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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Smooth-muscle proliferation is the hallmark of lymphangioleiomyomatosis (LAM). Although little is known about the pathogenesis of LAM, nitric oxide (NO) is a key regulator of smooth-muscle proliferation. NO is linked to the pathogenesis of other lung diseases such as asthma, in part by the finding of higher-than-normal levels of exhaled NO. If NO were involved in the abnormal smooth-muscle proliferation in LAM, we reasoned that exhaled NO from individuals with LAM would also differ from that of healthy control subjects. To evaluate this hypothesis, we studied exhaled NO in individuals with LAM in comparison with healthy and asthmatic women using a chemiluminescent NO analyzer. Women with LAM had higher exhaled NO than did healthy women but lower than asthmatic women (NO [parts per billion] median (25 to 75%): LAM 8 [7 to 15] [n = 28], control 6 [5 to 8] [n = 21], asthma 14 [8 to 25] [n = 22]; Kruskal-Wallis P < 0.001). Immunohistochemical studies on formalin-fixed, paraffin-embedded sections of surgical and autopsy material from lungs of individuals with LAM showed diffuse NO synthase III (NOSIII) expression in the lesional smooth muscle of LAM similar to that in the vascular endothelium. NOSIII expression was limited to the vascular endothelium and bronchial smooth muscle in healthy control lungs. The increased NO and the presence of NOSIII expression in lesional smooth muscle warrants further study into the potential role for NO in the pathogenesis of LAM.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Lymphangioleiomyomatosis (LAM) is a rare disease that
affects women, primarily in their reproductive years. It is a
devastating illness that carries a very poor prognosis due
to progressive loss of lung function and ultimately leads to
death. The hallmark of the disease is the non-neoplastic proliferation of atypical smooth-muscle cells within the lung
parenchyma leading to the progressive loss of lung function (1). Because of the rarity of LAM, little is known
about its pathogenesis or effective therapies (1, 5, 10).
Corticosteroids and cytotoxic agents offer no benefit (1).
Given the occurrence of the disease in women of child-bearing years (1), reports of clinical worsening with exogenous estrogen (6), and the presence of estrogen and
progesterone receptors in the proliferating LAM cells (1,
7), hormones
particularly estrogenhave been implicated in the pathogenesis of LAM. Hormonal manipulation, such as antiestrogen therapy, has been used with some success in treatment of LAM but the response to
such therapy has been variable (1, 3, 10, 11).
Smooth-muscle proliferation is regulated by numerous cytokines and mediators, but is profoundly affected by nitric oxide (NO) (12). NO is a diffusible gas that is produced endogenously in the lung by a group of enzymes known collectively as NO synthases (NOSs) (EC 1.14.13.39) (18). These enzymes convert the amino acid L-arginine to NO and L-citrulline in the presence of oxygen and other cofactors (20). Three isoforms of NOS have been identified: neuronal (NOSI), inducible (NOSII), and endothelial (NOSIII) (18). Interestingly, NOSIII expression is regulated by estrogen (12, 22). In this context, we questioned whether NOSIII and NO are involved in the abnormal smooth-muscle proliferation in LAM. NO is implicated in the pathophysiology of lung diseases such as asthma (23, 24) and pulmonary hypertension (25), in part due to the findings of higher or lower levels of NO in exhaled gases. Thus, we reasoned that if NO/NOSIII plays a role in LAM, exhaled NO of individuals with LAM would differ from that of healthy control subjects. To evaluate this hypothesis, exhaled NO of individuals with LAM was compared with exhaled NO of healthy and asthmatic women, and NOS expression in the lung was evaluated.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Patient Selection
Individuals with LAM were selected based on a pathologic diagnosis by open lung biopsy or transbronchial biopsy. They were identified from membership in the LAM Foundation and evaluated during a LAM Foundation meeting. Five individuals had
previously undergone a lung transplant at the time of NO measurement. The median duration since transplant in these individuals was 2 yr (range, 1.5 to 8 yr). Because LAM is a disease that
affects women in childbearing age, healthy control subjects and
individuals with asthma were selected on the basis of female gender and being of childbearing age. Asthma diagnosis was based
on American Thoracic Society guidelines (26). Asthmatic individuals had mild to moderate asthma controlled with 
2 agonists
and/or inhaled but not oral corticosteroids. Control individuals
were identified by the absence of pulmonary symptoms or history
of pulmonary disease. The study was approved by the Cleveland
Clinic Foundation Institutional Review Board, and informed
consent was obtained from all individuals.
Exhaled NO Measurement
Levels of exhaled NO were determined by two methods. In the
offline method, exhaled gas was measured by having the individuals inhale to total lung capacity (TLC) followed by exhaling
against 10 cm of water pressure into a Mylar balloon while wearing nose clips. This maneuver was repeated twice with no breath
hold, and again two times with a 15-s inspiratory breath hold at
TLC. In the online method, individuals inhaled via the mouth to
TLC and exhaled without delay against resistance into a mouthpiece connected directly to the analyzer while targeting a constant pressure of 12 to 13 torr. This combination of pressure and
resistance resulted in a flow rate of 50 ml/s. The exhalation was
maintained until a steady NO plateau of 3 s duration was detected on the computer screen (after ~ 10 to 15 s). Repeated exhalations were performed until three NO plateaus were obtained
which agreed within 10% of the average value but no more than
five times (27). NO was quantitated by a chemiluminescence NO
analyzer (NOA 280; Sievers, Boulder, CO). The analyzer was
calibrated daily using NO-free gas (zero air; Praxair, Cleveland,
OH) and 8.7 parts per million NO gas (Praxair) (18). Data from
the analyzer were transferred in real time to a laptop computer
via a modem connection and analyzed utilizing Microsoft Excel.
NO levels were performed on individuals breathing room air, or
while breathing air with no NO (zero air; Praxair) when ambient
NO levels were 
20 parts per billion (ppb).
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections (5 µM) were used for immunohistochemistry staining with rabbit polyclonal epitope- purified immunoglobulin (Ig) G antibody directed against human NOSIII (ABR-PA 1-037; Affinity Bioreagents, Golden, CO) after 16 min of proteinase digestion and with a mouse monoclonal IgG antibody against melanoma-specific antigen HMB-45, which has previously been reported to be present in the characteristic LAM spindle cells (4), (ENZO, Farmingdale, NY) after 8 min of proteinase digestion. Immunohistochemical staining was performed by an automated biotin-avidin peroxidase system (Ventana-ES-320) with amino-ethyl-carbonyl (Ventana, Tucson, AZ) as a chromogen. Positive controls for the NOSIII consisted of the adjacent pulmonary vasculature of LAM or control lungs. Positive controls for HMB-45 consisted of a tissue section of a malignant melanoma. Immunohistochemical studies for estrogen and progesterone receptor were performed using mouse monoclonal antibodies to estrogen receptor and progesterone receptor (Ventana) after 20 min of microwave antigen retrieval. Positive controls for estrogen and progesterone receptors consisted of a tissue section of a breast carcinoma. Negative controls of secondary antibody only were performed on each section of tissue studied. Lungs from five patients with LAM were obtained from explanted lung or autopsy.
Statistical Analysis
The data are reported as means ± standard error of the mean (SEM) for normally distributed data and as median and interquartile ranges for data that were not normally distributed. Two-tailed t test statistics, analysis of variance, Kruskal-Wallis, and Mann-Whitney rank sum test were used as appropriate at a significance level of 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Clinical Characteristics
The LAM, control, and asthmatic individuals were similar
in age (yr, LAM 46 ± 2 [n = 28], control 41 ± 2 [n = 21],
asthma 47 ± 3 [n = 22]; P = 0.23]) The time since diagnosis
with LAM in individuals at entry into the study varied from
1 to 17 yr (Table 1). Control and asthmatic subjects were all
women in their reproductive years, to control for gender
and hormonal differences. Five of the 28 LAM individuals
had received lung transplantation, with the median duration
since transplant 2 yr (range, 1.5 to 8 yr) (Table 1). None of
the participants in the study were current users of tobacco
products. Eleven individuals with LAM were ex-smokers (16 ± 4 pack-years). Three control individuals were ex-smokers (12 ± 6 pack-years). Seven asthmatic individuals
were ex-smokers (20 ± 10 pack-years). Individuals with
LAM had airflow limitation and decreased diffusion capacity (DLCO) (Table 1). Pulmonary function testing of asthmatics revealed mild airway obstruction (forced vital capacity [FVC] % predicted), 90 ± 4; forced expiratory volume in
1 s [FEV1] % predicted, 76 ± 4; FEV1/FVC %, 70 ± 4). One
control was on levothyroxine and a -blocker, but the rest of controls were on no medications. All asthmatic subjects
were on intermittent 2 agonists and 13 out of 22 were receiving inhaled corticosteroids.
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In addition to the 28 women with LAM evaluated for exhaled NO, explanted lungs from a separate group of five LAM individuals, three from transplant and two from autopsy, were obtained for NOSIII immunostaining.
Exhaled NO
Offline and online exhaled NO measurements had a positive correlation (R = 0.74, P < 0.001) (Figure 1). Offline measurements with and without an inspiratory breath-hold also had a positive correlation (data not shown; R = 0.87, P < 0.001). Online NO was higher than offline. Offline measurements with an inspiratory breath-hold were used for analysis and comparison among groups. This method is reproducible over time. In four control subjects measured on two to seven separate occasions over a span of 1 mo to 3 yr, the coefficient of variation was 6.5%.
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Exhaled NO from women with LAM was higher than from female control subjects but lower than levels in female asthmatics (NO [ppb] median [25 to 75%]: LAM 8 [7 to 15; n = 28], control 6 [5 to 8; n = 21], asthma 14 [8 to 25; n = 22]; Kruskal-Wallis P < 0.001) (Figure 2). Individuals with asthma who were receiving inhaled corticosteroids had significantly lower exhaled NO levels than did asthmatics not receiving inhaled corticosteroids (NO [ppb] means ± SEM: asthma off corticosteroids 25 ± 5 [n = 9] versus asthma on corticosteroids 14 ± 2 [n = 13]; P = 0.05) (Figure 2). In contrast, NO levels were similar among individuals with LAM on or off corticosteroids (NO [ppb] means ± SEM: LAM off corticosteroids 11 ± 1 [n = 22] versus LAM on corticosteroids 12 ± 3 [n = 6]; P = 0.8) (Figure 2). LAM individuals with lung transplantation had NO levels similar to other LAM individuals (NO [ppb] means ± SEM: LAM with transplantation 15 ± 3 [n = 5] versus LAM 11 ± 1 [n = 23]; P = 0.2).
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Twenty of the 28 LAM patients were on hormonal therapy which included one or more of the following: progesterone, leuprolide, norethidone, or megesterol acetate.
There was no significant difference in exhaled NO between the two groups (NO [ppb] median [25 to 75%]: LAM receiving hormonal therapy 8 [6 to 16; n = 20] versus LAM not receiving hormonal therapy 10 [7 to 14; n = 8];
P = 0.78). Six of the healthy controls were postmenopausal. There was no difference in NO between the pre-
and postmenopausal women (NO [ppb] median [25 to 75]:
premenopausal 6 [5 to 8; n = 15] versus postmenopausal 6 [5 to 8; n = 6], P = 0.85). Exhaled NO did not correlate
with time since diagnosis (NO versus years since diagnosis: R = 
0.155 [P = 0.596]) or with disease severity (NO versus FVC: R = 0.308 [P = 0.214], NO versus FEV1: R = 0.0882 [P = 0.728], NO versus DLCO: R = 0.223 [P = 0.390]).
NOSIII Immunostaining
Semiquantitative evaluation revealed that the lesional smooth muscle of LAM uniformly showed diffuse positive cytoplasmic immunoreactivity for NOSIII. However, LAM spindle cells were less immunoreactive than the adjacent vascular endothelium and airway smooth muscle (Figure 3), which was diffusely strongly positive. The density of NOSIII staining was heterogeneous within the lesions, with a suggestion of perinuclear clearing that was not seen in the bronchial smooth muscle. Figure 3C illustrates a cross-section through a fascicle of LAM spindle cells that accentuates the presence of sarcoplasm around the myofiber, which may explain the clearing. Also, LAM spindle cells stained with less intensity than did the normal smooth-muscle cells, which may account for focal loss of staining as well. Control lungs were also evaluated for NOSIII immunostaining for comparison (n = 3). NOSIII staining was identified in the vascular and bronchial smooth muscles, and in the vascular endothelium. Unfortunately, commercially available NOSII and NOSI antibodies were not specific or sensitive enough to allow a confident interpretation of immunoreactivity of LAM cells (data not shown).
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The lesional smooth muscle of LAM showed moderate to strong focal cytoplasmic immunoreactivity for HMB-45 in all five patients (data not shown) as previously reported (4). HMB-45 immunohistochemistry was performed on subsequent tissue sections of lung which were stained for NOSIII. As previously reported, HMB-45 immunoreactivity was present only in the characteristic LAM spindle cells and was not seen in the bronchial smooth-muscle cells. The cells staining positive for HMB-45 were also positive for NOSIII. Lesional smooth-muscle cells also showed focal nuclear immunoreactivity for estrogen receptors as previously described (7). In contrast, LAM smooth-muscle cells did not show immunoreactivity for progesterone receptors (data not shown).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Currently, 200 to 400 individuals are diagnosed with LAM in the United States (28). The poor understanding of pathogenesis is compounded by the rarity of LAM and the unique pathology (28). However, previous studies have shown that LAM cells have estrogen and progesterone receptors, whereas normal bronchiolar or vascular smooth muscle do not stain for either receptor (1, 7). In this context, it has been postulated that estrogens play a role in LAM smooth-muscle proliferation.
LAM is characterized by the non-neoplastic proliferation of atypical smooth-muscle cells (LAM cells). The stimulus for proliferation is not known. However, NO is well known to mediate smooth-muscle proliferation in a variety of experimental systems (12). In general, NO inhibits smooth-muscle tone and cell proliferation through the activation of cyclic guanidine monophosphate (12, 21), through negative feedback control on calcium responses to growth factors (29), or by effects on programmed cell death (30). Although most studies support an inhibitory role for NO, others suggest that NO can stimulate the proliferation of smooth muscles (12). For example, physiologic levels of NO (µM) stimulate rat fetal pulmonary artery smooth-muscle cell and aortic smooth-muscle cell proliferation (12, 17).
In this study, higher-than-normal NO is demonstrated in
a relatively large cohort of individuals with LAM, i.e., 5 to
10% of the total population. This raises the possibility that
NO may play some role in the pathogenesis of LAM. However, the significance of high NO to pathogenesis of LAM is
unclear. NO is present in the normal human lung, evidenced
by NO in the exhaled air of humans and NO as S-nitrosothiol and NO2
in the airway aspirate and bronchoalveolar lavage fluid from human lungs (18, 24, 25, 31). NO is
produced by NOSs, which convert L-arginine to NO and
L-citrulline in a reaction that requires oxygen and nicotinamide adenine dinucleotide phosphate, and the cofactors flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, and calmodulin (20). Studies have identified the presence of the three NOS isoforms in the human lung
(12, 31). NOSI is located in inhibitory nonadrenergic noncholinergic neurons in the lung, NOSIII in endothelial cells
(12, 31), and NOSII is expressed in normal human airway
epithelium (32). In general, NOSI and NOSIII activity is dependent on increases in calcium to bind calmodulin, which
results in enzyme activation and picomolar levels of NO production (21). NOSII is regulated at the level of transcription
and messenger RNA (mRNA) stability (32), is calcium-independent, and produces nanomolar levels of NO (20).
Interpretation of the increased levels of NO in lung diseases, such as LAM or asthma, requires an understanding of the distribution of NO in tissues. The spatial distribution of NO in an organ such as the lung will be dependent on the solubility of NO (gas-liquid interfaces), NO diffusibility, the rate at which NO is synthesized, and the rate at which NO is consumed (18). In biologic systems, up to one-third of the NO synthesized may be consumed by chemical reactions. Because NO is freely diffusible, consumption of NO can occur at different sites within the cell, extracellular fluids, and intravascular compartments. Primary reactions that may consume NO intra- and extracellularly include its reaction with oxygen, superoxide, hemoglobin, another molecule of NO, enzymes containing iron-sulfur centers, heme-containing proteins, and thiol proteins (33). In general, however, exhaled NO reflects the concentration of total NO in liquids/tissues in the lung, because at atmospheric pressures over 97% of NO is rapidly distributed from the liquid to the gaseous phase (18).
Exhaled NO from individuals with LAM was significantly lower than NO of asthmatic individuals. High NO levels in asthma are closely associated with airway inflammation (23, 24) and are attributed to increased NOSII expression in airway epithelial cells. NOSII produces 1,000-fold higher levels of NO than does NOSIII (18), and is inhibited at the transcriptional and translational levels by corticosteroids (18, 32). In this context, lower NO in LAM than in asthma suggests that the mechanism of increased NO may be different. Importantly, inhaled corticosteroids significantly reduce NO in asthma whereas NO levels in LAM are similar regardless of corticosteroid use. Interestingly, NO levels in transplanted LAM individuals were also elevated; however, transplantation itself increases exhaled NO which may be related to rejection and NOSII expression (34). These observations and the fact that LAM is not an inflammatory lung process suggest that increased NO may not be related to NOSII. An alternative source of NO may be NOSIII, an isoform known to be expressed in smooth muscle and to be regulated hormonally. Both pregnancy and estradiol increase NOSIII mRNAs in several tissues (23). Strikingly, the expression of NOSIII in the fetal rodent lung increases during late gestation, which is mediated in part by estrogen (12, 35). In the present study, NOSIII was diffusely present in lesional smooth muscle of LAM. Although changes in NOS expression do not automatically mean there will be elevations in exhaled NO, on the basis of the uniformity of NOSIII expression in all smooth muscles in the lung, high levels of exhaled NO may be due to the fact that LAM lungs contain an abnormal amount of muscle with NOSIII expression. However, the lack of association between hormone therapy and NO levels does not support a role for estrogen in NOSIII expression in LAM. Further studies of regulation of NO in individuals with LAM may reveal insight into the role of NO in the progressive smooth-muscle proliferation in LAM.
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Footnotes |
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Address correspondence to: Raed A. Dweik, M.D., Cleveland Clinic Foundation, 9500 Euclid Ave./A-90, Cleveland, OH 44195.
(Received in original form February 8, 2000 and in revised form October 9, 2000).
Abbreviations: diffusion capacity, DLCO; forced expiratory volume in 1 s, FEV1; forced vital capacity, FVC; lymphangioleiomyomatosis, LAM; nitric oxide, NO; NO synthase, NOS; neuronal NOS, NOSI; inducible NOS, NOSII; endothelial NOS, NOSIII; parts per billion, ppb; standard error of the mean, SEM.Acknowledgments: The authors are indebted to Sue Byrnes and the LAM Foundation for identifying women with LAM for the study and providing their clinical information; to F. X. McCormack for use of NO analyzer; to J. Lang for artwork; and to E. J. Sullivan for helpful discussions. This work was supported by NIH grant HL60917.
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References |
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 B. D. Hoit, N. D. Dalton, S. C. Erzurum, D. Laskowski, K. P. Strohl, and C. M. Beall Nitric oxide and cardiopulmonary hemodynamics in Tibetan highlanders J Appl Physiol, November 1, 2005; 99(5): 1796 - 1801. [Abstract] [Full Text] [PDF]
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 S. C. Land and C. Rae iNOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core Am J Physiol Cell Physiol, October 1, 2005; 289(4): C918 - C933. [Abstract] [Full Text] [PDF]
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 F. A. Masri, S. A. A. Comhair, T. Koeck, W. Xu, A. Janocha, S. Ghosh, R. A. Dweik, J. Golish, M. Kinter, D. J. Stuehr, et al. Abnormalities in Nitric Oxide and Its Derivatives in Lung Cancer Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 597 - 605. [Abstract] [Full Text] [PDF]
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ATS/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005 Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 912 - 930. [Full Text] [PDF]
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 R A Dweik Nitric oxide, hypoxia, and superoxide: the good, the bad, and the ugly! Thorax, April 1, 2005; 60(4): 265 - 267. [Full Text] [PDF]
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S. B. Khatri, J. Hammel, M. S. Kavuru, S. C. Erzurum, and R. A. Dweik Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge J Appl Physiol, July 1, 2003; 95(1): 436 - 440. [Abstract] [Full Text] [PDF]
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R. F. Machado, J. K. Stoller, D. Laskowski, S. Zheng, J. A. Lupica, R. A. Dweik, and S. C. Erzurum Low levels of nitric oxide and carbon monoxide in alpha 1-antitrypsin deficiency J Appl Physiol, December 1, 2002; 93(6): 2038 - 2043. [Abstract] [Full Text] [PDF]
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S. B. KHATRI, M. OZKAN, K. MCCARTHY, D. LASKOWSKI, J. HAMMEL, R. A. DWEIK, and S. C. ERZURUM Alterations in Exhaled Gas Profile during Allergen-induced Asthmatic Response Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): 1844 - 1848. [Abstract] [Full Text] [PDF]
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