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Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, X

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting Lα and Lβ phases. In the Lβ structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the Lβ composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The Lβ chains, however, lack the tilt of the Lβ′ phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the Lβ phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactant in situ reduce surface tension to levels at which surfactant monolayers collapse from the surface in vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes induced by the added vesicles. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and X-ray reflection showed that the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from enlargement by the additional material of the ordered phase.

The only known compositional change in the phospholipids (PL) of pulmonary surfactant in response to a physiologic stimulus occurs around the time of birth. In most species, the predominant anionic PL changes from phosphatidylinositol (PtdIns) to phosphatidylglycerol (PtdGro). Because prior studies have shown that the change in the headgroup itself is functionally insignificant, we tested the hypothesis that the PtdIns and PtdGro contain different diacyl pairs. Experiments used electrospray-ionization mass spectrometry to determine the molecular species in PtdIns, PtdGro, and phosphatidylcholine (PtdCho) in surfactant from newborn calves and cows. The profiles for the two anionic PL were distinct. The PtdIns contained long, unsaturated fatty acid chains and no disaturated species. The PtdGro more closely resembled the profile from PtdCho. For each headgroup, the molecular species for calf and cow were similar. The differences between the two anionic PL indicate that the switch from PtdIns to PtdGro during maturation involves more than simple substitution of the headgroup, and suggest that the functional significance of the shift may reflect the different pool of diacyl pairs.

To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used x-ray diffuse scattering (XDS) to determine an order parameter of the lipid chains (S(xray)) and the bending modulus of the lipid bilayers (K(C)). Samples contained different amounts of the proteins with two sets of lipids. Dioleoylphosphatidylcholine (DOPC) provided a simple, well characterized model system. The nonpolar and phospholipids (N&PL) from extracted calf surfactant provided the biological mix of lipids. For both systems, the proteins produced changes in S(xray) that correlated well with K(C). The dose-response to the proteins, however, differed. Small amounts of protein generated large decreases in S(xray) and K(C) for DOPC that progressed monotonically. The changes for the surfactant lipids were erratic. Our studies then tested whether the proteins produced correlated effects on adsorption. Experiments measured the initial fall in surface tension during adsorption to a constant surface area, and then expansion of the interface during adsorption at a constant surface tension of 40 mN/m. The proteins produced a sigmoidal increase in the rate of adsorption at 40 mN/m for both lipids. The results correlated poorly with the changes in S(xray) and K(C) in both cases. Disordering of the lipid chains produced by the proteins, and the softening of the bilayers, fail to explain how the proteins promote adsorption of lipid vesicles.

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting L(α) and L(β) phases. In the L(β) structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the L(β) composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The L(β) chains, however, lack the tilt of the L(β′) phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the L(β) phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.

The hydrophobic surfactant proteins, SP-B and SP-C, promote rapid adsorption by the surfactant lipids to the surface of the liquid that lines the alveolar air sacks of the lungs. To gain insights into the mechanisms of their function, we used X-ray diffuse scattering (XDS) and molecular dynamics (MD) simulations to determine the location of SP-B and SP-C within phospholipid bilayers. Initial samples contained the surfactant lipids from extracted calf surfactant with increasing doses of the proteins. XDS located protein density near the phospholipid headgroup and in the hydrocarbon core, presumed to be SP-B and SP-C, respectively. Measurements on dioleoylphosphatidylcholine (DOPC) with the proteins produced similar results. MD simulations of the proteins with DOPC provided molecular detail and allowed direct comparison of the experimental and simulated results. Simulations used conformations of SP-B based on other members of the saposin-like family, which form either open or closed V-shaped structures. For SP-C, the amino acid sequence suggests a partial α-helix. Simulations fit best with measurements of XDS for closed SP-B, which occurred at the membrane surface, and SP-C oriented along the hydrophobic interior. Our results provide the most definitive evidence yet concerning the location and orientation of the hydrophobic surfactant proteins.

Background Certolizumab pegol (CZP) treatment has been shown to significantly improve work and household productivity and social participation compared to placebo in active non-radiographic axial spondyloarthritis (nr-axSpA) patients up to 24 weeks. Here, we report the impact of CZP in combination with non-biologic background medication (NBBM) on signs and symptoms of nr-axSpA compared to placebo+NBBM. Methods C-axSpAnd (NCT02552212) is a 3-year, phase 3, multicentre study including a 52-week double-blind, placebo-controlled period (completed). Patients had active nr-axSpA, objective signs of inflammation (OSI; elevated CRP and/or positive MRI of the sacroiliac joint), previous inadequate response to ≥ 2 NSAIDs and were randomised 1:1 to CZP (400 mg at Weeks 0/2/4, then 200 mg every 2 weeks) or placebo. The validated arthritis-specific Work Productivity Survey (WPS) assessed the impact of nr-axSpA on work and household productivity and social participation. Missing data were imputed using last observation carried forward (LOCF) post hoc in the Full Analysis Set (randomised patients who received ≥1 dose of CZP). Results 317 patients were randomised (CZP: 159; placebo: 158). Mean age at baseline was 37.3 years and 51.4% of patients were female. At baseline, most patients were employed (CZP: 124 [77.8%]; placebo: 123 [78.0%]) and reported a mean 3.7 (CZP) and 3.5 (placebo) workdays missed per month due to disease (Table 1). By Week 12, work absenteeism substantially improved in the CZP group compared with placebo (0.9 vs 2.1 days missed per month, LOCF), with further improvements at Week 52 (0.3 vs 2.0 days missed per month, LOCF). Between Week 12 and Week 52, most placebo patients (104, 65.8%) switched to open-label CZP, impacting Week 52 imputed outcomes. Despite this, similar patterns of improvement following CZP treatment were seen for absenteeism, workdays with impaired productivity, household days with missed/reduced productivity and social participation between imputed and observed case data (Table 1). Improvements were similar between male and female patients (data not shown). Conclusion CZP treatment resulted in improvements in work and household productivity and social participation for nr-axSpA patients as early as Week 12 compared to background medication only, with benefits maintained to Week 52. Disclosures K. Gaffney: Other; Research Grants/Consultancy Fees from Abbvie, Biogen, Celgene, Gilead, Izana, Janssen, Lilly, Novartis, Pfizer, UCB Pharma. A. Deodhar: Consultancies; AbbVie, Amgen, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, Glaxo Smith and Klein, Janssen, Novartis, Pfizer and UCB. Grants/research support; BMS, Eli Lilly, Glaxo Smith & Kline, Janssen, Novartis, Pfizer and UCB. L. Gensler: Consultancies; Galapagos, Eli Lilly and Janssen. Grants/research support; AbbVie, Amgen, Novartis, UCB Pharma. J. Kay: Consultancies; AbbVie, Boehringer Ingelheim, Celltrion Healthcare, Horizon Therapeutics, Merck Sharp & Dohme, MorphoSys, Novartis, Pfizer, Samsung Bioepis, Sandoz and UCB Pharma. Grants/research support; Gilead Sciences, Novartis AG, Pfizer and UCB Pharma. W. Maksymowych: Other; Consultant and/or speaker fees and/or grants from AbbVie, Amgen, Eli Lilly, Janssen, Merck, Pfizer, Synarc, Sanofi and UCB Pharma. N. Haroon: Consultancies; Abbvie, Amgen, Eli Lilly, Janssen, Novartis and UCB Pharma. R. Landewé: Consultancies; Abbott, Ablynx, Amgen, Astra-Zeneca, Bristol Myers Squibb, Centocor, GlaxoSmithKline, Novartis, Merck, Pfizer, Roche, Schering-Plough, UCB Pharma, Wyeth. Member of speakers’ bureau; Abbott, Amgen, Bristol Myers Squibb, Centocor, Merck, Pfizer, Roche, Schering-Plough, UCB Pharma, Wyeth. Grants/research support; Abbott, Amgen, Centocor, Novartis, Pfizer, Roche, Schering-Plough, UCB Pharma, Wyeth. M. Rudwaleit: Consultancies; Abbott, Bristol-Myers Squibb, Janssen, MSD, Pfizer, Roche, UCB Pharma. S. Hall: Other; Consulting fees/ research grants from AbbVie, Eli Lilly, Novartis, and UCB Pharma. L. Bauer: Other; Employee of UCB Pharma. B. Hoepken: Other; Employee of UCB Pharma. N. de Peyrecave: Other; Employee of UCB Pharma. T. Kumke: Other; Employee of UCB Pharma. D. van der Heijde: Consultancies; AbbVie, Amgen, Astellas, AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Daiichi, Eli Lilly, Galapagos, Gilead, Janssen, Merck, Novartis, Pfizer, Regeneron, Roche, Sanofi, and UCB. Other; Director of Imaging Rheumatology BV.

To determine how different components affect the structure of pulmonary surfactant, we measured X-ray scattering by samples derived from calf surfactant. The surfactant phospholipids demonstrated the essential characteristics of the L(γ) phase: a unit cell with a lattice constant appropriate for two bilayers; crystalline chains detected by wide angle X-ray scattering (WAXS). The electron density profile, obtained from scattering by oriented films at different relative humidities (70–97%), showed that the two bilayers, arranged as mirror images, each contain two distinct leaflets with different thicknesses and profiles. The detailed structures suggest one ordered leaflet that would contain crystalline chains, and one disordered monolayer likely to contain the anionic compounds, which constitute ~10% of the surfactant phospholipids. The spacing and temperature-dependence detected by WAXS fit with an ordered leaflet composed of dipalmitoyl phosphatidylcholine. Physiological levels of cholesterol had no effect on this structure. Removing the anionic phospholipids prevented formation of the L(γ) phase. The cationic surfactant proteins inhibited L(γ) structures, but at levels unlikely related to charge. Because the L(γ) phase, if arranged properly, could produce a self-assembled ordered interfacial monolayer, the structure could have important functional consequences. Physiological levels of the proteins, however, inhibit formation of the L(γ) structures at high relative humidities, making their physiological significance uncertain.

There are now eight approved biological disease-modifying antirheumatic drugs (bDMARDs), two biosimilars and one targeted synthetic DMARD in Australia with a number of new products and biosimilars in the pipeline. bDMARDs have excellent efficacy, especially when combined with traditional DMARDs, and a well characterised but manageable safety profile. These expanded therapeutic options have revolutionised patient care and made remission (including drug free remission) a realistic goal. Evidence of a "window of opportunity" that changes the long term phenotype of the disease has been well established, so therapy should be commenced as early as possible in the disease process and a shared care model between general practitioner and rheumatologist provides the best outcomes. While there is no cure for rheumatoid arthritis, treatment has improved to the point where many patients can achieve a normal quality of life.

When compressed by the shrinking alveolar surface area during exhalation, films of pulmonary surfactantin situreduce surface tension to levels, at which surfactant monolayers collapse from the surfacein vitro. Vesicles of pulmonary surfactant added below these monolayers slow collapse. X-ray scattering here determined the structural changes that improve stability. Grazing incidence X-ray diffraction on monolayers of extracted calf surfactant detected an ordered phase. Mixtures of dipalmitoyl phosphatidylcholine and cholesterol, but not the phospholipid alone, mimic that structure. At concentrations that stabilize the monolayers, vesicles in the subphase had no effect on the unit cell, and the film remained monomolecular. The added vesicles, however, produced a concentration-dependent increase in the diffracted intensity. These results suggest that the enhanced resistance to collapse results from components of an ordered interfacial phase which partition from subphase to the surface, increasing the area of the ordered structure.SIGNIFICANCELow alveolar surface tensions are essential for maintaining the integrity of the pulmonary air-sacks during normal breathing. Films of pulmonary surfactant cause the low tensions. The interfacial structures required for the low surface tensions remain uncertain. These studies used X-ray scattering to determine the initial structure of pulmonary surfactant monolayers, and to establish how vesicles of pulmonary surfactant enhance the ability of those initial monolayers to sustain low tensions. The initial monolayers contained ordered structures that differ from the crystalline forms widely speculated to occur in alveolar films. The added vesicles had no effect on the local structure of the initial monolayer, but substantially increased the area of the ordered regions. This structural change reasonably explains the functional improvement.

[Tahir, Hasan] Barts Hlth NHS Trust, Rheumatol, London, England. [Mease, Philip J.] Swedish Med Ctr, Dermatol, Seattle, WA USA. [Mease, Philip J.] Univ Washington, Seattle, WA 98195 USA. [Kavanaugh, Arthur] Univ Calif La Jolla, San Diego Sch Med, La Jolla, CA USA. [Reimold, Andreas] Dallas Vet Affairs Med Ctr, Dallas, TX USA. [Reimold, Andreas] Univ Texas Southwestern Med Ctr Dallas, Dallas, TX 75390 USA. [Rech, Juergen] Friedrich Alexander Univ Erlangen Nurnberg, Univ Klinikum Erlangen, Dept Internal Med Rheumatol & Immunol, Erlangen, Germany. [Hall, Stephen] Emeritus Res, Rheumatol, Camberwell, Australia. [Geusens, Piet] Univ Hasselt, Rheumatol, Hasselt, Belgium. [Pellet, Pascale; Mpofu, Shephard] Novartis Pharma AG, Immunol & Dermatol, Basel, Switzerland. [Delicha, Eumorphia Maria] Novartis Pharma AG, Biostat, Basel, Switzerland. [Pricop, Luminita] Novartis Pharmaceut, Immunol Hepatol & Dermatol Franchise, E Hanover, NJ USA.

Background:Upadacitinib (UPA) 15 mg once daily (QD) has demonstrated efficacy in phase 3 studies of patients with rheumatoid arthritis (RA).1–4 Early prediction of response to treatment with UPA could help to optimize therapy.Objectives:To identify baseline (BL) characteristics or Week (Wk) 12 disease activity measures that may predict the achievement of remission (REM) or low disease activity (LDA) at 6 months in patients with RA receiving UPA 15 mg.Methods:This ad hoc analysis included patients who were randomized to UPA 15 mg QD, as monotherapy in methotrexate (MTX)-naïve patients (SELECT-EARLY) or in combination with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), in patients with an inadequate response (IR) to MTX (SELECT-COMPARE) or ≥1 tumor necrosis factor inhibitors (TNFis) (SELECT-BEYOND and SELECT-CHOICE). The association of BL characteristics (including age, disease duration, prior/concomitant treatments, C-reactive protein [CRP], seropositivity, and disease activity) and Wk 12 disease activity parameters with the achievement of Clinical Disease Activity Index (CDAI) REM (≤2.8) or LDA (≤10) at Wk 24 (or Wk 26 in SELECT-COMPARE) was assessed by concordance statistics (C-statistics), or area under the receiver operator characteristic curve. C-index values and 95% confidence intervals were calculated by fitting a univariate logistic regression model for: demographic and BL characteristics, Wk 12 disease activity measures, and change from BL at Wk 12 in disease activity measures. A multivariate logistic regression with stepwise model selection was also performed. The proportion of patients achieving Wk 24/26 CDAI REM/LDA was stratified by ≥50% improvement from BL in swollen and/or tender joint count in 66/68 joints (SJC66/TJC68).Results:A total of 1377 patients were included in the analysis. Across the 4 studies, CDAI REM and LDA were achieved in 11.0–28.4% and 50.0–58.6% of patients, respectively (Table 1). BL demographics and disease characteristics were weakly predictive (C-index 0.80) of Wk 24/26 CDAI REM or LDA. Disease Activity Score in 28 joints using CRP and pain at Wk 12 were strongly predictive of Wk 24/26 CDAI REM (except in SELECT-CHOICE). Physician’s global assessment at Wk 12 was the only common predictor in the multivariate regression models for CDAI REM/LDA at Wk 24/26 across the 4 studies. A greater proportion of patients achieving ≥50% improvement in SJC66 and TJC68 at Wk 12 achieved CDAI REM (16.5–37.8% vs 0–9.4%) or LDA (66.0–72.8% vs 20.9–35.7%) at Wk 24/26 than those who did not.Table 1.Achievement of CDAI LDA and REM at Wk 24/26aSELECT-EARLYSELECT-COMPARESELECT-BEYONDSELECT-CHOICEPatient populationMTX-naïveMTX-IRTNFi-IRTNFi-IRTreatmentUPA 15 mg monotherapy (n=317)UPA 15 mg + MTX(n=651)UPA 15 mg + csDMARD(n=146)UPA 15 mg + csDMARD(n=263)Efficacy at Wk 24/26a, n (%)CDAI REM (≤2.8)90 (28.4)150 (23.0)16 (11.0)60 (22.8)CDAI LDA (≤10)178 (56.2)343 (52.7)73 (50.0)154 (58.6)a Wk 26 for SELECT-COMPARE onlyConclusion:BL characteristics did not strongly predict response to UPA, but composite disease activity scores at Wk 12 predicted Wk 24/26 REM/LDA with UPA 15 mg QD across MTX-naïve, MTX-IR, and TNFi-IR patients. ≥50% improvement in SJC/TJC at Wk 12 was also associated with Wk 24/26 REM/LDA.References:[1]van Vollenhoven R, et al. Arthritis Rheumatol 2020;72:1607–20; 2. Genovese MC, et al. Lancet 2018;391:2513–24; 3. Fleischmann R, et al. Arthritis Rheumatol 2019;71:1788–800; 4. Rubbert-Roth A, et al. N Engl J Med 2020;383:1511–21.Acknowledgements:AbbVie funded this study; contributed to its design; participated in data collection, analysis, and interpretation of the data; and participated in the writing, review, and approval of the abstract. No honoraria or payments were made for authorship. Medical writing support was provided by Laura Chalmers, PhD, of 2 the Nth (Cheshire, UK), and was funded by AbbVie.Disclosure of Interests:Arthur Kavanaugh Consultant of: Janssen, Grant/research support from: Janssen, Zoltán Szekanecz: None declared, Edward C. Keystone Speakers bureau: Abbott, Amgen, AstraZeneca, Biotest, Bristol-Myers Squibb, Eli Lilly, Genentech, Janssen, MSD, Novartis, Pfizer, Roche, Sanofi, and UCB, Consultant of: Abbott, Amgen, AstraZeneca, Biotest, Bristol-Myers Squibb, Eli Lilly, Genentech, Janssen, MSD, Novartis, Pfizer, Roche, Sanofi, and UCB, Grant/research support from: Abbott, Amgen, AstraZeneca, Biotest, Bristol-Myers Squibb, Eli Lilly, Genentech, Janssen, MSD, Novartis, Pfizer, Roche, Sanofi, and UCB, Andrea Rubbert-Roth Consultant of: AbbVie, Amgen, Bristol-Myers Squibb, Chugai, Eli Lilly, Janssen, Novartis, Pfizer, Roche, and Sanofi, Stephen Hall Grant/research support from: Pfizer, Ricardo Xavier: None declared, Joaquim Polido-Pereira: None declared, In-Ho Song Shareholder of: May own stock or options in AbbVie, Employee of: AbbVie, Naomi Martin Shareholder of: May own stock or options in AbbVie, Employee of: AbbVie, Yanna Song Shareholder of: May own stock or options in AbbVie, Employee of: AbbVie, Samuel Anyanwu Shareholder of: May own stock or options in AbbVie, Employee of: AbbVie, Peter Nash Grant/research support from: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Gilead, Janssen, MSD, Novartis, Pfizer, Roche, Sanofi, and UCB.

Aim. Examine the efficacy of once-weekly subcutaneous tocilizumab (SC-TCZ) on joint damage at 24 weeks based on radiography of the hands and feet and magnetic resonance imaging (MRI) of the hand in subjects with moderate to severe rheumatoid arthritis (RA). Methods. In this Australian open-label, multicentre, prospective, single-arm study, subjects received 162 mg SC-TCZ weekly. Primary endpoint was change in radiographic Genant-modified Total Sharp Score (TSS) between baseline and Week 24. Secondary endpoints included change from baseline to Week 24 in RA MRI scoring (RAMRIS) of erosions, synovitis, and osteitis and Cartilage Loss Score (CARLOS) in the dominant hand and disease activity score 28 (DAS28). Results. 52 subjects were enrolled (80% female, mean (SD) age 57 (12) years). Radiography showed mild but not significant progression of joint damage (mean (SD) change in TSS 0.46 (1.29)). Synovitis reduced significantly on MRI; however, osteitis, erosion, and cartilage loss did not change significantly. DAS28 improved significantly by Week 24; 78% of subjects achieved DAS28 remission. SC-TCZ was generally well tolerated. Conclusion. Synovitis and DAS28 decreased significantly; however, no significant change in osteitis or joint damage was observed at Week 24. Trial registration. This trial is registered with Clinicaltrials.gov registration number NCT01951170 (ML28703).

Roche. F. Hoffmann-La Roche, Ltd (Roche) Objectives This study compared the efficacy and safety of subcutaneous (SC) versus intravenous (IV) formulations of tocilizumab in patients with rheumatoid arthritis with an inadequate response to disease-modifying antirheumatic drugs (DMARD).Methods Patients (n=1262) were randomly assigned to receive tocilizumab-SC 162mg weekly+placebo-IV every 4weeks or tocilizumab-IV 8mg/kg every 4weeks+placebo-SC weekly in combination with traditional DMARD. the primary outcome was to demonstrate the non-inferiority of tocilizumab-SC to tocilizumab-IV with regard to the proportion of patients in each group achieving an American College of Rheumatology (ACR) 20 response at week 24 using a 12% non-inferiority margin (NIM). Secondary outcomes were disease activity score using 28 joints (DAS28), ACR responses, health assessment questionnaire scores and safety assessments.Results At week 24, 69.4% (95% CI 65.5 to 73.2) of tocilizumab-SC-treated patients versus 73.4% (95% CI 69.6 to 77.1) of tocilizumab-IV-treated patients achieved an ACR20 response (weighted difference between groups -4.0%, 95% CI -9.2 to 1.2); the 12% NIM was met. ACR50/70 responses, DAS28 and physical function improvements were comparable between the tocilizumab-SC and tocilizumab-IV groups. the safety profiles of tocilizumab-SC and tocilizumab-IV were similar, and the most common adverse event was infection. Injection-site reactions (ISR) occurred more frequently in the tocilizumab-SC group than in the tocilizumab-IV (placebo-SC) group. No anaphylaxis was reported over the 24weeks.Conclusions Tocilizumab-SC 162mg weekly demonstrated comparable efficacy to tocilizumab-IV 8mg/kg. the safety profile of tocilizumab-SC is consistent with the known and well-established safety profile of tocilizumab-IV, with the exception of a higher incidence of ISR, which were more common with tocilizumab-SC administration. Charite, D-10117 Berlin, Germany Free Univ Berlin, Berlin, Germany Humboldt Univ, D-10099 Berlin, Germany Klinikum Univ Koln, Cologne, Germany Ctr Hosp Univ Toulouse, Toulouse, France Cabrini Med Ctr, Malvern, Australia Poznan Univ Med Sci, J Strus Poznan Municipal Hosp, Poznan, Poland Universidade Federal de São Paulo, São Paulo, Brazil Arthrit & Diabet Clin Inc, Monroe, LA USA Rheumatol Associates PA, Charleston, SC USA East Penn Rheumatol Associates PC, Bethlehem, PA USA Hoffmann La Roche Inc, Nutley, NJ 07110 USA Roche Prod Ltd, Welwyn Garden City AL7 3AY, Herts, England Genentech Inc, San Francisco, CA 94080 USA Org Med Invest, Buenos Aires, DF, Argentina Universidade Federal de São Paulo, EPM, São Paulo, Brazil Web of Science

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. Previous studies have shown that SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce faster adsorption and formation of the bicontinuous structures by similar mechanisms, then they should also have different abilities to form the cubic phases. To test this hypothesis, we measured small-angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the dose-related ability of the combined proteins to induce the cubic phases at temperatures more than 25 °C below the point at which POPE alone forms the curved inverse-hexagonal phase. With SP-C, diffraction from cubic structures was either absent or present at very low intensities only with larger amounts of protein. The correlation between the structural effects of inducing curved structures and the functional effects on the rate of adsorption fits with the model in which SP-B promotes adsorption by facilitating formation of an inversely curved, rate-limiting structure.

Bubbles and droplets offer multiple advantages over Langmuir troughs for compressing interfacial films. Experiments, however, that manipulate films to maintain constant surface tension (γ) present problems because they require feedback. Measurements of bubbles and droplets calculate γ from the shape of the interface, and calculations in real time based on finding the Laplacian shape that best fits the interface can be difficult. Faster methods obtain γ from only the height and diameter, but the bubbles and droplets rest against a solid support, which obscures one section of the interface and complicates measurements of the height. The experiments here investigated a series of optical variables that affect the visualized location of the different surfaces for captive bubbles. The pitch of the support and camera as well as the collimation of illuminating light affected the accuracy of the measured dimensions. The wavelength of illumination altered the opacity of turbid subphases and hydrated gel used to form the solid support. The width of all visualized edges depended on the spectral width and collimation of the illuminating light. The intensity of illumination had little effect on the images as long as the grayscale remained within the dynamic range of the camera. With optimization of these optical factors, the width of all edges narrowed significantly. The surfaces away from the solid support approached the infinite sharpness of the physical interface. With these changes, the grayscale at the upper interface provided the basis for locating all surfaces, which improved real-time measurements based on the height and diameter.

Systemic lupus erythematosus is a heterogeneous autoimmune disease that is associated with B-cell hyperactivity, autoantibodies, and increased concentrations of B-lymphocyte stimulator (BLyS). The efficacy and safety of the fully human monoclonal antibody belimumab (BLyS-specific inhibitor) was assessed in patients with active systemic lupus erythematosus.Patients (aged ≥18 years) who were seropositive with scores of at least 6 on the Safety of Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) were enrolled in a multicentre phase 3 study, which was done in Latin America, Asia-Pacific, and eastern Europe. Patients were randomly assigned by use of a central interactive voice response system in a 1:1:1 ratio to belimumab 1 mg/kg or 10 mg/kg, or placebo by intravenous infusion in 1 h on days 0, 14, and 28, and then every 28 days until 48 weeks, with standard of care. Patients, investigators, study coordinators, and sponsors were masked to treatment assignment. Primary efficacy endpoint was improvement in the Systemic Lupus Erythematosus Responder Index (SRI) at week 52 (reduction ≥4 points in SELENA-SLEDAI score; no new British Isles Lupus Assessment Group [BILAG] A organ domain score and no more than 1 new B organ domain score; and no worsening [0·3 increase] in Physician's Global Assessment [PGA] score) versus baseline. Method of analysis was by modified intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00424476.867 patients were randomly assigned to belimumab 1 mg/kg (n=289) or 10 mg/kg (n=290), or placebo (n=288). 865 were treated and analysed in the belimumab (1 mg/kg, n=288; 10 mg/kg, n=290) and placebo groups (n=287). Significantly higher SRI rates were noted with belimumab 1 mg/kg (148 [51%], odds ratio 1·55 [95% CI 1·10-2·19]; p=0·0129) and 10 mg/kg (167 [58%], 1·83 [1·30-2·59]; p=0·0006) than with placebo (125 [44%]) at week 52. More patients had their SELENA-SLEDAI score reduced by at least 4 points during 52 weeks with belimumab 1 mg/kg (153 [53%], 1·51 [1·07-2·14]; p=0·0189) and 10 mg/kg (169 [58%], 1·71 [1·21-2·41]; p=0·0024) than with placebo (132 [46%]). More patients given belimumab 1 mg/kg (226 [78%], 1·38 [0·93-2·04]; p=0·1064) and 10 mg/kg (236 [81%], 1·62 [1·09-2·42]; p=0·0181) had no new BILAG A or no more than 1 new B flare than did those in the placebo group (210 [73%]). No worsening in PGA score was noted in more patients with belimumab 1 mg/kg (227 [79%], 1·68 [1·15-2·47]; p=0·0078) and 10 mg/kg (231 [80%], 1·74 [1·18-2·55]; p=0·0048) than with placebo (199 [69%]). Rates of adverse events were similar in the groups given belimumab 1 mg/kg and 10 mg/kg, and placebo: serious infection was reported in 22 (8%), 13 (4%), and 17 (6%) patients, respectively, and severe or serious hypersensitivity reactions on an infusion day were reported in two (1%), two (1%), and no patients, respectively. No malignant diseases were reported.Belimumab has the potential to be the first targeted biological treatment that is approved specifically for systemic lupus erythematosus, providing a new option for the management of this important prototypic autoimmune disease.Human Genome Sciences and GlaxoSmithKline.

This research explores the relationship between rheumatoid arthritis (RA) severity measures, clinical characteristics, and patient preference-based health-related quality of life (ie, utility) in Australian RA patients. A secondary objective was to investigate the relationship between a range of disease severity measures, clinical characteristics, and the cost of RA-related resource use.This was a cross-sectional, multicenter study of consecutive patients with RA agedor =18 years attending routine clinical appointments. Patients completed a questionnaire comprising general demographic, resource use, and disease-specific questions; the RA-specific Health Assessment Questionnaire (HAQ); and 2 multiattribute preference-based quality-of-life (utility) instruments, the Health Utilities Index Mark 3 (HUI3) and the EuroQol 5 Dimensions (EQ-5D). A second questionnaire was completed by the patient's rheumatologist, with questions on key clinical data pertinent to RA, including the number and location of tender joints and swollen joints, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) concentration, current and previous treatments, and details of important comorbid conditions. Data on RA-specific resource use were also collected, including health-professional visits (eg, general practitioner, specialist, nurse, occupational therapist, paid caregivers), hospital admissions, surgery, and RA-associated home modifications. Spearman nonparametric correlation, simple linear, multiple, and stepwise regression analyses were used to explore the relationship between utility scores (HUI3 and EQ-5D) and variables including HAQ, RA-related resource use, and key demographic, physical, and biochemical measures.A total of 170 patients were recruited from 4 centers in Australia. Consistent with the epidemiology of RA, the ratio of women to men was approximately 3:1 (126 vs 44, respectively). Male and female patients were of similar age (mean [SD], 59.2 [12.9] and 58.9 [12.2] years, respectively). Time since diagnosis of RA was significantly shorter for men than for women (mean difference, -4.52 years; 95% CI, -8.65 to -0.38; P = 0.03). Of all the disease severity measures and clinical characteristics investigated, patients' HAQ scores predicted their utility most closely (for HUI3, R(2) = 0.626, P0.001; for EQ-5D, R(2) = 0.403, P0.001). The Disease Activity Score 28 severity index provided the next best relationship with a patient's utility; however, its explanatory power was poor (for HUI3, R(2) = 0.085, P0.001; for EQ-5D, R(2) = 0.042, P = 0.008). ESR, CRP, and, RA-affected joint counts had negligible explanatory power for patient utility. In analyses of the relationship between a range of key variables and the direct costs associated with RA, the HAQ score explained 22% of the variability in log costs (P0.001).This study found that of all the disease severity measures and clinical characteristics investigated, patients' HAQ scores predicted their utility most closely.

ObjectiveTo evaluate the efficacy and safety of golimumab to 52 weeks in patients with active rheumatoid arthritis despite methotrexate.MethodsPatients were randomly assigned to receive placebo plus methotrexate (group 1), golimumab 100 mg plus placebo (group 2), golimumab 50 mg plus methotrexate (group 3) and golimumab 100 mg plus methotrexate (group 4). At week 16, patients in groups 1, 2 and 3 who had less than 20% improvement in tender and swollen joints entered early escape. At week 24, patients in group 1 who had not entered early escape crossed over to 50 mg golimumab plus methotrexate.ResultsAt week 16, 31%, 27% and 17% of patients in groups 1, 2 and 3, respectively, entered early escape. At week 52, 44%, 45%, 64% and 58% of patients in groups 1, 2, 3 and 4, respectively, achieved 20% improvement in the American College of Rheumatology criteria; and 34%, 31%, 42% and 53%, respectively, achieved low disease activity (≤3.2) according to the 28-joint disease activity score. Patients in group 4 appeared to have an increased risk of serious adverse events and serious infections.ConclusionThe results of various outcome measures showed that the response rates achieved by patients receiving golimumab to 24 weeks were sustained to 52 weeks. The safety profile appeared to be consistent with the known safety profile of tumour necrosis factor inhibitors.

The hydrophobic surfactant proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface. Previous evidence suggests that they achieve this effect by facilitating the formation of a rate-limiting negatively curved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipid leaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by 1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transition at 71 degrees C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57 degrees C, the proteins induce formation of bicontinuous inverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidence of other structures above 0.1% and 62 degrees C, but no change in the lattice-constants of the lamellar or cubic phases. The induction of the bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypothetical stalk-intermediate, supports the proposed model of how the surfactant proteins promote adsorption.

Summary Background Remission and radiographic non-progression are goals in the treatment of early rheumatoid arthritis. The aim of the combination of methotrexate and etanercept in active early rheumatoid arthritis (COMET) trial is to compare remission and radiographic non-progression in patients treated with methotrexate monotherapy or with methotrexate plus etanercept. Methods 542 outpatients who were methotrexate-naive and had had early moderate-to-severe rheumatoid arthritis for 3–24 months were randomly assigned to receive either methotrexate alone titrated up from 7·5 mg a week to a maximum of 20 mg a week by week 8 or methotrexate (same titration) plus etanercept 50 mg a week. Coprimary endpoints at 52 weeks were remission measured with the disease activity score in 28 joints (DAS28) and radiographic non-progression measured with modified total Sharp score. Treatment was allocated with a computerised randomisation and enrolment system, which masked both participants and carers. Analysis was done by modified intention to treat with last observation carried forward for missing data. This study is registered with ClinicalTrials.gov, number NCT00195494). Findings 274 participants were randomly assigned to receive combined treatment and 268 methotrexate alone. 132 of 265 (50%, 95% CI 44–56%) patients who took combined treatment and were available for assessment achieved clinical remission compared with 73 of 263 (28%, 23–33%) taking methotrexate alone (effect difference 22·05%, 95%CI 13·96–30·15%, p 5·1). 196 of 246 (80%, 75–85%) and 135 of 230 (59%, 53–65%), respectively, achieved radiographic non-progression (20·98%, 12·97–29·09%, p Interpretation Both clinical remission and radiographic non-progression are achievable goals in patients with early severe rheumatoid arthritis within 1 year of combined treatment with etanercept plus methotrexate. Funding Wyeth Research.

Objective. To assess the efficacy, safety, and pharmacology of subcutaneous administration of golimumab in patients with active rheumatoid arthritis (RA) despite treatment with methotrexate (MTX). Methods. Patients were randomly assigned in a double-blinded manner to receive injections of placebo plus MTX or 50 mg or 100 mg golimumab every 2 or 4 weeks plus MTX through week 48. Patients originally assigned to receive injections every 2 weeks had the interval increased to every 4 weeks starting at week 20. The primary end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at week 16. The study was powered to detect a difference in the primary end point when the combined golimumab groups and at least I of the individual dose groups were compared with placebo. Results. The primary end point was attained. Sixty-one percent of patients in the combined golimumab plus MTX dose groups achieved an ACR20 response at week 16 compared with 37% of patients in the placebo plus MTX group (P = 0.010). In addition, 79% of patients in the group receiving 100 mg golimumab every 2 weeks achieved an ACR20 response (P < 0.001 versus placebo). Through week 20 (after which patients receiving placebo were switched to active infliximab therapy), serious adverse events were reported in 9% of patients in the combined golimumab groups and in 6% of patients in the placebo group. Conclusion. Golimumab plus MTX effectively reduces the signs and symptoms of RA and is generally well tolerated in patients with an inadequate response to MTX.

Roche F. Hoffmann-La Roche, Ltd. Objectives To evaluate the long-term efficacy and safety of subcutaneous (SC) tocilizumab (TCZ) versus intravenous (IV) TCZ, including switching formulations, in patients with rheumatoid arthritis (RA) and inadequate response to disease-modifying antirheumatic drugs (DMARDs). Methods Patients (n=1262) were randomised 1: 1 to receive TCZ-SC 162 mg weekly (qw)+placebo-IV every four weeks (q4w) or TCZ-IV 8 mg/kg q4w+placebo-SC qw in combination with DMARD(s). After a 24-week double-blind period, patients receiving TCZ-SC were re-randomised 11: 1 to TCZ-SC (n=521) or TCZ-IV (TCZ-SCIV, n=48), and patients receiving TCZ-IV were re-randomised 2: 1 to TCZ-IV (n=372) or TCZ-SC (TCZ-IV-SC n=186). Maintenance of clinical responses and safety through week 97 were assessed. Results The proportions of patients who achieved American College of Rheumatology (ACR) 20/50/70 responses, Disease Activity Score in 28 joints remission and improvement from baseline in Health Assessment Questionnaire Disability Index >= 0.3 were sustained through week 97 and comparable across arms. TCZ-SC had a comparable safety profile to TCZ-IV through week 97, except that injection site reactions (ISRs) were more common with TCZ-SC. Safety profiles in patients who switched were similar to those in patients who received continuous TCZ-SC or TCZ-IV treatment. The proportion of patients who developed anti-TCZ antibodies remained low across treatment arms. No association between anti-TCZ antibody development and clinical response or adverse events was observed. Conclusions The long-term efficacy and safety of TCZ-SC was maintained and comparable to that of TCZ-IV, except for ISRs. Profiles in patients who switched formulations were comparable to those in patients who received TCZ-IV or TCZ-SC. TCZ-SC provides additional treatment options for patients with RA. Free Univ Berlin, Berlin, Germany Humboldt Univ, D-10099 Berlin, Germany Klinikum Univ Koln, Cologne, Germany Ctr Hosp Univ Toulouse, Toulouse, France Cabrini Med Ctr, Malvern, Vic, Australia Poznan Med Univ, Poznan, Poland Univ Fed Sao Paulo, Sao Paulo, Brazil Arthrit & Diabet Clin Inc, Monroe, LA USA Rheumatol Associates South Carolina, Charleston, SC USA East Penn Rheumatol Associates, Bethlehem, PA USA Genentech Inc, San Francisco, CA 94080 USA Roche Prod Ltd, Welwyn Garden City AL7 3AY, Herts, England Org Med Invest, Buenos Aires, DF, Argentina Universidade Federal de São Paulo, São Paulo, Brazil Web of Science

To determine if hydrophobic surfactant proteins affect the stability of pulmonary surfactant monolayers at an air/water interface, the studies reported here compared the kinetics of collapse for the complete set of lipids in calf surfactant with and without the proteins. Monomolecular films spread at the surface of captive bubbles were compressed at 37 degrees C to surface pressures above 46 mN/m, at which collapse first occurred. The rate of area-compression required to maintain a constant surface pressure was measured to directly determine the rate of collapse. For films with and without the proteins, higher surface pressures initially produced faster collapse, but the rates then reached a maximum and decreased to values0.04 min(-1) above 53 mN/m. The maximum rate for the lipids with the proteins (1.22 +/- 0.28 min(-1)) was almost twice the value for the lipids alone (0.71 +/- 0.15 min(-1)). Because small increments in surface pressure produced large shifts in the rate close to the fastest collapse, compressions at a series of constant speeds also established the threshold rate required to achieve high surface pressure as an indirect indication of the fastest collapse. Both samples produced a sharply defined threshold that occurred at slightly faster compression with the proteins present, supporting the conclusion of the direct measurements that the proteins produce a faster maximum rate of collapse. Our results indicate that at 47-53 mN/m, the hydrophobic surfactant proteins destabilize the compressed monolayers and tend to limit access to the higher surface pressures at which the lipid films become metastable.

To determine how the hydrophobic surfactant proteins promote insertion of the surfactant lipids into an air/water interface, we measured the effect of lysophosphatidylcholine (LPC) on adsorption. Existing models contend that the proteins function either by disordering the lipids or by stabilizing a negatively curved structure located between the adsorbing vesicle and the interface. Because LPC produces greater disorder but positive curvature, the models predict opposite effects. With vesicles containing either dioleoyl phosphatidylcholine (DOPC) or the neutral and phospholipids isolated from calf surfactant, LPC increased the initial rate at which surface tension fell. The final surface tension, however, remained well above the value of approximately 25 mN/m expected for a saturated surface. With two preparations, dioleoyl phosphatidylethanolamine and gramicidin A-DOPC, which form the negatively curved hexagonal-II (H(II)) phase and adsorb rapidly, LPC instead had little effect on initial adsorption but delayed the fall of surface tension below approximately 30 mN/m. LPC produced a similar inhibition of the late adsorption for extracted calf surfactant. Unlike dioleoyl phosphatidylethanolamine and gramicidin A-DOPC, small-angle x-ray scattering and (31)P-nuclear magnetic resonance for extracted calf surfactant detected no evidence for the H(II) phase. Our results indicate that although LPC can promote the initial adsorption of vesicles containing only lamellar lipids, it inhibits the facilitation by the hydrophobic proteins of late adsorption. Our findings support a model in which the surfactant proteins accelerate adsorption by producing a focal tendency to stabilize a negatively curved kinetic intermediate without a general shift to the H(II) phase.

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air–water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (HII) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and 31P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the HII phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.

Unlike surfactants considered in previous studies, when phosphatidylcholine (PC) monolayers collapse at constant surface tension to form a 3D bulk phase, surface area decreases at rates that slow. The different kinetics could result from collapse by a distinct mechanism. Rather than the transfer of molecules all along the interface between the monolayer and bulk phase, PC films can collapse by the folding and subsequent sliding of a bilayer over the monolayer. By this mechanism, molecules can transfer to collapsed trilayers through a locus of constant size. In this article, we use the theory of nucleation and growth to show analytically that during collapse, the area can decrease at rates that decelerate when each individual structure grows through a region of fixed dimensions. We also show that binary films of 30% dihydrocholesterol (dchol) and dipalmitoyl phosphatidylcholine (DPPC), which have previously been shown to form a homogeneous monolayer from which trilayer disks grow through a point, collapse with rates of area decay that slow, in agreement with our analytical expressions.

Prior studies have shown that during and after slow compressions of monomolecular films containing the complete set of purified phospholipids (PPL) from calf surfactant at an air/water interface, surface pressures (pi) reach and sustain values that are remarkably high relative to expectations from simple systems with model lipids. Microscopy shows that the liquid-expanded, tilted-condensed, and collapsed phases are present together in the PPL films between 45 and 65 mN/m. The Gibbs phase rule restricts equilibrium coexistence of three phases to a single pi for films with two components but not for more constituents. We therefore determined if the surprising stability of PPL reflects release from the thermodynamic restrictions of simple model systems by the presence of multiple components. Experiments with binary films containing dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine first tested the predictions of the phase rule. The onset of three-phase coexistence, determined by fluorescence microscopy, and its termination, established by relaxation of collapsing films on a captive bubble, occurred at similar pi. Experiments for PPL using the same methods suggested that the three phases might coexist over a range of pi, but limited to approximately 2 mN/m, and extending below rather than above the coexistence pi for the binary films. Our results show that the PPL films at high pi must deviate from equilibrium and that they must then be metastable.

Fluid monolayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine collapse from an air/water interface to form a three-dimensional bulk phase at the equilibrium spreading pressure (pie) of approximately 47 mN/m. This phase transition limits access to higher surface pressures under equilibrium conditions or during slow continuous compressions. We have shown previously that these films avoid collapse and become metastable when compressed on a captive bubble to surface pressures above 60 mN/m and that the metastability persists during expansion at least to pie. Here, we first documented the extent of this persistent metastability. Rates of isobaric collapse during expansion of the metastable films were up to 3 orders of magnitude slower than those during the initial compression to high surface pressures. Recovery of the ability to collapse depended on the surface pressure to which the films were expanded and how long they were held there. Films reverted after brief exposure to 20 mN/m and after 1 h at 35 mN/m. At pie, films remained capable of reaching high surface pressures during slow compressions after 65 h, although an increase in compressibility above 55 mN/m suggested somewhat increased rates of collapse. We also determined if the films remained metastable when they acquired sufficient free area to allow reinsertion of collapsed material. Faster isobaric expansion in the presence of more collapsed material and with further deviation below pie supported the existence of reinsertion. The persistence of metastability to pie shows that films with sufficient free area to allow reinsertion remain resistant to collapse. Observations that suggest heterogeneous reinsertion, however, argue that free area may be distributed heterogeneously and leave open the possibility that metastability persists because significant regions retain a restricted free area.

Captive bubbles are commonly used to determine how interfacial films of pulmonary surfactant respond to changes in surface area, achieved by varying hydrostatic pressure. Although assumed to be isothermal, the gas phase temperature (Tg) would increase by >100°C during compression from 1 to 3 atm if the process were adiabatic. To determine the actual change in temperature, we monitored pressure (P) and volume (V) during compressions lasting g. P · V fell during and after the rapid compressions, consistent with reductions in n, the moles of gas phase molecules, because of increasing solubility in the subphase at higher P. As expected for a process with first-order kinetics, during 1 h after the rapid compression P · V decreased along a simple exponential curve. The temporal variation of n moles of gas was determined from P · V >10 min after the compression when the two phases should be isothermal. Back extrapolation of n then allowed calculation of Tg from P · V immediately after the compression. Our results indicate that for bubbles with or without interfacial films compressed to >3 atm within 1 s, the change in Tg is

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.

Pulmonary surfactant functions by first flowing rapidly into the alveolar air/water interface, but then resisting collapse from the surface when the adsorbed interfacial film is compressed during exhalation. Widely accepted models emphasize the importance of phase behavior in both processes. Recent studies show, however, that fluidity is a relatively minor determinant of adsorption and that solid films, which resist collapse, can form by kinetic processes unrelated to equilibrium phase behavior.

Films of pulmonary surfactant in the lung are metastable at surface pressures well above the equilibrium spreading pressure of 45 mN/m but commonly collapse at that pressure when compressed in vitro. The studies reported here determined the effect of compression rate on the ability of monolayers containing extracted calf surfactant at 37 degrees C to maintain very high surface pressures on the continuous interface of a captive bubble. Increasing the rate from 2 A(2)/phospholipid/min (i.e., 3% of (initial area at 40 mN/m)/min) to 23%/s produced only transient increases to 48 mN/m. Above a threshold rate of 32%/s, however, surface pressures reached > 68 mN/m. After the rapid compression, static films maintained surface pressures within +/- 1 mN/m both at these maximum values and at lower pressures following expansion at < 5%/min to > or = 45 mN/m. Experiments with dimyristoyl phosphatidylcholine at 37 degrees C produced similar results. These findings indicate that compression at rates comparable to values in the lungs can transform at least some phospholipid monolayers from a form that collapses readily at the equilibrium spreading pressure to one that is metastable for prolonged periods at higher pressures. Our results also suggest that transformation of surfactant films can occur without refinement of their composition.

The ability of surfactant aggregate−H2O interfaces to control the regioselectivity of Diels−Alder reactions has been investigated. Cycloadditions of surfactant 1,3-dienes 2-[[3-(dimethyldodecylsilyl)-1,3-butadien-2-yl]thio]-N,N,N-trimethyl-1-ethanaminium iodide (1a) and 6-[[3-(dimethyloctylsilyl)-1,3-butadien-2-yl]thio]-N,N,N-trimethyl-1-hexanaminium iodide (1b) with surfactant dienophiles (E)-2-[[[2-(dodecoxycarbonyl)ethenyl]carbonyl]oxy]-N,N,N-trimethyl-1-ethanaminium iodide (2a) and (E)-6-[[[2-(octoxycarbonyl)ethenyl]carbonyl]oxy]-N,N,N-trimethyl-1-hexanaminium bromide (2b) within their aqueous mixed micelles have been performed at 25(35) °C. The cycloaddition of 1a and 2a gave a 30:1 ratio of trans-1-[(2-trimethylammonio)ethylthio]-2-(dimethyldodecylsilyl)-4-(dodecoxycarbonyl)-5-[(2-trimethylammonio)ethoxycarbonyl]-1-cyclohexene dihalide (15a) and trans-1-[(2-trimethylammonio)ethylthio]-2-(dimethyldodecylsilyl)-4-[(2-trimethylammonio)ethoxycarbonyl]-5-(dodecoxycarbonyl)-1-cyclohexene dihalide (16a), r...

The phase behavior of monolayers containing the complete set of purified phospholipids (PPL) obtained from calf surfactant was investigated as a model for understanding the phase transitions that precede compression of pulmonary surfactant to high surface pressure. During compression, both fluorescence microscopy and Brewster angle microscopy (BAM) distinguished domains that separated from the surrounding film. Quantitative analysis of BAM grayscales indicated optical thicknesses for the PPL domains that were similar to the liquid condensed phase for dipalmitoyl phosphatidylcholine (DPPC), the most abundant component of pulmonary surfactant, and higher and less variable with surface pressure than for the surrounding film. BAM also showed the optical anisotropy that indicates long-range orientational order of tilted lipid chains for the domains, but not for the surrounding film. Fluorescence microscopy shows that addition of DPPC to the PPL increased the area of the domains. At fixed surface pressures from 20 - 40 mN/m, the total area of each phase grew in proportion with the mol fraction of DPPC. This constant variation allowed analysis of the DPPC mol fraction in each phase, construction of a simple phase diagram, and calculation of the molecular area for each phase. Our results indicate that the phase surrounding the domains is more expanded and compressible, and contains reduced amounts of DPPC in addition to the other phospholipids. The domains contain a mol fraction for DPPC of at least 96%. The structure of the interfacial film of pulmonary surfactant is a critical determinant of its physiological function. Pul- monary surfactant is the mix of lipids and proteins that coats the alveolar air spaces. It adsorbs to the surface of the thin liquid layer that lines the alveoli, and the resulting film lowers interfacial tension and stabilizes small alveoli. Dur- ing the expiratory phase of the respiratory cycle, the de- creasing alveolar surface area compresses the film, and measurements in the lung indicate that its surface pressure achieves high values approaching 70 mN/m (Schurch, 1982). Lung surfactant's physiological function then de- pends on the formation of a film that remains stable at such high pressures without collapse from the interface. Most investigators have attributed this stability to the presence of a highly ordered liquid-condensed (LC) phase (Keough, 1992). The most prevalent component of surfactant, di- palmitoyl phosphatidylcholine (DPPC), occurs as the LC phase in single-component monolayers at physiologic tem- peratures and can achieve the pressures measured in the lung. This constituent, however, represents only a third of surfactant (Kahn et al., 1995), and both the structure and the composition of the functional film in situ remain uncertain. We have undertaken studies of the phase behavior of surfactant monolayers at pressures below the equilibrium spreading pressures of approximately 45 mN/m. Phase be- havior under these conditions provides important informa- tion for understanding the development of the structure and composition of the functional film at higher pressures. Our studies emphasize the full complexity of the surfactant

We have shown previously that lateral compression of pulmonary surfactant monolayers initially induces separation of two phases but that these remix when the films become more dense (1). In the studies reported here, we used fluorescence microscopy to examine the role of the different surfactant constituents in the remixing of the separated phases. Subfractions containing only the purified phospholipids (PPL), the surfactant proteins and phospholipids (SPPL), and the neutral and phospholipids (NPL) were obtained by chromatographic separation of the components in extracted calf surfactant (calf lung surfactant extract, CLSE). Compression of the different monolayers produced nonfluorescent domains that emerged for temperatures between 20 and 41 degreesC at similar surface pressures 6-8 mN/m higher than values observed for dipalmitoyl phosphatidylcholine (DPPC), the most prevalent component of pulmonary surfactant. Comparison of the different preparations showed that the neutral lipid increased the total nonfluorescent area at surface pressures up to 25 mN/m but dispersed that total area among a larger number of smaller domains. The surfactant proteins also produced smaller domains, but they had the opposite effect of decreasing the total nonfluorescent area. Only the neutral lipids caused remixing. In images from static monolayers, the domains for NPL dropped from a maximum of 26 +/- 3% of the interface at 25 mN/m to 4 +/- 2% at 30 mN/m, similar to the previously reported behavior for CLSE. During continuous compression through a narrow range of pressure and molecular area, in NPL, CLSE, and mixtures of PPL with 10% cholesterol, domains became highly distorted immediately prior to remixing. The characteristic transition in shape and abrupt termination of phase coexistence indicate that the remixing caused by the neutral lipids occurs at or close to a critical point.