Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by bacterial elastase in a murine null-infection model.

Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 10(7) or 10(9) CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular surfaces cleared both P. aeruginosa strains efficiently, even when 10(9) CFU was used: e.g., <0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in SP-D gene-targeted (SP-D(-/-)) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D(-/-) mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.

Expression of surfactant protein D in human corneal epithelial cells is upregulated by Pseudomonas aeruginosa.

We reported previously that surfactant protein D (SP-D) was present in human tears and corneal epithelial cells, and that it contributed to tear fluid protection of those cells against Pseudomonas aeruginosa invasion. This suggested a role in ocular innate immunity. Here, we explored the effects of bacterial challenge on SP-D expression by human corneal epithelial cells. Results showed that these cells produced and secreted SP-D constitutively in culture, and that production (mRNA, protein) and secretion of SP-D were upregulated after exposure to heat-killed P. aeruginosa or to purified flagellin or lipopolysaccharide. To begin exploring the mechanism for flagellin-mediated SP-D induction, cells were exposed to purified flagellin or flagellin mutated in the TLR-5-binding domain (L94A, L88A) which reduces IL-8 secretion by A549 respiratory cells. Mutated flagellin did not upregulate IL-8 expression in corneal epithelial cells, but did induce SP-D responses. Mitogen-activated protein kinase inhibitors, especially the JNK inhibitor SP600125, reduced secretion of SP-D, but not production, in the presence of P. aeruginosa. These data show that while SP-D and IL-8 corneal responses are each induced by P. aeruginosa or its antigens, they can involve different regions of the same ligand. The data suggest that separate mechanisms may regulate SP-D secretion and production by human corneal epithelia.

Integrin beta6 mediates phospholipid and collectin homeostasis by activation of latent TGF-beta1.

Surfactant lines the alveolar surface and prevents alveolar collapse. Derangements of surfactant cause respiratory failure and interstitial lung diseases. The collectins, surfactant proteins A and D, are also important in innate host defense. However, surfactant regulation in the postnatal lung is poorly understood. We found that the epithelial integrin, alphavbeta6, regulates surfactant homeostasis in vivo by activating latent transforming growth factor (TGF)-beta. Adult mice lacking the beta-subunit of alphavbeta6 (Itgb6-/-) developed increased bronchoalveolar lavage phospholipids and surfactant proteins A and D, and demonstrated abnormal-appearing alveolar macrophages, reminiscent of the human disease pulmonary alveolar proteinosis. Using lung-specific expression of constitutively active TGF-beta1 in Itgb6-/- mice, we found that TGF-beta1 was sufficient to normalize these abnormalities. Tgfbeta1-deficient mice also demonstrated increased phospholipids and surfactant proteins A and D, but mice lacking the key TGF-beta signaling molecule, SMAD3, did not. Therefore, integrin-mediated activation of latent TGF-beta1 regulates surfactant constituents independent of intracellular SMAD3. In vivo increases in surfactant protein A and D were not associated with increases in mRNA for these proteins in alveolar tissue from Itgb6-/- mice. On the other hand, isolated alveolar macrophages from Itgb6-/- mice were defective in processing phospholipids in vitro, suggesting that reduced surfactant clearance contributes to altered surfactant homeostasis in these mice in vivo. These findings show that alphavbeta6 and TGF-beta1 regulate homeostasis of phospholipids and collectins in adult mouse lungs and may have implications for anti-fibrotic therapeutics that inhibit active TGF-beta in the lung.

A randomized trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia.

BACKGROUND: Experimental and clinical data suggest that fetal endoscopic tracheal occlusion to induce lung growth may improve the outcome of severe congenital diaphragmatic hernia. We performed a randomized, controlled trial comparing fetal tracheal occlusion with standard postnatal care. METHODS: Women carrying fetuses that were between 22 and 27 weeks of gestation and that had severe, left-sided congenital diaphragmatic hernia (liver herniation and a lung-to-head ratio below 1.4), with no other detectable anomalies, were randomly assigned to fetal endoscopic tracheal occlusion or standard care. The primary outcome was survival at the age of 90 days; the secondary outcomes were measures of maternal and neonatal morbidity. RESULTS: Of 28 women who met the entry criteria, 24 agreed to randomization. Enrollment was stopped after 24 patients had been enrolled because of the unexpectedly high survival rate with standard care and the conclusion of the data safety monitoring board that further recruitment would not result in significant differences between the groups. Eight of 11 fetuses (73 percent) in the tracheal-occlusion group and 10 of 13 (77 percent) in the group that received standard care survived to 90 days of age (P=1.00). The severity of the congenital diaphragmatic hernia at randomization, as measured by the lung-to-head ratio, was inversely related to survival in both groups. Premature rupture of the membranes and preterm delivery were more common in the group receiving the intervention than in the group receiving standard care (mean [+/-SD] gestational age at delivery, 30.8+/-2.0 weeks vs. 37.0+/-1.5 weeks; P<0.001). The rates of neonatal morbidity did not differ between the groups. CONCLUSIONS: Tracheal occlusion did not improve survival or morbidity rates in this cohort of fetuses with congenital diaphragmatic hernia.

Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice.

BACKGROUND: Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. METHODS: SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. MAIN RESULTS: After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. CONCLUSION: Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Abnormal vascular tone in infants and children with lung hypoplasia: Findings from cardiac catheterization and the response to chronic therapy.

OBJECTIVE: We describe four cases of chronic pulmonary hypertension in infants and children with chronic lung disease and pulmonary hypoplasia due to severe congenital diaphragmatic hernia (CDH) or congenital cystic adenomatoid malformation (CCAM). We report data from cardiac catheterization under various conditions: baseline respiratory support and room air, hyperoxic and inhaled nitric oxide challenge. We further report cardiac catheterization measures after chronic pulmonary vasodilator therapy with sildenafil alone or a combination of sildenafil and inhaled nitric oxide (three patients). DESIGN: Case series. SETTING: Tertiary academic center. PATIENTS: Infants and children ages 0-11 yrs with CDH (n = 3) or CCAM (n = 1) with evidence of chronic pulmonary hypertension by echocardiogram and cor pulmonale (n = 3). INTERVENTIONS: Catheterization and pulmonary vasodilator therapy. MEASUREMENTS AND MAIN RESULTS: Pulmonary vascular resistance, pulmonary arterial pressure, and changes in these measures were assessed. A 20% change in pulmonary vascular resistance was considered a clinically significant response. Ten catheterizations were performed in four patients. All patients had elevated pulmonary vascular resistance and pulmonary arterial pressures at initial catheterizations and significant vasodilation during inhaled nitric oxide. CONCLUSIONS: Chronic lung disease following pulmonary hypoplasia from CDH and CCAM is associated with abnormal pulmonary vascular tone in infants and children with evidence of chronic pulmonary hypertension. Chronic pulmonary vasodilator therapy may improve pulmonary vascular function and enhance lung growth in infants and children who are treated during their period of potential for rapid lung growth.

Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli.

To investigate the effects of surfactant proteins A and D (SP-A and SP-D, respectively) in urinary tract infection (UTI), SP-A and SP-D double knockout (SP-A/D KO) and wild type (WT) C57BL/6 female mice were infected with uropathogenic Escherichia coli by intravesical inoculation. Compared with WT mice SP-A/D KO mice showed increased susceptibility to UTI, as evidenced by higher bacterial CFU, more infiltrating neutrophils and severe pathological changes. Keratinocyte-derived chemokine increased in the kidney of WT mice but not in SP-A/D KO mice 24 h post-infection. Compared with control, the level of IL-17 was elevated in the kidney of infected WT and SP-A/D KO mice and the level of IL-17 was higher in the infected SP-A/D KO mice than in infected WT mice 24 and 48 h post-infection. The basal level of p38 MAPK phosphorylation in SP-A/D KO mice was higher than in WT mice. The phosphorylated p38 level was elevated in the kidney of WT mice post infection but not in SP-A/D KO mice. Furthermore, in vitro growth of uropathogenic E. coli was inhibited by SP-A and SP-D. We conclude that SP-A and SP-D function as mediators of innate immunity by inhibiting bacterial growth and modulating renal inflammation in part by regulating p38 MAPK-related pathway in murine UTI.

Design-based stereological analysis of the lung parenchymal architecture and alveolar type II cells in surfactant protein A and D double deficient mice.

Alveolar epithelial type II cells synthesize and secrete surfactant. The surfactant-associated proteins A and D (SP-A and SP-D), members of the collectin protein family, participate in pulmonary immune defense, modulation of inflammation, and surfactant metabolism. Both proteins are known to have overlapping as well as distinct functions. The present study provides a design-based stereological analysis of adult mice deficient in both SP-A and SP-D (A(-)D(-)) with special emphasis on parameters characterizing alveolar architecture and surfactant-producing type II cells. Compared to wild-type, A(-)D(-) mice have fewer and larger alveoli, an increase in the number and size of type II cells, as well as more numerous and larger alveolar macrophages. More surfactant-storing lamellar bodies are seen in type II cells, leading to a threefold increase in the total volume of lamellar bodies per lung, but the mean volume of a single lamellar body remains constant. These results demonstrate that chronic deficiency of SP-A and SP-D in mice leads to parenchymal remodeling, type II cell hyperplasia and hypertrophy, and disturbed intracellular surfactant metabolism. The design-based stereological approach presented here provides a framework for the quantitative lung structure analysis in gene-manipulated mice as well as in human lung disease.

Protective Role of Surfactant Protein D in Ocular Staphylococcus aureus Infection.

Staphylococcus aureus is one of the most common pathogens causing keratitis. Surfactant protein D (SP-D) plays a critical role in host defense and innate immunity. In order to investigate the role of SP-D in ocular S. aureus infection, the eyes of wild-type (WT) and SP-D knockout (SP-D KO) C57BL/6 mice were infected with S. aureus (10(7) CFU/eye) in the presence and absence of cysteine protease inhibitor(E64).Bacterial counts in the ocular surface were examined 3, 6, 12, 24 hrs after infection. Bacterial phagocytosis by neutrophils and bacterial invasion in ocular epithelial cells were evaluated quantitatively. S. aureus-induced ocular injury was determined with corneal fluorescein staining. The results demonstrated that SP-D is expressed in ocular surface epithelium and the lacrimal gland; WT mice had increased clearance of S. aureus from the ocular surface (p<0.05) and reduced ocular injury compared with SP-D KO mice. The protective effects of SP-D include increased bacterial phagocytosis by neutrophils (p<0.05) and decreased bacterial invasion into epithelial cells (p<0.05) in WT mice compared to in SP-D KO mice. In the presence of inhibitor (E64), WT mice showed enhanced bacterial clearance (p<0.05) and reduced ocular injury compared to absent E64 while SP-D KO mice did not. Collectively, we concluded that SP-D protects the ocular surface from S. aureus infection but cysteine protease impairs SP-D function in this murine model, and that cysteine protease inhibitor may be a potential therapeutic agent in S. aureus keratitis.

Tissue distribution of surfactant proteins A and D in the mouse.

Surfactant proteins A and D, collagen-like lectins (collectins), were first isolated from the lung. In the lung, SP-A and SP-D have roles in surfactant homeostasis and innate immunity. In this study we show that SP-A and SP-D mRNA can be detected in a significant number of non-pulmonary tissues but the proteins have a more limited distribution. SP-D protein was detected in lung, uterus, ovary, and lacrimal gland, whereas SP-A protein was detected only in the lung. The results suggest that SP-D participates in mucosal immunity throughout the body.

A recombinant fragment of human surfactant protein D reduces alveolar macrophage apoptosis and pro-inflammatory cytokines in mice developing pulmonary emphysema.

Rapid removal of apoptotic cells is an important mechanism for immune homeostasis and the resolution of inflammation. Delayed clearance of apoptotic alveolar macrophages may cause activation of healthy bystander macrophages and contribute to high macrophage number and emphysema in surfactant protein D (SP-D) knock-out mice. Using flow cytometry and Annexin V and propidium iodide as markers for apoptosis and necrosis, respectively, SP-D-deficient mice were found to have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages in the lungs. SP-D-deficient mice accumulate apoptotic macrophages in the lung, and this accumulation can be reduced by treatment with recombinant SP-D (but not SP-A). The recombinant SP-D binds preferentially to apoptotic cells. The data are consistent with a specific role in vivo for SP-D in promoting apoptotic cell clearance in the lungs to limit macrophage-mediated inflammation and reveal a potential new mechanism for therapeutic targeting in the prevention of emphysema.

Surfactant protein D binds genomic DNA and apoptotic cells, and enhances their clearance, in vivo.

Collectins, such as surfactant protein D (SP-D), bind apoptotic cells; however, the ligands that they recognize on these cells are unknown. We hypothesized that SP-D binds to the DNA present on these cells. We show that SP-D binds and aggregates mouse alveolar macrophage DNA effectively. Alveolar macrophages of SP-D((-/-)) mice contained more nicked DNA than those of SP-A((-/-)) and wild type mice. Our results also suggest that carbohydrate recognition domains of SP-D may recognize DNA present on the apoptotic cells. Therefore, cell-surface DNA could be a ligand for recognition of apoptotic cells by collectins.

Treatment of rebound and chronic pulmonary hypertension with oral sildenafil in an infant with congenital diaphragmatic hernia.

OBJECTIVE: We describe a case of chronic pulmonary hypertension in a 7-wk-old infant with congenital diaphragmatic hernia and an oral teratoma. Our patient was dependent on low-dose inhaled nitric oxide and was still very unstable with systemic right ventricular pressures leading to frequent oxygen desaturations. We administered sildenafil therapy to stabilize the infant with discontinuation of inhaled nitric oxide. We describe successful discontinuation of the inhaled therapy as well as a period of stabilization and improvement with continued sildenafil administration. DESIGN: Case report. SETTING: Intensive care nursery in tertiary academic center. PATIENT: A 7-wk-old infant with congenital diaphragmatic hernia who was mechanically ventilated from birth. INTERVENTION: Oral sildenafil 0.3 mg/kg/dose every 12 hrs. MEASUREMENTS AND RESULTS: Right ventricular pressure (from tricuspid valve regurgitant flow) to systemic systolic arterial pressure was measured by echocardiogram. Right ventricular to systemic pressure ratio was marginally improved with the initiation of sildenafil therapy. Inhaled nitric oxide was successfully discontinued, and the patient clinically stabilized temporarily, but he ultimately succumbed to his pulmonary hypertension. CONCLUSION: Sildenafil may be a useful therapy for chronic pulmonary hypertension in congenital diaphragmatic hernia, but further studies of safety and efficacy need to be performed.

Mice deficient in surfactant protein A (SP-A) and SP-D or in TLR2 manifest delayed parturition and decreased expression of inflammatory and contractile genes.

Previously we obtained compelling evidence that the fetus provides a critical signal for the initiation of term labor through developmental induction of surfactant protein (SP)-A expression by the fetal lung and secretion into amniotic fluid (AF). We proposed that interactions of AF macrophage (Mϕ) Toll-like receptors (TLRs) with SP-A, at term, or bacterial components, at preterm, result in their activation and migration to the pregnant uterus. Herein the timing of labor in wild-type (WT) C57BL/6 mice was compared with mice homozygous null for TLR2, SP-A, SP-D, or doubly deficient in SP-A and SP-D. Interestingly, TLR2(-/-) females manifested a significant (P < 0.001) delay in timing of labor compared with WT as well as reduced expression of the myometrial contraction-associated protein (CAP) gene, connexin-43, and Mϕ marker, F4/80, at 18.5 d postcoitum (dpc). Whereas in first pregnancies, SP-A(-/-), SP-D(-/-), and SP-A/D(-/-) females delivered at term (∼19.5 dpc), in second pregnancies, parturition was delayed by approximately 12 h in SP-A(-/-) (P = 0.07) and in SP-A/D(-/-) (P <0.001) females. Myometrium of SP-A/D(-/-) females expressed significantly lower levels of IL-1ß, IL-6, and CAP genes, connexin-43, and oxytocin receptor at 18.5 dpc compared with WT. F4/80(+) AF Mϕs from TLR2(-/-) and SP-A/D(-/-) mice expressed significantly lower levels of both proinflammatory and antiinflammatory activation markers (e.g. IL-1ß, IL-6, ARG1, YM1) compared with gestation-matched WT AF Mϕs. These novel findings suggest that the pulmonary collectins acting via TLR2 serve a modulatory role in the timing of labor; their relative impact may be dependent on parity.

Surfactant protein d deficiency in mice is associated with hyperphagia, altered fat deposition, insulin resistance, and increased basal endotoxemia.

Pulmonary surfactant protein D (SP-D) is a host defence lectin of the innate immune system that enhances clearance of pathogens and modulates inflammatory responses. Recently it has been found that systemic SP-D is associated with metabolic disturbances and that SP-D deficient mice are mildly obese. However, the mechanism behind SP-D's role in energy metabolism is not known.Here we report that SP-D deficient mice had significantly higher ad libitum energy intake compared to wild-type mice and unchanged energy expenditure. This resulted in accumulation but also redistribution of fat tissue. Blood pressure was unchanged. The change in energy intake was unrelated to the basal levels of hypothalamic Pro-opiomelanocortin (POMC) and Agouti-related peptide (AgRP) gene expression. Neither short time systemic, nor intracereberoventricular SP-D treatment altered the hypothalamic signalling or body weight accumulation.In ad libitum fed animals, serum leptin, insulin, and glucose were significantly increased in mice deficient in SP-D, and indicative of insulin resistance. However, restricted diets eliminated all metabolic differences except the distribution of body fat. SP-D deficiency was further associated with elevated levels of systemic bacterial lipopolysaccharide.In conclusion, our findings suggest that lack of SP-D mediates modulation of food intake not directly involving hypothalamic regulatory pathways. The resulting accumulation of adipose tissue was associated with insulin resistance. The data suggest SP-D as a regulator of energy intake and body composition and an inhibitor of metabolic endotoxemia. SP-D may play a causal role at the crossroads of inflammation, obesity, and insulin resistance.

Susceptibility of mice genetically deficient in SP-A or SP-D gene to invasive pulmonary aspergillosis.

Pulmonary surfactant proteins, SP-A and SP-D, are carbohydrate pattern recognition molecules of innate immunity, which significantly enhance phagocytosis and killing of Aspergillus fumigatus, a pathogenic fungus, by neutrophils and macrophages. The present study examined the susceptibility of immunosuppressed SP-A gene deficient (SP-A(-/-)) or SP-D gene deficient (SP-D(-/-)) mice to A. fumigatus conidia challenge compared to wild-type (WT) mice. A. fumigatus-challenged SP-A(-/-) (SP-A(-/-) IPA) mice showed less mortality (40%) than the WT-IPA mice (100%) and increased mortality (60%) following administration of SP-A with decreased TNF-alpha and IFN-gamma to IL-4 ratio than SP-A(-/-) IPA mice. The SP-D(-/-) IPA mice (57.14%) showed similar mortality as WT-IPA mice (60%). However, the SP-D (-/-) IPA mice (42.86% mortality on day 2) died earlier than the WT-IPA mice (20% mortality on day 2), showed a higher hyphal density and tissue injury in lungs. Treatment with SP-D or a recombinant fragment of human SP-D rhSP-D reduced the mortality to 50% and 33%, respectively, concomitant with higher IFN-gamma to IL-4 ratios in treated SP-D(-/-) mice, compared to untreated control group. The results showed that SP-D gene deficient mice are more susceptible to IPA while SP-A gene deficient mice acquire resistance to IPA.

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice.

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.

A recombinant fragment of human surfactant protein D lacking the short collagen-like stalk fails to correct morphological alterations in lungs of SP-D deficient mice.

Emphysema-like pathology is a characteristic feature of surfactant protein D (SP-D) knock-out mice. Treatment with a recombinant fragment of human SP-D consisting of a short collagen-like stalk (but not the entire collagen-like domain of native SP-D), neck, and carbohydrate recognizing domain (CRD) inhibits development of emphysema-like pathology in SP-D deficient mice. On the other hand, it has been shown that the entire collagen-like domain is necessary for preventing SP-D knock-out mice from pulmonary emphysema development. Thus, in the present study, we aimed to elucidate the role of the short collagen-like stalk for the function of the recombinant fragment of human SP-D. We treated SP-D knock-out mice with a fragment of human SP-D lacking the short collagen-like stalk and compared the effects on lung morphology with results from untreated wild-type and SP-D knock-out mice and from SP-D knock-out mice treated with a recombinant fragment of human SP-D including the short collagen-like stalk. The fragment of SP-D lacking the short collagen-like stalk failed to correct pulmonary emphysematous alterations demonstrating the importance of the short collagen-like stalk for the biological activity of the recombinant fragment of human SP-D.

Genetic analysis of congenital cystic adenomatoid malformation reveals a novel pulmonary gene: fatty acid binding protein-7 (brain type).

The pathogenesis of congenital cystic adenomatoid malformation (CCAM) is unknown and its natural history is unpredictable. Fatty acid binding protein-7 (FABP-7) has been previously described in brain and breast development, but never before in the lung. We investigate gene expression in CCAM, and hypothesize that CCAM results from an aberration in the signaling pathway during lung development. Under IRB approval, tissue specimens of fetal CCAM, fetal control, postnatal CCAM, and postnatal control were examined and microarray analysis was performed. Candidate differentially expressed genes were selected with log-odds ratio (B) >0 and false discovery rate <0.05. Validation of differential expression was achieved at the RNA and protein levels. FABP-7 was underexpressed in fetal CCAM compared with fetal lung in both the microarray and by RT-PCR. Findings were duplicated by Western Blot analysis and immunohistochemistry. This is the first description of FABP-7 in the human lung. Decreased expression of FABP-7 in fetal CCAM compared with normal fetal lung at both the RNA and protein levels suggests FABP-7 may have a role in pulmonary development and in the pathogenesis of CCAM.