Genetic variants of surfactant proteins A, B, C, and D in bronchopulmonary dysplasia.

BPD_28D (O2 dependency at 28 days of life) and BPD_36W (O2 dependency at 36 wks post-menstrual age) are diseases of prematurely born infants exposed to mechanical ventilation and/or oxygen supplementation. In order to determine whether genetic variants of surfactant proteins (SPs-A, B, C, and D) and SP-B-linked microsatellite markers are risk factors in BPD, we performed a family based association study using a Greek study group of 71 neonates (<30 wks gestational age) from 60 families with, 52 BPD_28D and 19 BPD_36W, affected infants. Genotyping was performed using newly designed pyrosequencing assays and previously published methods. Associations between genetic variants of SPs and BPD subgroups were determined using Transmission Disequilibrium Test (TDT) and Family Based Association Test (FBAT). Significant associations (p

Rare SP-A alleles and the SP-A1-6A(4) allele associate with risk for lung carcinoma.

Next to cigarette smoking, genetic factors may contribute to lung cancer risk. Pulmonary surfactant components may mediate response to inhaled carcinogenic substances and/or play a role in lung function and inflammation. We studied associations between surfactant protein (SP) genetic variants and risk in lung cancer subgroups. Samples (n=308) were genotyped for SP-A1, -A2, -B, and -D marker alleles. These included 99 patients with small cell lung carcinoma (SCLC, n=31), or non-SCLC (NSCLC, n=68) consisting of squamous cell carcinoma (SCC, n=35), and adenocarcinoma (AC) (n=23); controls (n=99) matched by age, sex, and smoking status (clinical control) to SCLC and NSCLC; and 110 healthy individuals (population control). We found (a) no significant marker associations with SCLC, (b) rare SP-A2 (1A9) and SP-A1 (6A11) alleles associate with NSCLC risk when compared with population control, (c) the same alleles (1A9, 6A11) associate with risk for AC when compared with population (6A11) or clinical control (1A9), and (d) the SP-A1-6A4 allele (found in approximately 10% of the population) associates with SCC, when compared with population or clinical control. A correlation between SP-A variants and lung cancer susceptibility appears to exist, indicating that SP-A alleles may be useful markers of lung cancer risk.

Structural analysis and lipid-binding properties of recombinant human surfactant protein a derived from one or both genes.

Surfactant protein A (SP-A) constitutes an important part of the innate immune defense in the lung. In humans there are two functional genes (SP-A1 and SP-A2). The functional importance of having two distinct chain types in human SP-A is undefined. Amino acid substitutions in the primary structure of the protein may have effects on structural stability or on activity. To address this issue, SP-A1, SP-A2, and coexpressed SP-A1/SP-A2 variants were in vitro expressed in insect cells, purified, and used for study. We found the following: (1) Human SP-A variants expressed in insect cells, derived from one gene (SP-A1 or SP-A2) or both genes, differ in the relative extent and heterogeneity of oligomerization. SP-A1 and SP-A2 exist in small oligomeric forms, whereas coexpressed SP-A1/SP-A2 products favor the formation of larger oligomers. (2) Circular dichroic and fluorescence spectroscopic studies identified structural differences between SP-A variants in the collagen domain, with SP-A2 being more stable than SP-A1 but not in the calcium binding region. Recombinant human SP-A variants expressed in insect cells exhibit a lower melting temperature compared to native human SP-A. Oligomerization does not increase the thermal stability of the collagen domain of coexpressed SP-A1/SP-A2. (3) The ability of SP-A to undergo self-aggregation and induce phospholipid and bacterial lipopolysaccharide aggregation is greater for SP-A2 than for coexpressed SP-A1/SP-A2, which in turn is greater than that observed for SP-A1. The presence of SP-A1 polypeptide chains in coexpressed products modulates functional capabilities of SP-A, which depend on both the collagen and globular domains.

Surfactant protein gene A, B, and D marker alleles in chronic obstructive pulmonary disease of a Mexican population.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation. It is most likely the result of complex interactions of environmental and genetic factors. Because pulmonary surfactant components play important roles in normal lung function, innate host defence, and inflammation in the lung, this study investigated the hypothesis that the surfactant protein genes are involved in certain cases of COPD. Genotype analysis of surfactant protein (SP)-A, SP-B, SP-B-linked microsatellite, and SP-D marker alleles was performed in patients with COPD (n=97) and smoker (n=82) or nonsmoker (n=99) controls. Univariate and multiple logistic regression analyses were performed. The regression analysis results between COPD and smokers revealed several COPD susceptibility alleles (AA62_A, B1580_C, D2S388_5), based on an odds ratio (OR >2.5). The predictive ability of this model for developing COPD is good (c=0.926). Allele-allele (B1580_C and D2S388_5) and allele-environment (i.e. smoking) interactions were detected. When smoker controls were compared to nonsmoker controls, marker D2S388 5 appeared to be smoking-independent (p=0.874), whereas marker alleles AA62_A (p=0.045) and B1580_5 (p=0.007) were smoking-dependent. Males were at higher risk (OR=6.05, p=0.001), and smoking (>50 packs x yr(-1)) increased risk (OR=5.38, p=0.007). Males and alleles of loci flanking SP-B were associated with more severe cases (forced expiratory volume in one second/forced vital capacity < or = 40%). The present results indicate that the surfactant protein alleles may be useful in chronic obstructive pulmonary disease by either predicting the disease in a subgroup and/or by identifying disease subgroups that may be used for therapeutic intervention. These observations should now be confirmed in a larger study, designed according to strict epidemiological criteria.

Both human SP-A1 and Sp-A2 genes are expressed in small and large intestine.

The human SP-A locus consists of two functional genes and one pseudogene, and SP-A is shown to play a role in local host defense and the regulation of inflammation in lung. Because the intestine, like the lung, is constantly exposed to foreign and potentially harmful substances, we investigated the hypothesis that both human SP-A genes are expressed in intestine. We demonstrate that both SP-A genes are expressed in human small and large intestine. The presence of SP-A mRNA in human intestine was detected by reverse transcription polymerase chain reaction (RT-PCR), Northern blot analysis, and immunohistochemistry. The size of intestinal SP-A mRNA is the same as that in human lung, but the level of expression, compared with that in the lung, is very low in both the small and large intestine. Immunohistochemical analysis revealed positive reactivity for SP-A in a subgroup of epithelial cells in the intestine. Expression of both SP-A1 and SP-A2 genes was established by gene-specific PCR amplification, PCR-based converted RFLP discrimination, and direct sequencing of RT-PCR products. We speculate that SP-A in the intestine plays a role in local host defense and inflammation.

Response surface modeling for the inactivation of Escherichia coli O157:H7 on green peppers (Capsicum annuum L.) by chlorine dioxide gas treatments.

The effects of chlorine dioxide (ClO2) gas concentration (0.1 to 0.5 mg/liter), relative humidity (RH) (55 to 95%), treatment time (7 to 135 min), and temperature (5 to 25 degrees C) on inactivation of Escherichia coli O157:H7 on green peppers were studied using response surface methods. A four-factor, central, composite, rotatable design was used. The microbial log reduction was measured as a response. A direct membrane-surface-plating method with tryptic soy agar and sorbitol MacConkey agar was used to resuscitate and enumerate ClO2-treated E. coli O157:H7 cells. The statistical analysis and the predictive model developed in this study suggest that ClO2 gas concentration, treatment time, RH, and temperature all significantly (P < 0.01) increased the inactivation of E. coli O157:H7. ClO2 gas concentration was the most important factor, whereas temperature was the least significant. The interaction between ClO2 gas concentration and RH indicated a synergistic effect. The predictive model was validated, and it could be used to determine effective ClO2 gas treatments to achieve a 5-log reduction of E. coli O157:H7 on green peppers.

Surfactant protein A and B genetic variants and respiratory distress syndrome: allele interactions.

The contribution of multiple genetic components in disease pathogenesis is relevant to both diseases of multifactorial and/or multigenic etiology such as the respiratory distress syndrome (RDS) and to diseases where a single gene has been identified as the disease-causing gene. An example of the latter is cystic fibrosis (CF) where the disease-causing gene has been clearly identified as the CF transmembrane conductance regulator gene, but genetic variants of the mannose binding protein and surfactant protein A have been associated with disease severity in CF. The overall rationale for considering genetic contribution to disease pathogenesis is based on the premise that all diseases or deaths (except perhaps those resulting from trauma) have a genetic component. The difference in genetic contribution among various diseases is the percent contribution and the number of factors that make this contribution. Therefore, if the number of genetic contributors is small and the percentage of genetic contribution is high it may be less challenging to identify such factors. In this paper we summarize allele associations and discuss allele interaction of the surfactant protein genes in relation to RDS (the term allele and genetic variant will be used interchangeably).

Induction of AP-1 binding to intron 1 of SP-A1 and SP-A2 is implicated in the phorbol ester inhibition of human SP-A promoter activity.

A deletional analysis of the SP-A1 promoter in NCI-H441 cells was performed to identify potential cis-acting elements involved in phorbol ester-mediated repression of human SP-A transcription. The phorbol ester TPA reduced SP-A1 and SP-A2 promoter activity to approximately 35% to 45% compared to that of control cells. The inhibitory effect of TPA was significantly reduced upon removal of the region +64/+394 relative to the SP-A1 transcription start site. Using NCI-H441 nuclear proteins, electromobility shift assay analysis showed that the intron region +309/+329 of SP-A1 and the corresponding region of SP-A2 formed sequence-specific DNA/protein complexes that were induced by TPA exposure. The region +318/+324 of SP-A1 contains sequences similar to a consensus AP-1 binding site, TGACTGA (TCACTGA for SP-A2), which when mutated (TGAGAGT) prevented the formation of the TPA-induced DNA/protein complex. The TPA-induced complex was supershifted in the presence of antibody against the Jun family of proteins, but not the Fos family of proteins. These results suggest that the binding of AP-1 or an AP-1--like factor to the first intron of SP-A1 and SP-A2 may be involved in the phorbol ester inhibition of human SP-A gene expression.

Human SP-A protein variants derived from one or both genes stimulate TNF-alpha production in the THP-1 cell line.

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-A gene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-alpha production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-alpha production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-alpha production, and 3) the increases in TNF-alpha production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1 and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-alpha levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.

An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances.

SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression.

The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.

Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells.

Glucocorticoids have complex effects on human surfactant protein (SP) SP-A1 and SP-A2 gene expression that occur at both transcriptional and post-transcriptional levels. In the lung adenocarcinoma cell line NCI-H441, dexamethasone causes a dose-dependent decrease in total SP-A mRNA levels and inhibits SP-A gene transcription. In this study, a deletional analysis of the SP-A1 promoter was performed in order to identify cis-acting elements that mediate dexamethasone responsiveness in NCI-H441 cells. The region -32/+63 relative to the start of SP-A1 transcription mediated both basal promoter activity and dexamethasone repression of transcription. Removal of the region +18/+63 abolished dexamethasone responsiveness, indicating that sequences within this region are necessary for the inhibitory effect. Furthermore, the region -32/+63 formed a sequence-specific DNA-protein complex with NCI-H441 nuclear extract. This DNA-protein complex was induced by dexamethasone exposure and its formation was mediated partially by sequences within the region +26/+63.

Regulation of expression of human SP-A1 and SP-A2 genes in fetal lung explant culture.

Human pulmonary surfactant protein A (SP-A) is genetically complex and its regulation may also be complex, reflecting genotypic variability. Fetal lung explants were used to study the regulation of the SP-A genes, SP-A1 and SP-A2, by dexamethasone, interferon, gamma (IFN gamma), cyclic 3',-5' adenosine monophosphate (cAMP), and tumor necrosis factor alpha (TNF alpha). For comparison, the mRNA levels of surfactant protein B (SP-B) and its response to test substances were also examined. Results showed: (a) In control culture total SP-A mRNA varied widely among explants (C.V. = 0.70) compared with SP-B (C.V. = 0.26) (b) IFN gamma significantly increased total SP-A mRNA but there were marked differences among fetal lungs in response to all treatments. (c) SP-A1 mRNA concentration is higher than SP-A2 in both control and treated explants. (d) SP-A1 alleles are inhibited to a greater degree by dexamethasone than SP-A2 alleles. The relative effect of cAMP and IFN gamma on SP-A1 and SP-A2 mRNA varied widely among explants. We conclude that SP-A genotype may account in part for the marked differences in SP-A mRNA concentration among fetal lungs and that the SP-A genes and/or alleles may be differentially regulated.

Organization of the human SP-A and SP-D loci at 10q22-q23. Physical and radiation hybrid mapping reveal gene order and orientation.

The human surfactant protein (SP) A locus has been assigned to chromosome 10q22-q23 and consists of two very similar genes, SP-A1 and SP-A2, as well as a truncated pseudogene. SP-A belongs to the family of collagenous C-type lectins along with mannose binding protein (MBP) and SP-D, both of which have also been mapped to the long arm of chromosome 10. In this article we report the relative location and orientation of each of the SP-A and SP-D genomic sequences. Characterization of two overlapping genomic clones revealed that the SP-A pseudogene lies in a reverse orientation 15 kb away from the 5' side of SP-A1. This finding was verified by the amplification of the entire SP-A pseudogene/SP-A1 intergenic region using long-range polymerase chain reaction. The relative location of SP-A2 and SP-D was then ascertained by testing a number of sequence tagged sites against the Stanford TNG3 and G3 radiation hybrid panels. The radiation hybrid mapping data showed that both SP-A2 and SP-D are on the 5' side of SP-A1 at approximate distances of 40 kb and 120 kb, respectively. The SP-A and SP-D loci were also oriented relative to the centromere, with the overall order being: centromere-SP-D-SP-A2-pseudogene-SP-A1- telomere.

Surfactant protein A activates NF-kappa B in the THP-1 monocytic cell line.

The expression of many genes for which products are involved in inflammation is controlled by the transcriptional regulator nuclear factor (NF)-kappa B. Because surfactant protein (SP) A is involved in local host defense in the lung and alters immune cell function by modulating the expression of proinflammatory cytokines as well as surface proteins involved in inflammation, we hypothesized that SP-A exerts its action, at least in part, via activation of NF-kappa B. We used gel shift assays to determine whether SP-A activated NF-kappa B in the THP-1 cell line, a human monocytic cell line. Activation of NF-kappa B in THP-1 cells by SP-A doses as low as 1 microgram/ml occurred within 30 min of SP-A treatment, peaked at 60 min, and then declined. This activation is inhibited by known inhibitors of NF-kappa B or by simultaneous treatment of the cells with surfactant lipids. Moreover, the NF-kappa B inhibitors blocked SP-A-dependent increases in tumor necrosis factor-alpha mRNA levels. These observations suggest a mechanism by which SP-A plays a role in the pathogenesis of some lung conditions and point to potential therapeutic measures that could be used to prevent SP-A induced inflammation in the lung.

Conditional evaluation of broncho-alveolar lavage in mechanically ventilated patients with suspected unilateral lobar pneumonia.

OBJECTIVE: In an attempt to improve our ability to diagnose the cause of ventilator-associated pneumonia (VAP), we explore the usefulness of the conditional evaluation of bronchoalveolar lavage (BAL) samples from the involved and non-involved areas in patients with suspected unilateral lobar VAP (UL-VAP). DESIGN: Prospective study. SETTING: University teaching hospital intensive care unit. PATIENTS: We studied 19 consecutive patients with suspected UL-VAP. MEASUREMENTS AND MAIN RESULTS: Nine of the 12 patients (47%) developed UL-VAP. There was a significant difference between the involved and non-involved areas in UL-VAP patients (P < 0.001) in respect of the quantitative bacterial cultures (QBCs) of BAL samples for each micro-organism, whereas there was no difference in patients without UL-VAP. When we applied the criterion of usual BAL (one micro-organism in concentrations > 10(5) colony-forming units per millilitre) for UL-VAP diagnosis, the sensitivity was 100%, the specificity 70%, the positive predictive value 75%, and the negative predictive value 100%. When we used the conditional evaluation of the BAL results for UL-VAP diagnosis, in the involved and non-involved areas, the sensitivity was 78%, the specificity 90%, the positive predictive value 87.5% and the negative predictive value 82%. A statistically significant difference was found when we compared the difference in QBCs between the BAL samples for each micro-organism, between the involved and non-involved areas in patients with and without VAP (P < 0.001). CONCLUSION: These data suggest that utilisation of the conditional evaluation of the QBCs of BAL samples improves significantly our ability to diagnose the cause of UL-VAP.

In vitro analysis of rat surfactant protein A gene expression.

Previous findings suggest that the rat proximal promoter segment (rPPS) of the SP-A gene is important in the regulation of the lung-specific expression of the gene. In this report, two regions within the rPPS containing thyroid transcription factor-1 (TTF-1) binding sites are identified that form strong lung- and thyroid- specific DNA-protein complexes. These regions bind nuclear polypeptides with similar apparent molecular mass to TTF-1, suggesting that TTF-1 binds to these regions. Two regions within the rPPS that form weaker lung-specific DNA-protein complexes are also identified. The transcription start site is mapped, and a functional analysis shows that the sequences of the 5' flanking region are sufficient to support in vitro transcription but are not sufficient to reproduce tissue-specific expression. Together, these results show that lung- and thyroid-specific, as well as lung-specific, DNA-protein interactions occur within the rPPS but are not sufficient for the lung-specific expression of the rat SP-A gene to be duplicated in vitro.

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.