Search

In the acute respiratory distress syndrome (ARDS), alveolar surface tension,T, may be elevated. ElevatedTshould increase ventilation-induced lung injury. Exogenous surfactant therapy, intended to lowerT, has not reduced mortality. Sulforhodamine B (SRB) might, alternatively, be employed to lowerT. We test whether substances suspected of elevatingTin ARDS raiseTin the lungs and test the abilities of exogenous surfactant and SRB to reduceT. In isolated rat lungs, we micropuncture a surface alveolus and instill a solution of a purportedT-raising substance: control saline, cell debris, secretory phospholipase A2(sPLA2), acid or mucins. We test each substance alone; with albumin, to model proteinaceous edema liquid; with albumin and exogenous surfactant; or with albumin and SRB. We determineT in situin the lungs by combining servo-nulling pressure measurement with confocal microscopy, and applying the Laplace relation. With control saline, albumin does not alterT, additional surfactant raisesTand additional SRB lowersT. The experimental substances, without or with albumin, raiseT. Excepting under aspiration conditions, addition of surfactant or SRB lowersT. Exogenous surfactant activity is concentration and ventilation dependent. Sulforhodamine B, which could be delivered intravascularly, holds promise as an alternative therapeutic.New and NoteworthyIn the acute respiratory distress syndrome (ARDS), lowering surface tension,T, should reduce ventilation injury yet exogenous surfactant has not reduced mortality. We show with directT-determination in isolated lungs that substances suggested to elevateTin ARDS indeed raiseT, and exogenous surfactant reducesT. Further, we extend our previous finding that sulforhodamine B (SRB) reducesTbelow normal in healthy lungs and show that SRB, too, reducesTunder ARDS conditions.

Whether alveolar liquid surface tension, T, is elevated in the acute respiratory distress syndrome (ARDS) has not been demonstrated in situ in the lungs. Neither is it known how exogenous surfactant, which has failed to treat ARDS, affects in situ T. We aim to determine T in an acid-aspiration ARDS model before and after exogenous surfactant administration. In isolated rat lungs, we combine servo-nulling pressure measurement and confocal microscopy to determine alveolar liquid T according to the Laplace relation. Administering 0.01 N (pH 1.9) HCl solution by alveolar injection or tracheal instillation, to model gastric liquid aspiration, raises T. Subsequent surfactant administration fails to normalize T. Furthermore, in normal lungs, tracheal instillation of control saline or exogenous surfactant raises T. Lavaging the trachea with saline and injecting the lavage solution into the alveolus raises T, suggesting that tracheal instillation may wash T-raising airway contents to the alveolus. Adding 0.01 N HCl or 5 mM CaCl2—either of which aggregates mucins—to tracheal lavage solution reduces or eliminates the effect of lavage solution on alveolar T. Following tracheal saline instillation, liquid suctioned directly out of alveoli through a micropipette contains mucins. Additionally, alveolar injection of gastric mucin solution raises T. We conclude that 1) tracheal liquid instillation likely washes T-raising mucins to the alveolus and 2) even exogenous surfactant that could be delivered mucin-free to the alveolus might not normalize T in acid-aspiration ARDS. NEW & NOTEWORTHY We demonstrate in situ in isolated lungs that surface tension is elevated in an acid-aspiration acute respiratory distress syndrome (ARDS) model. Following tracheal liquid instillation, also in isolated lungs, we directly sample alveolar liquid. We find that liquid instillation into normal lungs washes mucins to the alveolus, thereby raising alveolar surface tension. Furthermore, even if exogenous surfactant could be delivered mucin-free to the alveolus, exogenous surfactant might fail to normalize alveolar surface tension in acid-aspiration ARDS.

Lymphangions, the segments of lymphatic vessels between two adjacent lymphatic valves, actively pump lymph. Acute changes in transmural pressure and lymph flow have profound effects on lymphatic pump function in vitro. Chronic changes in pressure and flow in vivo have also been reported to lead to significant changes in lymphangion function. Because changes in pressure and flow are both cause and effect of adaptive processes, characterizing adaptation requires a more fundamental analysis of lymphatic muscle properties. Therefore, the purpose of the present work was to use an intact lymphangion isovolumetric preparation to evaluate changes in mesenteric lymphatic muscle mechanical properties and the intracellular Ca2+ in response to sustained mesenteric venous hypertension. Bovine mesenteric veins were surgically occluded to create mesenteric venous hypertension. Postnodal mesenteric lymphatic vessels from mesenteric venous hypertension (MVH; n = 6) and sham surgery (Sham; n = 6) animals were isolated and evaluated 3 days after the surgery. Spontaneously contracting MVH vessels generated end-systolic active tension and end-diastolic active tension lower than the Sham vessels. Furthermore, steady-state active tension and intracellular Ca2+ concentration levels in response to KCl stimulation were also significantly lower in MVH vessels compared with those of the Sham vessels. There was no significant difference in passive tension in lymphatic vessels from the two groups. Taken together, these results suggest that following 3 days of mesenteric venous hypertension, postnodal mesenteric lymphatic vessels adapt to become weaker pumps with decreased cytosolic Ca2+ concentration.

752 Background: Pancreatic ductal adenocarcinomas (PDAC) often exhibit desmoplasia, elevated CTGF (connective tissue growth factor) expression and inflammation. The influence of inflammation on patient outcomes was examined in a dose-ranging trial of the anti-CTGF antibody pamrevlumab in combination with a fixed regimen of gemcitabine and erlotinib in locally advanced or metastatic PDAC patients (NCT01181245; Picozzi et al. J Cancer Clin Trials 2017 2:123; n = 75). Methods: The prognostic utility of pre-treatment plasma levels of C-reactive protein (CRP), transforming growth factor β1 (TGFβ1), albumin, CTGF and CA 19-9 were assessed by univariate and multivariate Cox analysis. Demographic parameters, treatment cohort, and pamrevlumab exposure determined on treatment day 15 were also evaluated, as were changes in biomarker levels over the first four weeks of treatment. Results: Elevated baseline CTGF and CRP were prognostic for shorter overall survival (OS) by univariate analysis (HR = 3.2 for CRP > 10 mg/L, p= 0.00002 and HR = 1.6 for CTGF > 10 ng/mL, p= 0.045). In a five-factor multivariate Cox model that included CRP and TGFβ1 as continuous Ln-transformed variables, performance status, age, and pamrevlumab treatment cohort, cohort assignments associated with increasing pamrevlumab exposure predicted improved OS (HR = 0.87, p= 0.03). Removing CRP from this model reduced the prognostic utility of pamrevlumab cohort assignment and exposure, indicating an important contribution of inflammation to interpretation of treatment outcome. Changes in inflammation biomarkers over the course of treatment were also evaluated, but were not prognostic in this study. Conclusions: In multivariate Cox models, assessment of pre-treatment CRP levels improved ability to detect significant differences in PDAC patient survival outcomes associated with pamrevlumab treatment. Our results emphasize the utility of accounting for pre-treatment CRP levels as an independent prognostic factor in PDAC treatment effect models.

Edematous lungs contain regions with heterogeneous alveolar flooding. Liquid is trapped in flooded alveoli by a pressure barrier—higher liquid pressure at the border than in the center of flooded alveoli—that is proportional to surface tension, T. Stress is concentrated between aerated and flooded alveoli, to a degree proportional to T. Mechanical ventilation, by cyclically increasing T, injuriously exacerbates stress concentrations. Overcoming the pressure barrier to redistribute liquid more homogeneously between alveoli should reduce stress concentration prevalence and ventilation injury. In isolated rat lungs, we test whether accelerated deflation can overcome the pressure barrier and catapult liquid out of flooded alveoli. We generate a local edema model with normal T by microinfusing liquid into surface alveoli. We generate a global edema model with high T by establishing hydrostatic edema, which does not alter T, and then gently ventilating the edematous lungs, which increases T at 15 cmH2O transpulmonary pressure by 52%. Thus ventilation of globally edematous lungs increases T, which should increase stress concentrations and, with positive feedback, cause escalating ventilation injury. In the local model, when the pressure barrier is moderate, accelerated deflation causes liquid to escape from flooded alveoli and redistribute more equitably. Flooding heterogeneity tends to decrease. In the global model, accelerated deflation causes liquid escape, but—because of elevated T—the liquid jumps to nearby, aerated alveoli. Flooding heterogeneity is unaltered. In pulmonary edema with normal T, early ventilation with accelerated deflation might reduce the positive feedback mechanism through which ventilation injury increases over time. NEW & NOTEWORTHY We introduce, in the isolated rat lung, a new model of pulmonary edema with elevated surface tension. We first generate hydrostatic edema and then ventilate gently to increase surface tension. We investigate the mechanical mechanisms through which 1) ventilation injures edematous lungs and 2) ventilation with accelerated deflation might lessen ventilation injury.

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.

In vitro studies have revealed that acute increases in transmural pressure increase lymphatic vessel contractile function. However, adaptive responses to prolonged changes in transmural pressure in vivo have not been reported. Therefore, we developed a novel bovine mesenteric lymphatic partial constriction model to test the hypothesis that lymphatic vessels exposed to higher transmural pressures adapt functionally to become stronger pumps than vessels exposed to lower transmural pressures. Postnodal mesenteric lymphatic vessels were partially constricted for 3 days. On postoperative day 3, constricted vessels were isolated, and divided into upstream (UP) and downstream (DN) segment groups, and instrumented in an isolated bath. Although there were no differences between the passive diameters of the two groups, both diastolic diameter and systolic diameter were significantly larger in the UP group than in the DN group. The pump index of the UP group was also higher than that in the DN group. In conclusion, this is the first work to report how lymphatic vessels adapt to prolonged changes in transmural pressure in vivo. Our results suggest that vessel segments upstream of the constriction adapt to become both better fluid conduits and lymphatic pumps than downstream segments.

Determination of the structure of the unliganded monomeric state of neurophysin is central to an understanding of the allosteric relationship between neurophysin peptide-binding and dimerization. We examined this state by NMR, using the weakly dimerizing H80E mutant of bovine neurophysin-I. The derived structure, to which more than one conformer appeared to contribute, was compared with the crystal structure of the unliganded des 1-6 bovine neurophysin-II dimer. Significant conformational differences between the two proteins were evident in the orientation of the 3,10 helix, in the 50-58 loop, in beta-turns, and in specific intrachain contacts between amino- and carboxyl domains. However, both had similar secondary structures, in independent confirmation of earlier circular dichroism studies. Previously suggested interactions between the amino terminus and the 50-58 loop in the monomer were also confirmed. Comparison of the observed differences between the two proteins with demonstrated effects of dimerization on the NMR spectrum of bovine neurophysin-I, and preliminary investigation of the effects of dimerization on H80E spectra, allowed tentative distinction between the contributions of sequence and self-association differences to the difference in conformation. Regions altered by dimerization encompass most binding site residues, providing a potential explanation of differences in binding affinity between the unliganded monomeric and dimeric states. Differences between monomer and dimer states in turns, interdomain contacts, and within the interdomain segment of the 50-58 loop suggest that the effects of dimerization on intrasubunit conformation reflect the need to adjust the relative positions of the interface segments of the two domains for optimal interaction with the adjacent subunit and/or reflect the dual role of some residues as participants both at the interface and in interdomain contacts.

The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.

Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, DeltaE47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.

Purpose To evaluate and model the pharmacokinetic and pharmacodynamic behavior in rats of FG-3019, a human monoclonal antibody targeting connective tissue growth factor (CTGF). Methods FG-3019, human CTGF (rhCTGF), or the N-terminal domain of rhCTGF were administered intravenously to rats and concentrations of these proteins as well as endogenous CTGF were determined by immunoassays. FG-3019, or 125I-labeled FG-3019, and human CTGF (rhCTGF) were co-administered to assess the impact of CTGF on the elimination rate and tissue localization of FG-3019, which was further characterized by immunohistochemical analysis. A PK/PD model for target-mediated elimination of FG-3019 was developed to fit the kinetic data. Results FG-3019 exhibited non-linear pharmacokinetics in rats. Circulating concentrations of the N-terminal half of CTGF increased after dosing with FG-3019, reached maximal levels after 1–5 days, and returned toward baseline levels as FG-3019 cleared from the circulation, whereas the concentration of intact CTGF was unaffected by administration of FG-3019. Co-administration of rhCTGF dramatically enhanced the rate of FG-3019 elimination, redistributing the majority of 125I-labeled FG-3019 from the blood to the liver, kidney, spleen and adrenal gland. FG-3019 co-administered with CTGF was found along the sinusoids of the liver and adrenal glands, the capillaries of the kidney glomeruli and in the spleen. A pharmacokinetic model for target-mediated elimination of FG-3019 was used to fit the time courses of FG-3019 and endogenous CTGF plasma concentrations, as well as time courses of rhCTGF and rhCTGF N-fragment after intravenous administration of these species. Conclusions FG-3019 is subject to target mediated elimination in rats. Electronic supplementary material The online version of this article (doi:10.1007/s11095-016-1918-0) contains supplementary material, which is available to authorized users.