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Am J Physiol Lung Cell Mol Physiol 292: L448-L453, 2007. First published October 27, 2006; doi:10.1152/ajplung.00307.2006 Free Article
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The C/A(–18) polymorphism in the surfactant protein B gene influences transcription and protein levels of surfactant protein B

Wendy K. Steagall,1 Jing-Ping Lin,2 and Joel Moss1

1Pulmonary-Critical Care Medicine Branch and 2Office of Biostatistics Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

Submitted 10 August 2006 ; accepted in final form 26 October 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Surfactant protein B (SP-B) is an essential component of surfactant that promotes adsorption and spreading of surfactant phospholipids and stabilizes the phospholipid monolayer. SP-B is essential for respiratory function in newborn humans and mice; adult mice with levels of SP-B below 25% of wild-type develop fatal respiratory distress syndrome. A potential regulatory function of the C/A(–18) single nucleotide polymorphism (SNP) in the promoter of the SP-B gene was examined. Transcriptional analysis and ELISA on bronchoalveolar lavage fluid revealed that the presence of the C allele correlated with more SP-B promoter activity and protein. There was approximately threefold difference in amounts of SP-B in bronchoalveolar lavage fluid from CA(–18) and AA(–18) individuals. By EMSA, Sp1 bound more tightly to the C allele sequence than to the A allele sequence, perhaps accounting for the differences in transcription. Genotyping of a normal volunteer population showed ~31% of the population were AA homozygotes, suggesting that these individuals produce less SP-B. Differences in amounts of SP-B resulting from the promoter SNP could affect the clinical presentation of pulmonary disease.

electrophoretic mobility shift assay; enzyme-linked immunosorbent assay; respiratory function; SFTPB

PULMONARY SURFACTANT is a complex mixture of phospholipids and four surfactant proteins, A, B, C, and D, that resists surface tension and prevents the collapse of the pulmonary alveoli (6, 7). Surfactant protein B (SP-B) is an essential hydrophobic protein encoded on chromosome 2, which is synthesized as a 381-amino acid precursor and processed to a mature 8-kDa form coded by exons 6 and 7 (7, 31). Expression of SP-B is apparently restricted to respiratory alveolar type II epithelial cells and Clara cells, nonciliated bronchiolar epithelial cells (32). It promotes adsorption and spreading of surfactant phospholipids and stabilizes the phospholipid monolayer (9, 31). Hereditary SP-B deficiency is an autosomal recessive disorder, which may result in severe respiratory failure and lung transplantation or death (15, 22).

The gene for SP-B (SFTPB) (24) has many polymorphisms, of which four single nucleotide polymorphisms (SNPs) [C/A(–18), C/A(1013), C/T(1580), and A/G(9306)] have been the subject of many studies (5, 8, 10, 16). The C allele of the C/T(1580) polymorphism, encoding threonine at amino acid 131 in the SP-B precursor protein and promoting N-linked glycosylation of asparagine 129, is a susceptibility factor in infant and adult respiratory distress syndromes, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD) (8, 16, 25, 27). Functions for the other SP-B SNPs have not been reported. C/A(–18) is located in the SP-B promoter between the TATA box and the transcription initiation site (24). Because of the critical location of the polymorphism in the promoter, we investigated whether it would affect promoter function, and we report here that the SNP affects Sp1 binding as well as promoter activity both in vitro and in vivo.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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Luciferase assays. Nucleotides –218 to 435 of the SP-B promoter were PCR-amplified (primers SP-BPS/SP-BPAS) (Table 1) and cloned into pGL3-basic (containing the luciferase gene) (Promega, Madison, WI). pSPBC contains the C allele; pSPBA the A allele. As a control, the A of pSPBA was mutated to C by site-directed mutagenesis, producing pSPBAC [QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)] using primers SP-BA/CS and SP-BA/CAS. All constructs were verified by sequencing. NCI-H441 cells (ATCC, Rockville, MD) were transfected with SP-B constructs and the control plasmid pRL-TK (Promega). Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega).


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  		Table 1. Primer sequences

 
EMSA. Double-stranded oligos (SPBCS and SPBCAS, SPBAS and SPBAAS; Table 1) were synthesized and end-labeled [Ready-To-Go T4 Polynucleotide Kinase (Amersham Pharmacia Biotech, Piscataway, NJ)]. Recombinant human Sp1 (Promega) was incubated with unlabeled octamer transcription factor 1 (Oct1) and Sp1 oligos (Promega) as indicated. Probe was added, and mixtures were subjected to electrophoresis in Novex 6% DNA-retardation gels (Invitrogen). EMSA was also performed with the NuShift Sp1 kit and NCI-H441 nuclear extract (ActiveMotif, Carlsbad, CA).

Transcription factor knockdowns. NCI-H441 cells were transfected with either plasmid pSPBC or pSPBA and 200 pmol/well of a six-well plate of either siGenome SMARTpool reagent human YY1, Sp1, Sp3, or the siCONTROL nontargeting small interfering RNA (siRNA) no. 1 (Dharmacon, Lafayette, CO). Cells were collected 96 h after transfection: samples were used to determine protein concentrations (DC Protein Assay; Bio-Rad, Hercules, CA) and for Western blot analysis to verify knockdown of protein; a second sample was used for luciferase assays as above. Because the pRL-TK vector contained Sp1 sites, luciferase activity was normalized to protein concentration. Antibodies used for immunoblotting included rabbit polyclonal antibodies to Sp1 (H-225), Sp3 (D-20), and YY1 (H-414) from Santa Cruz Biotechnology (Santa Cruz, CA).

ELISA. ELISAs on samples of bronchoalveolar lavage fluid (BALF) from normal volunteers and patients with lymphangioleiomyomatosis (LAM) were performed as described (11). Epithelial lining fluid corrections were made by measuring the concentrations of urea in plasma, taken at the time of the lavage, and in the BALF. An epithelial lining fluid dilution factor was determined by dividing the concentration of urea in the plasma by the concentration of urea in the lavage fluid. The concentration of SP-B, as determined by the ELISA, was then multiplied by the epithelial lining fluid dilution factor to correct for differences in the lavage volumes. Infasurf (Forest Pharmaceuticals, St. Louis, MO) was used as an SP-B standard.

Study population and genotyping. The study population comprised 218 normal volunteers, all female and primarily Caucasian (198 individuals), with 8 Asian, 10 African-American, and 2 Hispanic volunteers. Patients with LAM were matched to healthy volunteers based on ethnicity, sex, and age (±5 yr). The research was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (NHLBI protocols 96-H-100 and 95-H-0186), and informed consent was obtained from all participants. Genomic DNA was prepared from whole blood using the PureGene kit (Gentra Systems, Minneapolis, MN). Genotype at –18 was determined as described (16) using primers 535, 536, 556, and 95A (Table 1).

Statistics. A 1:1 age-, sex-, and ethnicity-matched case-control study was designed to compare the SP-B promoter genotypes in LAM patients and controls, using Multinomial Logistic Regression in the statistical software SPSS. The t-tests were performed with Microsoft Excel.


   RESULTS
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 MATERIALS AND METHODS
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DISCUSSION
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Promoter activity. To analyze effects of C/A(–18) on SP-B promoter activity, a fragment (nucleotides –218 to 435) of the SP-B promoter containing either the A or C allele was cloned into pGL3-basic to generate pSPBA or pSPBC, respectively, with a firefly luciferase gene reporter. These constructs contain the minimal promoter sequence shown to contain binding sites for both specific lung cell and general transcription factors (19, 30). pSPBA or pSPBC plus pRL-TK was transfected into NCI-H441 cells, a human adenocarcinoma cell line with properties of Clara cells. Firefly luciferase was quantified and normalized to renilla luciferase activity produced by the control plasmid pRL-TK. Lysates of cells transfected with pSPBC contained significantly more luciferase activity than those of cells transfected with pSPBA (23.1 ± 4.4 vs. 12.7 ± 1.4 units, means ± SE, n = 4 each; P = 0.049; Fig. 1A).


Figure 1
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  		Fig. 1. A: luciferase activity of pSPBA (containing the A allele), pSPBC (with the C allele), and pSPBAC (pSPBA mutagenized to the C allele) after transfection into NCI-H441 cells. Experiments were performed 4 times. P = 0.049 pSPBA vs. pSPBC. B: surfactant protein B (SP-B) concentrations in bronchoalveolar lavage fluid (BALF) from normal volunteers or patients with lymphangioleiomyomatosis (LAM) grouped by C/A(–18) genotype. Line indicates the mean for each group; {blacklozenge} represents the concentration of SP-B in the BALF from 1 healthy volunteer or LAM patient.

 
To ensure that the differences in luciferase activity produced by different promoters were due entirely to the allelic difference, the A allele in pSPBA was mutagenized to a C allele to produce the plasmid, pSPBAC; constructs were sequenced to confirm that the only difference was at the polymorphic site. Luciferase activity directed by pSPBAC (23.1 ± 3.1 units, n = 4; Fig. 1A) was the same as that with pSPBC, confirming that the polymorphism was responsible for the difference seen in luciferase activity.

Transcription factor binding. The sequence of the promoter C allele is similar to a binding site for the transcription factor, Sp1 (Fig. 2A), and the replacement of C with A abolishes this motif. To determine whether Sp1 binds this sequence, recombinant human Sp1 (rhSp1) was incubated with a C allele probe, and the interaction was evaluated by EMSA. rhSp1 bound the probe containing the C allele, causing a shift in band mobility (Fig. 2B). Binding was proportional to rhSp1 concentration (Fig. 2C) and was inhibited by addition of excess unlabeled C allele probe but not by the unlabeled Oct1 oligonucleotide. Binding to the A allele probe was much less than to the C allele probe and was affected little, if at all, by increasing concentration of rhSp1 (Fig. 2C). These data suggest that the nucleotide sequences of SP-B promoter alleles influence Sp1 binding, with the presence of the C nucleotide necessary for Sp1 binding.


Figure 2
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  		Fig. 2. EMSA with recombinant human Sp1 (rhSp1) and C and A promoter allele oligonucleotides (oligo). A: sequence of the putative Sp1 binding site (nucleotides –25 to –16). B: C and A alleles were incubated with rhSp1 and competitor oligonucleotides where appropriate. O, octamer transcription factor 1 (Oct1) consensus oligonucleotide; S, Sp1 consensus oligonucleotide. C: C and A alleles were incubated with increasing amounts of rhSp1. D: A and C alleles were incubated with nuclear extract from NCI-H441 cells, competitor oligonucleotides, and antibody to Sp1 where appropriate. Arrow indicates supershifted band.

 
To determine whether the interaction between the C allele and Sp1 occurred also in cells, nuclear extract from NCI-H441 cells was incubated with the probes, and EMSA was performed (Fig. 2D). Both the A and C allele probes specifically bound nuclear factors. A supershifted band was detected on addition of an anti-Sp1 antibody, which was more with the C allele than the A allele.

Knockdown of transcription factors by siRNA. Because several bands were seen in the EMSA experiments, we decided to establish whether other transcription factors involved in SP-B promoter activation could be affected by the polymorphism. Sp3, as another SP-related GC-box-binding protein, could be involved, or, alternatively, YY1, which binds the sequence CCATG (28), could have a role. NCI-H441 cells were transfected with the luciferase vectors pSPBA or pSPBC and siRNA for Sp1, YY1, or Sp3. Ninety-six hours after transfection, protein levels of the transcription factors were checked by Western blot analysis and were shown to be less than those seen in cells treated with a control nontargeting siRNA (Fig. 3A). Luciferase assays were performed, and results were normalized to protein content of the cells, as the renilla luciferase construct pRL-TK contained Sp1 sites. Knockdown of Sp3 and YY1 led to a decrease in luciferase activity for both pSPBA and pSPBC compared with levels seen with the control siRNA, whereas knockdown of Sp1 led to an increase in luciferase activity for both plasmids (n = 6 for each condition) (Fig. 3B). The ratio of luciferase activity of pSPBC to pSPBA was found to be 1.8 (Fig. 1A) when no siRNA were used. Knockdown of YY1 resulted in a pSPBC-to-pSPBA ratio of 1.6 (P = 0.187 pSPBC activity vs. pSPBA activity), suggesting that whereas YY1 may play a role in SPB promoter activation, it probably did not play a major role in determining the differences in promoter activity based on the polymorphism, as the luciferase activities of the two constructs were almost significantly different. Knockdown of either Sp1 or Sp3 led to a C-to-A allele ratio of 1.2, with no significant difference between the activity of the constructs (P = 0.556 for Sp1; P = 0.729 for Sp3), suggesting that these transcription factors are responsible for the differences in transcription from the promoters of the two alleles.


Figure 3
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  		Fig. 3. A: representative Western blot showing protein levels of YY1, Sp1, or Sp3 with or without small interfering RNA (siRNA) treatment in NCI-H441 cells also transfected with an SP-B promoter/luciferase construct. C allele and A allele refer to the C/A(–18) luciferase constructs used, whereas "+" indicates the presence of the appropriate siRNA and NS indicates that a nonspecific control siRNA was used. B: relative amounts of luciferase activity for the C allele or A allele constructs in the presence of a siRNA compared with activity in the presence of the control siRNA. Fold numbers indicate the difference in activity between the C allele construct and the A allele construct. Error bars are SE for n = 6 for each condition.

 
Measurement of SP-B protein by ELISA. To determine whether an effect of the polymorphism was detectable in vivo, SP-B levels in BALF from normal volunteers and patients with LAM were measured by ELISA. LAM is a multisystem disease affecting women that is characterized by proliferation of abnormal smooth muscle-like cells, leading to cystic lung lesions, lymphatic abnormalities, and abdominal tumors. The amount of SP-B in BALF from CA controls (n = 8) was 2.6 times that measured in AA controls (n = 6) (P = 0.039; 6,280 ± 1,500 ng/ml vs. 2,400 ± 560 ng/ml, means ± SE; Fig. 1B). The amount of SP-B in BALF from LAM patients who were heterozygous for CA (n = 14) was 3.8 times that of the LAM patients who were AA homozygotes (n = 18) (P = 0.0015; 5,460 ± 1,000 ng/ml vs. 1,450 ± 230 ng/ml, means ± SE). As CC individuals are relatively rare [accounting for only 16% of a normal volunteer population (see SP-B genotyping below)], only two normal volunteers and one LAM patient were homozygous for C. The concentration of SP-B in BALF from the CC individuals was not significantly greater than that of the CA heterozygotes. These data might imply that the effect of the alleles is not additive; however, due to the wide range of SP-B concentrations of the CA heterozygotes, a better explanation may be that the concentrations of the CC individuals sampled fall in the lower end of the CC homozygote range. Thus the presence of the C allele in the SP-B promoter was associated with higher protein levels. These data are consistent with an in vivo as well as in vitro effect of the SP-B promoter polymorphism.

SP-B genotyping. Normal volunteers (n = 218) were genotyped for the SP-B promoter polymorphism. Sixty-eight (31.2%) were AA homozygotes, 115 (52.8%) were heterozygotes, and 35 (16.1%) were CC homozygotes. Genotyping of age-, sex-, and ethnicity-matched LAM patients revealed 82 (37.6%) AA homozygotes, 109 (50.0%) CA heterozygotes, and 27 (12.4%) CC homozygotes. Using any model of inheritance of genotypes (additive, dominant, or recessive), there was no difference between the normal volunteers and the LAM patients in the frequencies of the genotypes.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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The gene for SP-B has many polymorphisms; the four most studied SNPs are C/A(–18), C/A(1013), C/T(1580), and A/G(9306) (5, 8, 10, 16). We examined the function of the promoter SNP, C/A(–18), which is in a GC-rich repeat that flanks the TATAA box (24). The upstream half of the repeat has been reported to bind Sp1 (at position –35) (18, 19), but no specific protein is known to bind to the other half at position –24. The polymorphism C/A(–18) has not been widely studied; it has been ruled out as a susceptibility factor in COPD in both Chinese Han and Mexican populations (8, 10), and is not a factor in development of tuberculosis (5). Although found not to be a susceptibility gene in COPD in a Mexican population, the C allele of C/A(–18) was associated with more severe cases of COPD when the patient population was divided on the basis of pulmonary function [forced expiratory volume in 1 s divided by forced vital capacity (FEV1/FVC)] (8).

When a minimal promoter region containing the C/A(–18) polymorphism was amplified and cloned into a vector containing the firefly luciferase gene, the promoter containing the C allele yielded 1.8 times as much luciferase activity as did that with the A allele (P = 0.049), suggesting that the C allele of the SP-B promoter increased transcription more effectively than the A allele. We next investigated whether the effects seen with the luciferase assay had in vitro and in vivo correlates. As shown by EMSA (Fig. 2), rhSp1 bound to promoter region probes containing the C allele but only minimally to the A allele. When NCI-H441 nuclear extracts were used in EMSA, both alleles bound nuclear proteins, as would be expected since both alleles are found in healthy controls. The complex that bound to the C allele contained Sp1, as shown by the presence of a supershifted band after addition of an antibody to Sp1. This supershifted band was more intense than that seen in the presence of the A allele, suggesting that Sp1 does play a role in the difference in transcription levels between the two alleles. To determine whether the C allele altered lung SP-B content in vivo, SP-B was quantified by ELISA in BALF from normal volunteers (Fig. 1B). Levels of SP-B in BALF from CA normal volunteers were 2.6 times those from AA controls (P = 0.039), whereas there was a difference of 3.8-fold between BALF levels of SP-B of CA and AA LAM patients (P = 0.0015). The quantitative differences between luciferase assays and ELISAs in the magnitude of increases may simply reflect the absence of any necessary correlation between relative amounts of gene transcripts and protein products. In addition, the luciferase assays were done with transfected NCI-H441 cells, which are representative of Clara cells, whereas the ELISA quantified the protein in BALF. Effects of the SNP on protein synthesis may, of course, be influenced by cell-specific factors.

It is probable that C/A(–18) is one of many factors affecting the concentration of SP-B found in BALF. Those individuals used for the BALF study were also genotyped for three of the other SNPs of the SP-B gene (data not shown). Alleles of the C/T(1580) and A/G(9306) polymorphisms were not correlated to SP-B concentration in BALF. The polymorphism C/A(1013) did show an association with the amounts of SP-B, with the CC individuals having significantly less SP-B than the heterozygotes or those homozygous for the A allele. It is known that C/A(–18) is in linkage disequilibrium with C/A(1013), with the A allele of C/A(–18) linked to the C allele of C/A(1013) (17). It is probable that the association of SP-B concentrations with C/A(1013) is due to the linkage of the polymorphism to C/A(–18); however, a contribution of C/A(1013) to SP-B protein levels cannot be ruled out.

The sequence in which the polymorphism is embedded (GCCATGC C/A CC) is similar to the consensus GC box (G/A C/T C/T CCGCCC C/A) (24) that binds Sp1; it does, however, contain mismatches compared with the canonical sequence. Analysis of the sequence containing the polymorphism by the Transcription Element Search System (26) revealed a putative binding site for Sp1 with the C allele, but not with the A allele. Both alleles, when reduced to 26-nucleotide oligomers for EMSA experiments, did bind recombinant Sp1, with Sp1 binding being significantly higher for the C allele than the A allele (Fig. 2, B and C); however, the band generated on binding of recombinant Sp1 to the radiolabeled consensus Sp1 sequence oligonucleotide was many times stronger than that seen with the sequence found in the C allele. This sequence, even with the C present, does not appear to have the strong affinity for Sp1 seen with the classic Sp1 sequence.

Since the EMSA performed with extracts from NCI-H441 cells showed several other bands in addition to the Sp1-containing band (Fig. 2D), the possibility existed that other transcription factors could bind this sequence and play a role in the difference seen in transcription levels with the C and A alleles. Sp3 could be involved, as it is another GC-box-binding protein, while the sequence surrounding the polymorphism is also similar to the consensus sequence that binds YY1 (CCATG) (28). To try to determine which factor may be involved in the differences in promoter activity between the alleles, NCI-H441 cells were transfected with plasmids having either the A or C allele controlling firefly luciferase transcription and with siRNA for Sp1, Sp3, or YY1. Interestingly, whereas knockdown of YY1 and Sp3 protein levels decreased the amount of luciferase activity seen from both plasmids, knockdown of Sp1 increased the amount of luciferase activity from both A and C alleles. These results suggest that YY1 and Sp3 stimulate transcription, whereas Sp1 is inhibitory. The region of the SP-B promoter cloned into the firefly luciferase vector contains three Sp1/Sp3 binding sites (19) in addition to the putative Sp1 site containing the polymorphism. While Sp1 may participate in repressing transcription controlled by the SP-B promoter, it is also possible that in the context of the experiment, knockdown of Sp1 levels allowed the opportunistic binding of other transcription factors to any of the four possible Sp1 sites, leading to an erroneous conclusion regarding the function of Sp1 on the SP-B promoter.

Although Sp1 is thought of primarily as an activator of transcription, Sp1 or its binding sites have also been involved in repression of transcription of certain genes (33). This negative regulation may be the result of binding site competition, where Sp1 is replaced by another transcription factor, such as the competitive binding of Sp3 and Sp1 to the cardiac troponin T gene promoter (1), the transcobalamin II gene promoter (14), or the alcohol dehydrogenase gene promoter (12), leading to inhibition by Sp3 or activation by Sp1, depending on the relative ratios of the factors. Sp1 may also be involved in inhibition of transcription with the formation of inhibitory complexes with other trans factors, which may mask Sp1 activation domain sites (13, 21). Finally, Sp1 may directly inhibit transcription, as shown for the megakaryocyte-specific {alpha}IIb gene (29) and the metallothionein genes (23).

The plasmid containing the C allele controlling the luciferase gene produced 1.8 times the luciferase activity seen with the plasmid containing the A allele (P = 0.049). Comparison of luciferase activity generated when the protein levels of YY1 were decreased using siRNA knockdown revealed that the C allele had 1.6 times the activity of the A allele (P = 0.187), whereas the C allele yielded only 1.2 times the activity of the A allele with Sp1 (P = 0.556) and Sp3 (P = 0.729) knockdown. These data suggest that the difference in transcription seen between the C and A alleles is predominantly due to Sp1 or Sp3.

The SP-B promoter contains two parts, which differ in function. A specific lung cell region binds the specific transcription factors, thyroid transcription factor-1 and hepatocyte nuclear factor-3, and another region binds more ubiquitous transcription factors like Sp1 and Sp3 (2, 19). Sp1/Sp3 binds to the SP-B promoter at –162, –130, and –35 (19). Here, we show that Sp1 can bind to the site at –25 of the C allele but does not bind as well when C is replaced by A. This subtle difference in transcriptional machinery may account for the difference in amounts of SP-B recovered in BALF from AA and CA individuals. Although SP-B is certainly produced from the A allele, the addition of an extra Sp1 binding site may significantly boost protein production.

Even subtle differences in amount of SP-B between C/A(–18) genotypes may have an impact on survival in lung diseases, with larger amounts of SP-B contributing to survival. Approximately 31% of the population are AA homozygotes (this study, also refSNP ID: rs2077079), which is associated with less SP-B in BALF. It is well known that SP-B is essential for respiratory function in newborn humans and mice (4, 15, 22), and more recently reported that there is also a critical level of SP-B necessary for respiratory function in adult mice (20). Mice with heterozygous deletion of the SP-B gene had 50% less SP-B mRNA and protein than wild type as well as impaired lung compliance (3). In other experiments, transgenic mice engineered with doxycycline-dependent expression of SP-B exhibited an increased risk of respiratory failure when SP-B levels in BALF fell to less than 50% of normal; fatal respiratory distress syndrome occurred when SP-B levels were less than 25% of normal (20). The mice in respiratory distress had decreased amounts of surfactant phosphatidylglycerol in the alveoli and an accumulation of SP-C proprotein. We found an approximately threefold difference in amounts of SP-B produced from CA(–18) vs. AA(–18) individuals.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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The study was supported by the Intramural Research Program, National Institutes of Health/National Heart, Lung, and Blood Institute.


   ACKNOWLEDGMENTS
 
We thank Dr. Martha Vaughan and Dr. Vincent Manganiello for their constructive comments and revision of the manuscript. We thank the LAM Foundation and the Tuberous Sclerosis Alliance for patient referrals.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Moss, Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg. 10, Rm. 6D03, MSC 1590, Bethesda, MD 20892-1590 (e-mail: mossj{at}nhlbi.nih.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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