Mmol/ml, respectively) compared to N (7.99±0.60 Amol/ml).Phosphatidylcholine was decreased in A (62.64±2.20% PL) compared to N (76.27±2.05% PL). Phosphatidylglycerol was 11.58±1.21% PL in N and was decreased to 6.48±1.43% PL in A. SP-A was 123.64±20.66 zg/ml in N and was decreased to 49.28±21.68 jg/ml in AR and to 29.88±8.49 jg/ml in A. SP-B was 1.28±0.33 jig/ml in N and was decreased to 0.57±0.24 ,ug/ml in A. ST,,. was increased in AR (15.1±2.53 dyn/cm) and A (29.04±2.05 dyn/cm) compared to N (7.44±1.61 dyn/cm).

Lung surfactant is deficient in patients with acute respiratory distress syndrome (ARDS). We performed a randomized, prospective, controlled, open-label clinical study of administration of a bovine surfactant to patients with ARDS to obtain preliminary information about its safety and efficacy. Patients received either surfactant by endotracheal instillation in addition to standard therapy or standard therapy only. Three different groups of patients receiving surfactant were studied: patients receiving up to eight doses of 50 mg phospholipids/kg, those receiving up to eight doses of 100 mg phospholipids/kg, and those receiving up to four doses of 100 mg phospholipids/kg. Outcome measures included ventilatory support parameters, arterial blood gases, organ system failures, bronchoalveolar lavage (BAL) analyses, immunologic analyses, survival, and adverse events during the 28-d study period. Fifty-nine study patients were evaluable; 43 in the surfactant group and 16 in the control group. The FI(O2) at 120 h after treatment began was significantly decreased only for patients who received up to four doses of 100 mg phospholipids/kg surfactant as compared with control patients (p = 0.011). Mortality in the same group of patients was 18.8%, as compared with 43.8% in the control group (p = 0.075). The surfactant instillation was generally well tolerated, and no safety concerns were identified. This pilot study presents preliminary evidence that surfactant might have therapeutic benefit for patients with ARDS, and provides rationale for further clinical study of this agent.

CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.

Type II pneumocytes secrete pulmonary surfactant and are known to synthesize SP-35, a collagenous surfactant-associated protein. Freshly isolated type II cells also synthesize other bacterial collagenase-sensitive and hydroxyproline-containing proteins, including a glycoprotein designated CP4. CP4 was isolated from rat pneumocyte culture medium by immune precipitation with polyclonal antibodies to rat surfactant proteins or by DEAE chromatography and reverse-phase or gel permeation HPLC. CP4 did not cross-react with polyclonal antibodies to SP-35 and was completely resolved from SP-35 by SDS-PAGE (Mr 43K reduced) or isoelectric focusing. Unlike SP-35, which consists of acidic isoforms assembled as disulfide-bonded dimers and multimers, CP4 was secreted as basic isoforms assembled as disulfide-bonded trimers. Differences in primary structure were demonstrated by CNBr and V8 protease peptide mapping. The secretion of both proteins was inhibited by 2,2'-dipyridyl, an inhibitor of posttranslational prolyl and lysyl hydroxylation and collagen triple helix formation. CP4 was isolated from EDTA extracts of rat surfactant. These studies provide evidence for the heterogeneity of pneumocyte-derived collagenous surfactant-associated proteins.

The 5' flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.