Chylous Ascites as a Presentation of Lymphangioleiomyomatosis.

A 35-year-old woman presented to the hospital with a 4-week history of large-volume chylous ascites refractory to paracentesis and new-onset dyspnea. Thoracic computed tomography revealed diffuse pulmonary cystic lesions with pleural effusions, and abdominal computed tomography showed ascites with large bilateral retroperitoneal masses displaying positron emission tomography avidity. Biopsy of the masses demonstrated lymphatic invasion by a perivascular epithelioid cell neoplasm, a smooth muscle tumor.

New directions in interorganizational collaboration in dentistry: the CAMBRA Coalition model.

Caries management by risk assessment (CAMBRA) represents a paradigm shift in the management of dental decay. It treats dental caries as an infectious disease that is curable and preventable. The science supporting CAMBRA has been present for quite some time; however, its clinical adoption, until recently, remained slow.

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro.

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5.

Cell-cell communication in heterocellular cultures of alveolar epithelial cells.

The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections.

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix.

The pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture.

Polarity of alveolar epithelial cell acid-base permeability.

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined.

Na transport proteins are expressed by rat alveolar epithelial type I cells.

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 alpha-subunit of the rat (r) epithelial Na channel (alpha-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).

Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(2+) changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor.

Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

We investigated expression of the alpha(3)-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 alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane.

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels.

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double.

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h.

Modulation of t1alpha expression with alveolar epithelial cell phenotype in vitro.

T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture.

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells.

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF.

Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.

We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.

Na(+)-K(+)-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF.

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.

Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells.

The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies.

Effects of EGF on alveolar epithelial junctional permeability and active sodium transport.

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.

Basolateral localization of Na(+)-HCO3- cotransporter activity in alveolar epithelial cells.

We investigated the polarized distribution of Na(+)- and HCO3(-)-dependent recovery from intracellular acidification in alveolar epithelial cell monolayers. Rat alveolar type II cells were grown in primary culture on detachable tissue culture-treated Nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer.

Cl(-)-HCO3- exchanger isoform AE2 is restricted to the basolateral surface of alveolar epithelial cell monolayers.

We investigated the polarized distribution and isoform specificity of anion exchange (Cl(-)-HCO3- exchange) in alveolar epithelial cell monolayers. Rat alveolar type II epithelial cell monolayers were grown in primary culture on detachable tissue culture-treated nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer, the cells loaded with pH-sensitive dye, and intracellular pH (pHi) measured spectrofluorometrically.

Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells.

In this study, we investigated the polarized distribution of Na(+)-H+ antiport activity in alveolar epithelial cell monolayers. Rat alveolar type II pneumocytes were grown on detachable tissue culture-treated Nuclepore filters. The membrane filters, with their adherent intact alveolar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer.

Measurement of solute fluxes in isolated rat lungs.

Most previous studies in isolated perfused lungs have utilized measurements of solute flow from alveolar to vascular space to characterize the barrier and transport properties of the alveolar epithelium. In this study, we measured flux of a series of nonionic hydrophilic solutes and sodium across the alveolar epithelium of the isolated rat lung from perfusate to airspace (P-->A), as well as from airspace to perfusate (A-->P). Apparent permeability-surface area products (PS) were calculated from the rates of isotope appearance downstream in either the airspace or the perfusate.

Evidence for amiloride-sensitive sodium channels in alveolar epithelial cells.

To maintain alveolar air spaces relatively fluid free, the alveolar epithelium appears capable of vectorial transport of water and solutes. Active transepithelial transport of sodium by alveolar epithelial cell monolayers has previously been demonstrated, indicating that alveolar pneumocytes must possess ion transport mechanisms by which sodium can enter the cells apically for subsequent extrusion via Na(+)-K(+)-adenosinetriphosphatase activity at the basolateral surface. In this study, sodium entry mechanisms were investigated by directly measuring 22Na uptake into rat alveolar epithelial cells grown in primary culture.

Regulation of intracellular pH in alveolar epithelial cells.

Alveolar type II epithelial cells in adult mammalian lungs actively transport salt and water, secrete surfactant, and differentiate into type I cells under normal conditions and following lung injury. It has become increasingly apparent that, like all epithelial cells, alveolar pneumocytes have evolved specialized ion transport mechanisms by which they regulate their intracellular pH (pHi). pHi is an important biological parameter in all living cells whose regulation is necessary for normal cellular homeostasis.

Na(+)-HCO3- symport modulates intracellular pH in alveolar epithelial cells.

We investigated Na(+)-HCO3- cotransport as a mechanism for regulation of intracellular pH (pHi) in rat alveolar pneumocytes grown in primary culture. pHi was monitored using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells incubated in 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) medium at pH 7.