1), our present findings may have important pathological implications.

The assumed role of anti-hsp65 antibodies in the development of atherosclerosis is supported by several recent findings. Xu et al. (20) found an association of elevated level of mycobacterial hsp65 antibody with carotid atherosclerosis. More recently, Hoppichler et al. (12) measured significantly increased anti-hsp65 antibody titers in 114 patients with CHD as compared to 76 age- and sex-matched healthy controls. Similarly, Birnie et al. (16) demonstrated a positive correlation between the extent of coronary atherosclerosis and the titer of anti-hsp65 antibodies. At the same time, we also made this observation and found a marked increase in the titers of anti-hsp60 and anti-hsp65 antibodies in 357 patients with severe CHD who had undergone by-pass operation compared to healthy age- and sex-matched controls (Prohászka et al., manuscript in preparation). The possible pathological significance of anti-hsp60 antibodies is also highlighted by the recent finding of Latif et al. (21) who observed strong association between high titer antibodies against human hsp60 measured before cardiac transplantation with a more severe course as compared to patients with low amounts of these antibodies.

The second novel observation of the present work is the demonstration of differences of anti-hsp60 and anti-hsp65 antibodies in their antigen recognition and complement-activating ability. Antibodies to the hsp60 family are elevated in patients with several diseases as compared to healthy subjects. If antibodies against the different members of the chaperonin family are measured in parallel, usually but not always (22) strong correlation is found between their amounts. We have also found a significant correlation between the anti-hsp60 and anti-hsp65 antibodies in the sera of HIV patients (15) and patients with CHD (Prohászka et al., manuscript preparation).

Anti-hsp60/65 antibodies are widely referenced in the literature to be cross-reactive (2325) due to the evolutionary high conservation of the chaperonin 60 molecules. Not excluding the existence of such a cross-reaction, our findings pin-point another aspect of the nature of these antibodies, i.e. they can recognize different antigen moieties and may have distinct functional properties, as well. In contrast to the marked and dose-dependent complement activation by anti-hsp60 antibodies complexed to specific antigen, complement activation by hsp65 was not influenced by the amounts of specific antibodies present in the serum samples tested (Fig. 3Go). On the other hand, the results of cross-inhibitions (Fig. 4Go) indicate that marked differences exist between the antigen specificity of hsp60 and hsp65 antibodies: homologous proteins strongly inhibited the binding of antibodies to both hsp60 and hsp65, whereas addition of hsp60 did not reduce or only weakly inhibited the anti-hsp65 fixation and vice versa. Similar findings were previously reported by Handley et al. (22), who found that anti-human hsp60 antibodies present in the sera of healthy subjects and patients with different autoimmune diseases were competitively inhibited by soluble hsp60 but little or not with mycobacterial hsp65. The weak inhibition was probably due to shared epitopes on the two proteins. Recent studies of Metzler et al. (26) demonstrated that antibodies in human sera reacting with human hsp60 and/or mycobacterial hsp65 recognized at least three different epitopes on mycobacterial hsp65. Our findings presented in this study together with previous observations of Handley et al. (22) suggest that a mixture of antibodies to the various members of the hsp60 family may be present in the sera of healthy subjects and patients with various diseases. They are supposed to be distinct, only partially cross-reactive and differing from each other in some functional properties like complement-activating ability.


    Acknowledgments
 
We thank the subjects for their participation in the study. This work was performed in the framework of EU Concerted Action `The role of complement in the susceptibility to infections and chronic diseases' and supported financially by the EU-98-D9-124 (National Committee for Technological Development of Hungary), by the FKFP 0084/1997 (Ministry of Education), by the OTKA 7024141 and by the ETT 430/1996 (Ministry of Welfare) grants. We are highly indebted to Professor Dr Harvey Colten for his helpful suggestions, and to Professor Dr János Gergely and Dr Béla Fekete for critical reading of the manuscript. The excellent technical assistance of Erika Farkas and Margit Kovács is greatly acknowledged.


    Abbreviations
 
CHDcoronary heart disease
HIHSheat-inactivated human serum
hspheat shock protein
NHSnormal human serum

    Notes
 
Transmitting editor: H. Bazin

Received 30 July 1998, accepted 16 September 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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