Neonatal abstinence syndrome (NAS) is a clinical diagnosis based on the observation of symptoms attributed to postpartum substance withdrawal in a newborn with chronic in utero exposure. It is most frequently attributed to prescription and nonprescription maternal opioid use during pregnancy. 1 Both NAS and maternal opioid use during pregnancy are increasing in incidence. 2,3 Pediatricians can identify infants at risk for NAS on the basis of maternal history and drug screens.

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.

Surfactant protein B (SP-B)is essential to the function of pulmonary surfactant and to lamellar body genesis in alveolar epithelial type 2 cells. The bioactive, mature SP-B is derived from multistep post-translational proteolysis of a larger proprotein. The identity of the proteases involved in carboxyl-terminal cleavage of proSP-B remains uncertain. This cleavage event distinguishes SP-B production in type 2 cells from less complete processing in bronchiolar Clara cells. We previously identified pepsinogen C as an alveolar type 2 cell-specific protease that was developmentally regulated in the human fetal lung. We report that pepsinogen C cleaved recombinant proSP-B at Met 302 in addition to an amino-terminal cleavage at Ser 197

For alveolar type I cells, phenotype plasticity and physiology other than gas exchange await further clarification due to in vitro study difficulties in isolating and maintaining type I cells in primary culture. Using an established in vitro model of human fetal type II cells, in which the type II phenotype is induced and maintained by adding hormones, we assessed for transdifferentiation in culture toward a type I-like cell with hormone removal for up to 144 h, followed by electron microscopy, permeability studies, and RNA and protein analysis. T he distal alveolar epithelium contains type I and II cells. Type II cells comprise 75% of distal lung epithelium, 7% of the surface area, and produce surfactant. Type II cells have many well-characterized markers, including lamellar bodies, surfactant proteins A, B, C, and D (SP-A, B, C, and D) (1), and the aspartic protease pepsinogen C (PGC) (2).Type I cells, although less numerous, cover 93% of adult lung surface area and provide the gas exchange surface (1). Other type I functions, such as ion transport and fluid homeostasis, are less known. Type I cells form a tight monolayer with low permeability (3). However, the utility of previously described type I biochemical markers (4) has been limited by variable reproducibility and expression between species.Challenges in isolation and culture have limited progress in type I cell biology. Despite isolated reports (5,6), there are no widespread primary culture models. Instead, rat type II cells transdifferentiated toward a type I cell on tissue culture plastic (7) have served as a proxy for type I cells (8,9). There are limited data with regards to transdifferentiation in adult human alveolar epithelium (10), with no previous in vivo descriptions in human fetal lung. Rodent model transdifferentiation and the observation that type II cells serve as progenitors of type I cells after alveolar injury in mature lung (11) have led to the assumption that type I cells are derived from type II cells. However, the possibility that both are derived from a common precursor and retain some degree of plasticity has not been examined in the human fetal lung.Because of its potential plasticity, fetal alveolar epithelium provides unique opportunities to study differentiation/ transdifferentiation pathway(s) in the developing lung. We previously demonstrated that type II cells from human fetal lung can maintain a differentiated phenotype with dexamethasone, cAMP, and isobutylmethylxanthine (DCI) (12) and that these conditions induce differentiation of type II cells from naive human fetal lung epithelium (13). Here, we demonstrate that DCI withdrawal from cultured human fetal type II cells results in transdifferentiation toward type I-like cells. Transdifferentiation is associated with diminution of type II morphology, decreased expression of type II markers, and induction of type I cell markers. Importantly, transdifferentiated cells behave like type I cells, with decreased permeability and increased transepithelial resistance (T...