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Operational parallels in overall mechanisms of three-dimensional patterning of vertebrate organs are becoming increasingly apparent. Many key mediators, such as FGFs, BMPs, and sonic hedgehog, participate in organization of a number of organs, including the lungs, which exhibit a defined proximodistal (P-D) polarity. Recently, Wnt5a a member of the wingless family of signaling molecules involved in cell proliferation, differentiation, and organogenesis, was shown to underlie the outgrowth and P-D morphogenesis of the vertebrate limb. In the current study, we show that Wnt5a is expressed in the mouse lung and plays an important role in lung distal morphogenesis. Analysis of the mutant phenotype in mice carrying a targeted disruption of the Wnt5a locus shows distinct abnormalities in distal lung morphogenesis as manifested by distinct truncation of the trachea and overexpansion of the distal respiratory airways. In the face of deleted WNT5a activity, both epithelial and mesenchymal cell compartments of the Wnt5a(−/−) lungs exhibit increased cell proliferation. The overall architecture of the mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung maturation as reflected by persistence of thickened intersaccular interstitium. Absence of WNT5a activity in the mutant lungs leads to increased expression of Fgf-10, Bmp4, Shh, and its receptor Ptc, raising the possibility that WNT5a, FGF-10, BMP4, and SHH signaling pathways are functionally interactive.

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the α-subunit of the rat (r) epithelial Na channel (α-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump α1-subunit in AEC was assessed in situ. Na pump subunit isoform and α-rENaC expression was also evaluated by RT-PCR in highly purified (∼95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-α1and anti-β1Na pump subunit and anti-α-rENaC antibodies was detected, while reactivity with anti-α2Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-α1Na pump subunit antibody. Na pump α1- and β1- (but not α2-) subunits and α-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.

Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5f/−;Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5f/−;Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated β-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters, and CpG islands (CGIs) while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome. Citation Format: Jianxin Shi, Crystal Marconett, Jubao Duan, Paula Hyland, Peng Li, Zhaoming Wang, William Wheeler, Mihaela Campan, Jing Huang, Weiyin Zhou, Tim Triche, Laufey Amundadottir, Amy Hutchinson, Po-Han Chen, Beiyun Zhou, Brian Chung, Pier Alberto Bertazzi, Andrew W. Bergen, Mathew Freedman, Diane Lee, Kim Siegmund, Andrew Clay Warner, Ben Berman, Angela C. Pesatori, Zea Borok, Dario Consonni, Nilanjan Chatterjee, Margaret Tucker, Neil Caporaso, Stephen J. Chanock, Ite A. Laird-Offringa, Maria Teresa Landi. Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2014-287

Previous studies from this laboratory demonstrated that 4-hydroxy-2-nonenal (4HNE), a lipid peroxidation product, induces expression of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, in rat alveolar epithelial L2 cells. The present study demonstrates that 4HNE also induces GCS in primary cultured alveolar epithelial type II (AT2) cells. Enzyme activity, protein content, and messenger RNA levels of both the catalytic (GCS-HS) and regulatory (GCS-LS) subunits were significantly increased in AT2 cells treated with 5 or 10 microM 4HNE, the same concentrations that induced GCS expression in L2 cells. As in L2 cells, 4HNE induced a greater AT2-cell increase in GCS-LS than in GCS-HS, suggesting that modulation of GCS-LS may play a dominant role in regulating GSH concentration in response to oxidative stress. Additional studies using mitogen-activated protein kinase pathway inhibitors showed that induction by 4HNE of GCS-HS, but not GCS-LS, was mediated through activation of the extracellular regulated kinase pathway in L2 cells. The results demonstrate that L2 cells maintain the same responsiveness to oxidant challenge as do primary cultured AT2 cells in terms of increasing GSH synthetic capacity, and that different pathways are involved in the induction of two GCS subunits by 4HNE.

Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased ( P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion ([Formula: see text]) release by ELF inflammatory cells decreased after GSH therapy ( P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed [Formula: see text] release ( P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.

A recent study (Reducing Deaths due to Oxidative Stress, REDOXS) reported harm in critically ill patients who received glutamine supplementation [1]. This is in contrast to a number of earlier studies reporting beneficial effects or failing to demonstrate any effect [2, 3]. Naturally, this finding raises a number of questions, which are not answered by combining all existing studies into a large meta-analysis. This overview will discuss existing clinical data, including dosing and selection of patients. In addition, suggested mechanisms will be discussed from a clinical perspective. Finally, possibilities for future research will be outlined.

The molecular signals that control decisions regarding progenitor/stem cell proliferation versus differentiation are not fully understood. Differentiation of motile cilia from progenitor/stem cells may offer a simple tractable model to investigate this process. Wnt and Notch represent two key signaling pathways in progenitor/stem cell behavior in a number of tissues. Adenomatous Polyposis Coli, Apc is a negative regulator of the Wnt pathway and a well known multifunctional protein. Using the cre-LoxP system we inactivated the Apc locus via Foxj1-cre, which is expressed in cells committed to ciliated cell lineage. We then characterized the consequent phenotype in two select tissues that bear motile cilia, the lung and the testis. In the lung, Apc deletion induced β-catenin accumulation and Jag1 expression in ciliated cells and by lateral induction, triggered Notch signaling in adjacent Clara cells. In the bronchiolar epithelium, absence of Apc blocked the differentiation of a subpopulation of cells committed to the ciliogenesis program. In the human pulmonary adenocarcinoma cells, Apc over-expression inhibited Jag1 expression and promoted motile ciliogenic gene expression program including Foxj1, revealing the potential mechanism. In the testis, Apc inactivation induced β-catenin accumulation in the spermatogonia, but silenced Notch signaling and depleted spermatogonial stem cells, associated with reduced proliferation, resulting in male infertility. In sum, the present comparative analysis reveals the tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors by coordinating Wnt and Notch signaling.

We evaluated the effects of epidermal growth factor (EGF) on transepithelial resistance (Rt) and active ion transport by alveolar epithelial cell (AEC) monolayers on tissue culture-treated polycarbonate filters. Rat type II cells were cultured in completely defined serum-free medium (MDSF) or MDSF supplemented with EGF. The addition of EGF from either day 0 (chronic) or day 4 (subacute) resulted in significant increases in Rt and short-circuit current (ISC) on day 5. After subacute exposure, these effects were delayed in onset by 6-12 h and sustained for > 24 h. Basolateral (but not apical) EGF was responsible for these effects, which were prevented by preincubation with tyrphostin RG-50864, a reversible specific inhibitor of the EGF receptor tyrosine kinase. ISC decreased, with a sensitivity to apical inhibitors of sodium transport in the order benzamil > amiloride > 5-(N-ethyl-N-isopropyl) amiloride in MDSF +/- EGF, and was completely inhibited by the addition of basolateral ouabain. Net sodium flux and Na+, K+ -ATPase activity both increased approximately 50% in the presence of EGF. These results indicate that 1) EGF decreases tight junctional permeability and increases active sodium transport by AEC monolayers via basolaterally located EGF receptors, and 2) the pathways for AEC sodium entry and exit (+/- EGF) are apical high amiloride affinity sodium channels and basolateral sodium pumps.

Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76 +/- 0.14 K omega-cm2 by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71 +/- 0.35 microA/cm2. With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Farnoosh Fazlollahi1,8, Arnold Sipos1,2, Yong Ho Kim1,2, Sarah F Hamm-Alvarez6, Zea Borok1–3, Kwang-Jin Kim1,2,5–7, Edward D Crandall1,2,4,8 1Will Rogers Institute Pulmonary Research Center, 2Department of Medicine, 3Department of Biochemistry and Molecular Biology, 4Department of Pathology, 5Department of Physiology and Biophysics, 6Department of Pharmacology and Pharmaceutical Sciences, 7Department of Biomedical Engineering, 8Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA Background: In this study, primary rat alveolar epithelial cell monolayers (RAECM) were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms. Methods: Trafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate) of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-ß-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively). The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca2+ in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C. Results: Apical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-ß-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature decreased transmonolayer resistance by approximately 90% with a concomitant increase in quantum dot trafficking by about 80%; and 24 hours of treatment of RAECM with EGTA decreased transmonolayer resistance by about 95%, with increased quantum dot trafficking of up to approximately 130%. Conclusion: These data indicate that quantum dots do not injure RAECM and that quantum dot trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin, or dynamin. We conclude that quantum dot translocation across RAECM takes place via both transcellular and paracellular pathways and, based on comparison with our prior studies, interactions of nanoparticles with RAECM are strongly dependent on nanoparticle composition and surface properties. Keywords: alveolar epithelial barrier, transport, paracellular pathways, endocytosis

Cystic fibrosis (CF), a disorder characterized by mutations of the CF transmembrane regulator gene, is characterized in the lung by chronic inflammation, leading to progressive damage to the airway epithelium, bronchiectasis, and chronic obstructive lung disease. One process contributing to the airway derangement is the chronic burden of oxidants released by inflammatory cells on the respiratory epithelial surface. With this background, we hypothesized that glutathione in respiratory epithelial lining fluid (ELF) in CF patients might be oxidized and/or diminished in amount compared with that in normal subjects. Recovery of ELF by bronchoalveolar lavage from young adults with CF (n = 21) and normal subjects (n = 25) demonstrated marked neutrophil-dominated inflammation in ELF in CF patients. As predicted, ELF in CF patients was characterized by a deficiency of glutathione (P < 0.001), but this was secondary to a reduction in reduced glutathione (P < 0.001), inasmuch as there were no differences in ELF levels of oxidized glutathione (P > 0.2). Unexpectedly, there was also a marked deficiency of reduced glutathione in plasma (P < 0.02); i.e., the glutathione "deficiency" observed in ELF in CF patients is not limited to the site of the inflammation but is systemic. Although the etiology of this generalized deficiency of extracellular glutathione is unknown, it is important in considering options for treating the concomitant and devastating lung pathology in this disorder.

BACKGROUND--Concentrations of glutathione, a ubiquitous tripeptide with immune enhancing and antioxidant properties, are decreased in the blood and lung epithelial lining fluid of human immunodeficiency virus (HIV) seropositive individuals. Since the lung is the most common site of infection in those who progress to AIDS it is rational to consider whether it is possible to safely augment glutathione levels in the epithelial lining fluid of HIV seropositive individuals, thus potentially improving local host defence. METHODS--Purified reduced glutathione was delivered by aerosol to HIV seropositive individuals (n = 14) and the glutathione levels in lung epithelial lining fluid were compared before and at one, two, and three hours after aerosol administration. RESULTS--Before treatment total glutathione concentrations in the epithelial lining fluid were approximately 60% of controls. After three days of twice daily doses each of 600 mg reduced glutathione, total glutathione levels in the epithelial lining fluid increased and remained in the normal range for at least three hours after treatment. Strikingly, even though > 95% of the glutathione in the aerosol was in its reduced form, the percentage of oxidised glutathione in epithelial lining fluid increased from 5% before treatment to about 40% three hours after treatment, probably reflecting the use of glutathione as an antioxidant in vivo. No adverse effects were observed. CONCLUSIONS--It is feasible and safe to use aerosolised reduced glutathione to augment the deficient glutathione levels of the lower respiratory tract of HIV seropositive individuals. It is rational to evaluate further the efficacy of this tripeptide in improving host defence in HIV seropositive individuals.

After the initial infection with HIV, there is evidence of immune dysfunction despite an apparent normal clinical state. In the context that the lung is a major site affected by opportunistic infection during the progression of this immune dysfunction, and that some components of the immune system are activated during early HIV infection, we hypothesized that there may be activation of alveolar macrophages (AM), a key component of the pulmonary host defense system, during the asymptomatic phase of HIV infection. Compared to normals, in HIV-infected individuals the class II MHC molecules DR, DQ, and DP were all expressed more frequently and in greater cell surface density on AM (p0.03, all comparisons), and there was increased spontaneous release of superoxide anion (O2-.) by AM (p0.002). To gain insight into whether the activation of the AM was an inherent property of the cells or dependent on the in vivo milieu, AM were evaluated after 24 h in culture for O2-. release. In contrast to the findings in fresh AM, after 24 h in culture, O2-. release by HIV AM was not different from normals (p0.7), suggesting that these AM had been activated in vivo. To assess whether IFN-gamma could be mediating these effects, mRNA levels of the IP-10 gene (a gene specifically induced by increased concentrations of IFN-gamma) were quantified in AM. Strikingly, the IP-10 gene was expressed only in AM of HIV-seropositive individuals, suggesting the AM had been exposed to IFN-gamma in vivo. Overall, these observations are consistent with the concept that the HIV-seropositive state is associated with activation of AM, in part due to local exposure to IFN-gamma.

Prostaglandin E (PGE), a cyclooxygenase metabolite normally present in high concentrations in respiratory epithelial lining fluid (ELF), is capable of suppressing mesenchymal cell proliferation mediated by polypeptide-derived growth factors. Although PGE is normally abundant in respiratory ELF, PGE levels in ELF of individuals with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disorder characterized by intraalveolar mesenchymal cell accumulation and fibrosis, were found to be 50% lower than normal (p less than 0.01): that is, a relative PGE "deficiency" in ELF may enhance intraalveolar mesenchymal cell proliferation in IPF. With this background, it is rational to consider augmenting PGE levels in ELF as a future therapy for IPF. Since systemic administration of PGE is associated with significant adverse effects, in vitro and experimental animal studies were carried out to evaluate whether aerosol PGE administration could augment ELF PGE levels. Greater than 50% of a solution of PGE1 could be placed in droplets less than 3 microns mass median aerodynamic diameter without loss of function. Aerosolization of PGE1 to sheep (n = 14) resulted in a marked augmentation of ELF PGE1 levels (preaerosol 20 +/- 7 nM, 30 min postaerosol 1,150 +/- 210 nM; p less than 0.0 to 0.1). ELF PGE1 levels remained elevated for up to 2 h (p less than 0.05 compared with baseline) and returned to baseline by 3 h (p greater than 0.2). Lung interstitial fluid (lymph) PGE1 levels increased slightly, but to levels far less than ELF levels (preaerosol 7 +/- 1 nM, 30 min postaerosol 13 +/- 2 nM; p less than 0.01), and plasma PGE1 levels did not change (p greater than 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Idiopathic pulmonary fibrosis (IPF) is characterised by alveolar inflammation, exaggerated release of oxidants, and subnormal concentrations of the antioxidant glutathione in respiratory epithelial lining fluid (ELF). Glutathione (600 mg twice daily for 3 days) was given by aerosol to 10 patients with IPF. Total ELF glutathione rose transiently, ELF oxidised glutathione concentrations increased, and there was a decrease in spontaneous superoxide anion release by alveolar macrophages. Thus, glutathione by aerosol could be a means of reversing the oxidant-antioxidant imbalance in IPF.

Background Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. Methods Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. Results This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. Conclusion This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.

The use of steroids in treating acute respiratory obstruction is still controversial. In this double-blind controlled trial, we decided to examine the beneficial effects of a single large dose of methylprednisolone (MSSP), using objective criteria. In the emergency setting, methylprednisolone (30 mg/kg) has been shown to decrease the need for hospital admission in patients with acute bronchospasm. No difference in this improvement was seen among patients in the steroid-dependent or non-steroid-dependent populations. Based on our findings, we suggest that the early use of single-dose steroid therapy is appropriate treatment for patients with acute bronchospastic attacks.

The helicopter transport of acute cardiac patients has become increasingly common, although no study has examined solely the effect of such transport on outcome in this subset of patients. A combined air and ground critical care transport service provided the opportunity for a direct comparison of patients with acute cardiac conditions (myocardial infarction or unstable angina) transported either by our helicopter or by a specially equipped critical care ground vehicle. Both air and ground components were similarly equipped in terms of personnel and medical equipment. Seventy-eight (27 ground, 51 air) transport cases were studied. Both patient groups were comparable in terms of age, sex, Killip classification, and diagnosis. Serious untoward events, defined as arrhythmias, chest pain, hypotension, bradycardia, seizures, and cardiac arrest, occurred in 41% of air transports and 7.5% of ground transports (P less than .002). The overall incidence of untoward events was also significantly greater with air transports (25/51, or 49%) than with the ground vehicle (4/27, or 15%; P less than .005). The reasons for these differences are unknown.

Serial measurements of the plasma concentration of fibrinopeptide A, thrombin-increasable fibrinopeptide B (reflecting B beta 1–42), desarginyl fibrinopeptide B, beta thromboglobulin, and platelet factor 4 were made before, during, and after delivery in patients with preeclampsia/eclampsia. The data were correlated with routine coagulation studies, hematologic and renal status, as well as with the clinical manifestations. In 11 patients with mild preeclampsia, there were small increases in the fibrinopeptides at the time of delivery, but no other hematologic changes. In 5 patients with severe preeclampsia/eclampsia, there were marked increases in plasma levels of fibrinopeptides and platelet alpha granule proteins, which correlated in time with the clinical manifestations. When the changes in these patients were compared with those occurring in patients undergoing intraamniotic hypertonic saline infusion, it was noted that: (1) patients with severe preeclampsia/eclampsia usually presented when plasmin action on fibrinogen exceeded that of thrombin; (2) in patients with preeclampsia/eclampsia the increase in fibrinopeptides lasted from 3 to 7 days, rather than for several hours as occurred after the infusion of hypertonic saline, indicating a more persistent stimulus to intravascular coagulation in preeclampsia/eclampsia; (3) severe thrombocytopenia and increased platelet protein levels were seen in these patients and were disproportionate to the degree of increase in the fibrinopeptide A level, suggesting that a mechanism other than thrombin must have contributed to the platelet changes; and (4) in two patients with severe preeclampsia/eclampsia, high desarginyl fibrinopeptide B levels preceded renal insufficiency, possibly reflecting fibrin II formation in renal vessels.

It has been reported that patients with preeclampsia have decreased levels of antithrombin III heparin cofactor and that the degree of depression parallels the severity of disease. To determine the time course of development and resolution of this deficiency, and to gain insight into the cause of this deficiency, plasma antithrombin III antigen and heparin cofactor activity levels were measured serially in 11 women with mild preeclampsia. Although the ATIII antigen level remained stable during the antepartum period, the level of heparin cofactor was noted to fall progressively during the antepartum period. Both ATIII antigen and heparin cofactor increased promptly postpartum. The discrepancy between the levels of ATIII antigen and heparin cofactor activity suggests that in both mild and severe preeclampsia there is utilization of antithrombin III to form inactive protease inhibitor complexes.

It is often necessary to have a small animal model for hyperkalemia for use in electrolyte and acid base experiments. In reviewing the literature, we found a paucity of such animal models, especially for acute hyperkalemia. We have had difficulty in inducing acute hyperkalemia in rats using potassium chloride alone either intravenously or intraperitoneally and felt the need for an easily reproducible small animal model for hyperkalemia. We gave experimental animals a combination of intraperitoneal amiloride 3 mg/kg and potassium chloride 2 meq/kg in two divided doses while control animals received only the potassium chloride. Initial serum potassiums were similar but at 2 hr, the experimental group had significantly higher serum potassium levels which were sustained throughout the 8 hr of the experiment. Arterial blood gas revealed no significant difference in blood pH values at all time points during the experiment. We conclude that the combination of amiloride and potassium chloride is useful to produce acute hyperkalemia in rats and that this hyperkalemia is sustained beyond 6 hr. This model is convenient for use in metabolic experiments requiring the use of acutely hyperkalemic rats.

Generalized seizures may be associated with therapeutic or intentional theophylline overdose. Toxic levels of theophylline are also associated with a fall in potassium which could potentiate theophylline-induced seizures. To evaluate the role of serum potassium concentration in theophylline-induced seizures we investigated the seizure threshold in normokalemic and hyperkalemic rats during theophylline infusion to toxic levels. Hyperkalemic rats were prepared with intraperitoneal amiloride and potassium chloride and had a mean +/- SEM initial potassium of 5.29 +/- 0.15 mEq/L. Control animals received either amiloride or potassium and had initial serum potassium concentrations of 4.00 +/- 0.08 and 3.93 +/- 0.16 mEq/L, respectively. Potassium levels after 30 minutes of theophylline infusion were 4.11 +/- 0.18 mEq/L in the hyperkalemic rats and 3.47 +/- 0.06 and 3.51 +/- 0.12 mEq/L in the control animals. There were no significant differences in the serum theophylline concentrations at time of seizure, nor was there a correlation between serum potassium concentration and theophylline concentration at time of seizure. Since the preservation of normokalemia does not influence the onset of seizures in theophylline toxicity, this suggests that the potassium level has little effect on seizure activity in this model.

We investigated expression of the α3-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the α3-integrin subunit on AEC monolayer formation. α3-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. α3- and β1-integrin subunits coprecipitated in immunoprecipitation experiments with α3- and β1-subunit-specific antibodies, consistent with their association as the α3β1-integrin receptor at the cell membrane. Treatment with blocking anti-α3monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that α3- and β1-integrin subunits are expressed in AEC monolayers where they form the heterodimeric α3β1-integrin receptor at the cell membrane. Blockade of the α3-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the α3-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the α3β1-integrin receptor in AEC adhesion.

Background Epithelial-mesenchymal cross talk is centerpiece in the development of many branched organs, including the lungs. The embryonic lung mesoderm provides instructional information not only for lung architectural development, but also for patterning, commitment and differentiation of its many highly specialized cell types. The mesoderm also serves as a reservoir of progenitors for generation of differentiated mesenchymal cell types that include αSMA-expressing fibroblasts, lipofibroblasts, endothelial cells and others. Transforming Growth Factor β (TGFβ) is a key signaling pathway in epithelial-mesenchymal cross talk. Using a cre-loxP approach we have elucidated the role of the TGFβ type I receptor tyrosine kinase, ALK5, in epithelial-mesenchymal cross talk during lung morphogenesis. Results Targeted early inactivation of Alk5 in mesodermal progenitors caused abnormal development and maturation of the lung that included reduced physical size of the sub-mesothelial mesoderm, an established source of specific mesodermal progenitors. Abrogation of mesodermal ALK5-mediated signaling also inhibited differentiation of cell populations in the epithelial and endothelial lineages. Importantly, Alk5 mutant lungs contained a reduced number of αSMApos cells and correspondingly increased lipofibroblasts. Elucidation of the underlying mechanisms revealed that through direct and indirect modulation of target signaling pathways and transcription factors, including PDGFRα, PPARγ, PRRX1, and ZFP423, ALK5-mediated TGFβ controls a process that regulates the commitment and differentiation of αSMApos versus lipofibroblast cell populations during lung development. Conclusion ALK5-mediated TGFβ signaling controls an early pathway that regulates the commitment and differentiation of αSMApos versus LIF cell lineages during lung development. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0242-9) contains supplementary material, which is available to authorized users.

We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na+-K+-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current ( Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na+-K+-ATPase α1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas α2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the β1-subunit of Na+-K+-ATPase did not increase, whereas cellular α1- and β1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for α-, β-, and γ-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase α1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.