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L V Leak

Publications and source records attributed to L V Leak.

At least 19 recordsLinked to original sources
PubMed2000

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium.

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.

Animals↗
PubMed2000

A preliminary evaluation of emergency ultrasound in the setting of an emergency medicine training program.

In this article we seek to evaluate the diagnostic accuracy of emergency physicians performing emergency ultrasonography in the setting of an emergency medicine training program. A prospective observational study was performed at an inner city Level I trauma center with an emergency medicine residency training program. From July 1994 to December 1996 a convenience sample of ultrasound exams was recorded. The diagnostic quality ("acceptable or technically limited") was determined by a board-certified cardiologist or radiologist with fellowship training in ultrasonography. The emergency department interpretations were then compared to those of the blinded cardiologist or radiologist. Four hundred and fifty-six ultrasound examinations were videotaped and entered into the study; 408 (89%) of the studies performed were determined to be "acceptable." The diagnostic accuracy (sensitivity, specificity, positive and negative predictive values) of these studies were as follows: cardiac, to rule out effusion (n = 67; 0.83, 0.98, 0.88, 0.98); transabdominal, to rule out abdominal aortic aneurysms (AAA), cholelithiasis, or free peritoneal fluid (n = 263; 0.91, 0.89, 0.88, 0.92); renal, to rule out hydronephrosis (n = 45; 0.94, 0.96, 0.94, 0.96); pelvic, to rule in intrauterine pregnancy (n = 33; 1.0, 0.90, 0.96, 1.0). The 48 "technically limited studies" included: 39 transabdominal (33 gallbladder, 1 abdominal aortic aneurysm, 5 free peritoneal fluid), 6 cardiac, 2 renal, and 1 pelvic ultrasound. This study suggests that emergency physicians with a minimal amount of training display acceptable technical skill and interpretive acumen in their approach to emergency ultrasonography.

Clinical Competence↗
PubMed1999

Characterization of a transformed ovine lymphatic endothelial cell line.

OBJECTIVE: To develop a non-tumor-derived stable lymphatic endothelial cell line that exhibits rapid growth rate without serum and exogenous growth factors, while still maintaining key features characteristics of the non-transformed lymphatic endothelium. METHODS: Lymphatic endothelial cells were isolated from ovine mesenteric lymphatic vessels, grown to confluence and transfected with SV40 DNA using the calcium phosphate method. The resulting cell line was characterized using morphological, immunocytochemical, flow cytometric analysis, and immunoprecipatitation and Western blotting methods. RESULTS: The resulting cell line (sheep lymphatic endothelial transformed cell line, SLET-1) underwent rapid proliferation in the absence of growth factors and reduced concentrations of serum. In addition, key morphological and functional properties of the non-transformed lymphatic endothelium were retained. These include the ability to form confluent monolayer cultures, the expression of the lymphatic endothelial-specific VEGFR-3, FLT-4) tyrosine kinase receptor, the biosynthesis and secretion of von Willebrand factor and plasminogen activators. In addition, SLET-1 cells express cell surface antigens found on LEC that may act as antibody targets in various immune reactions. Monolayer cultures of the SLET-1 cells incubated with endothelial cell-growth factor formed tubular structures, indicating the retention of the capacity to differentiate. CONCLUSION: The SLET-1 cell line retained key morphological and functional properties characteristic of the non-transformed lymphatic endothelium. The ability to form capillary-like tubular structures provides an important cell line for defining the role of specific proteins that are involved in the lymphagiogenic (formation of new lymphatic vessels) process. Thus, this transformed lymphatic endothelial cell line provides an in citro model that may have widespread utility in studying regulatory mechanisms of lymphatic endothelial cell function and differentiation.

Animals↗
PubMed1997

Characterization of murine Flt4 ligand/VEGF-C.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

Amino Acid Sequence↗
PubMed1995

Nitric oxide production by lymphatic endothelial cells in vitro.

The present study demonstrates that confluent monolayer cultures of lymphatic endothelial cells produce and secrete NO. Immunofluorescent studies showed that eNOS activity can be stimulated with Ca ionophore to enhance the production of NO. Cells exposed to LPS and various cytokines stimulated the production of iNOS which showed the greatest increase in activity at 4 hrs and declined at 18 and 24 hrs. These studies provide evidence that, within the lymphatic vascular lumen, nitric oxide may be produced by the lymphatic endothelium which interact with various vasoactive substances to regulate lymphatic vascular tone. In addition, the production of NO by LEC may be important in the regulation of lymphatic vascular tone in order to more readily accommodate sudden fluctuations in lymph flow and pressure that normally occur during the process of lymph formation and propulsion.

Animals↗
PubMed1994

Lymphangiogenesis in vitro: formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells.

Lymphatic endothelial cells grown long term in culture form lymphatic capillary-like tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.

Animals↗
PubMed1993

Lymphatic endothelium isolation, characterization and long-term culture.

Using a collagenase trypsin-EDTA treatment, we have been able to successfully isolate and grow primary cultures of the lymphatic endothelium (LEC) that were subcultured, frozen for storage, subsequently thawed with good recovery and growth, and serially subcultured. The morphological features of cultured LEC were consistent with that observed for the endothelium of intact lymphatic vessels. A prominent feature of growing cultures was the appearance of large vacuoles in the perinuclear region of the cytoplasm, which became filled with fluid and cell debris engulfed from the culture medium. The basal cell surface lacked a well defined basal lamina and anchoring filaments were observed extending from the basal plasmalemmal surface into the underlying substratum. LEC in cultures were also positive for Factor VIII-related antigen. However, specific granules, characteristic of Weibel-Palade bodies were not observed in ultrathin sections of confluent cultures. F-actin was identified in LEC cultures using fluorescein phalloidin, and in confluent cultures actin filaments were located at the periphery of the cell as a continuous circumferential thin band and short filamentous bundles in the central part of the cell. By using heparin and endothelial cell growth supplement in the culture medium we have been able to grow stable cultures of lymphatic endothelial cells that could be maintained when serially subcultured for over two years. These LEC cultures provide an in vitro model for investigating the function and biochemical properties of the lymphatic endothelium.

Animals↗
PubMed1990

Milky spots of the omentum: a source of peritoneal cells in the normal and stimulated animal.

The topography and ultrastructure of the omentum in normal and stimulated mice were investigated with combined transmission (TEM) and scanning electron microscopy (SEM). The present study demonstrated that lymphocytes and monocytes were the principle cell types in the non-stimulated milky spot. Following stimulation with bacterial toxin and adjuvant there was an increased microvascular permeability to fluid, neutrophils, monocytes and fibrin deposits within the connective tissue matrix of milky spots, and a subsequent increased cellular migration across the mesothelial lining into the peritoneal cavity. Cellular migration from the milky spot to the peritoneal cavity is facilitated by the absence of a basal lamina from the submesothelial connective tissue layer, therefore, cells can migrate from the interstitial spaces of the milky spot to intercellular gaps between mesothelial cells without having to penetrate a fibrous barrier.

Animals↗
PubMed1987

Animal model of acute pericarditis and its progression to pericardial fibrosis and adhesions: ultrastructural studies.

To study the evolution of pericardial inflammation, we have developed a model of pericarditis in sheep by surgically injecting heat-killed staphylococci and Freund's adjuvant into the pericardial cavity under sterile conditions. The pericarditis evolved through the following phases: 1) inflammatory response, 2) mesothelial cell injury and desquamation, and 3) fibrotic phase. At 3-24 hr there was increased microvascular permeability, which resulted in the exudation of fluid, neutrophils, macrophages, and fibrin into the pericardial cavity and the pericardial interstitium. By 72 hr, large numbers of inflammatory cells were aggregated on the mesothelial surfaces and dispersed throughout the pericardial cavity, either as free-floating cells or located between strands of fibrin. At 6 days, fibrinolysis was apparent along the mesothelial surfaces; and newly formed collagen fibrils were deposited throughout the interstitial spaces and among the aggregated cells. These fibrils provided a matrix for the growth of new blood and lymphatic vessels into new connective tissue on both parietal and visceral pericardial surfaces. At 2 weeks, intrapericardial fibrosis had produced focal adhesions between the pericardial surfaces. At 1 month, extensive areas of the pericardial cavity were obliterated. By 9 months, there was a marked reduction in the numbers of cells and blood vessels and increased deposition of collagen and elastic fibers. The intrapericardial injection of heat-killed staphylococci and adjuvant provides a reproducible animal model to study the time course of pericardial inflammation.

Acute Disease↗
PubMed1986

Distribution of cell surface charges on mesothelium and lymphatic endothelium.

The distribution of anionic sites on the luminal surfaces of the peritoneal mesothelium and lymphatic endothelium was investigated by injecting cationized ferritin (CF) intraperitoneally. After washing with phosphate-buffered saline, the diaphragm was fixed and processed for electron microscopy. CF label occurred in discontinuous patches along the mesothelial surface. Microvilli were heavily marked and often closely applied to the mesothelial surface. The intercellular cleft was also heavily labeled. The luminal aspect of the lymphatic endothelium was more extensively labeled, with the marker occurring in long discontinuous dense bands. The clefts of lymphatic endothelial intercellular junctions were extensively labeled especially along regions where cells were loosely apposed. The existence of a high density of anionic sites on membranes at the intercellular junctions of both mesothelial and lymphatic endothelial cells represent a salient feature which is very different from binding in blood capillary endothelium. The presence of a high density of anionic sites along the intercellular clefts of adjacent cells may play a role in the rapid movement of small solutes and molecules from interstitial spaces into the lymphatic lumen.

Animals↗
PubMed1984

Effect of concanavalin A on lymph node macrophages: stimulation of endocytic cisternae.

Incubation of isolated lymph node macrophages with concanavalin A (Con A) resulted in a dense and continuous labeling of the plasmalemma and filopodia which were closely adherent to each other and the cell surface. Within a short time period (3-5 min), membranes of the closely apposed filopodia became invaginated into the cytoplasm to form numerous interconnecting cisternae. After 10 min the system of internalized membranes had migrated into the deeper cytoplasm and was closely associated with numerous actin filaments and other components of the cytoskeleton. The internalized plasmalemma remained in the cytoplasm up to 24 hr without fusing with lysosomes. Concomitant with plasmalemmal invagination and the formation of cisternae there were also changes in the Golgi apparatus. These appeared in the form of hypertrophied Golgi saccules and the accumulation of numerous vesicles around the Golgi. Treatment of isolated lymph node macrophages with either succinylated Con A, alpha-methyl-D-mannoside, or ferritin particles alone failed to produce the cisternal structures. The results suggested that the tetravalency of Con A may be responsible for the binding of adjacent Con A-labeled membranes to each other and for maintaining a crosslinking of membranes during invagination and internalization. It is suggested that this process of extensive membrane internalization represents a specialized form of endocytosis. At 24 hr after incubation with Con A, cisternal structures in close proximity to the Golgi vesicles showed signs of degradation. By 48 hr there was a breakdown of cisternal membranes with a release of Con A marker particles into large phagocytic vesicles, which also showed reaction product for acid phosphatase, suggesting a fusion with lysosomes.

Animals↗
PubMed1983

Interaction of mesothelium to intraperitoneal stimulation. I. Aggregation of peritoneal cells.

The effect of intraperitoneal injections of bacterial toxin and adjuvant on the diaphragmatic mesothelium and their interaction with peritoneal cells was investigated in mice. At 30 minutes to 8 hours after stimulation, large numbers of neutrophils were seen on the mesothelial surface. Many of these cells exhibited features characteristic of locomotion over the mesothelial surface, whereas others appeared to be in the process of entering and passing through stomata into lymphatic vessels. By 24 hours numerous neutrophils, macrophages, and a small number of lymphocytes formed cellular aggregates that were surrounded by fibrin filaments. At 48 hours, the peritoneal cells were more closely aggregated and formed several layers on the mesothelial surface. By 72 hours fibrin filaments appeared to be broken down in many areas, with a resultant electron-dense precipitate occupying large areas of the intercellular spaces and on the surfaces of cells. It is suggested that the fibrin provides a matrix for the adhesion and subsequent aggregation of peritoneal cells to the mesothelial surface. The separation of neighboring mesothelial cells which surrounded stomata caused a widening of mesothelial pores (stomata), thereby facilitating the egress of the increased fluid and cellular infiltrations caused by the stimulation. The presence of patent stomata underlying the cellular aggregate demonstrates the importance of the diaphragmatic stomata as a major passageway for the removal of fluids and cells in the unstimulated, as well as during the inflammatory, response.

Animals↗
PubMed1982

Concanavalin A receptor sites on lymph node cells in vivo and in vitro.

The distribution and density of receptors for concanavalin A (Con A) on the surfaces of cells of intact and isolated popliteal and axillary lymph nodes were investigated in the rabbit. Intact lymph nodes were perfused via the subcapsular (marginal) sinus with either Con A peroxidase or Con A ferritin, fixed with glutaraldehyde, and processed for electron microscopy. Both Con A peroxidase and Con A ferritin were distributed on the plasmalemma of lymphocytes, macrophages, neutrophils, plasma cells, reticular endothelial cells, and the vascular endothelium. Counts of Con A-conjugated ferritin particles indicated that the density of Con A receptors was generally similar for lymphocytes, macrophages, and neutrophils but lower on plasma cells. When lymph node cells were isolated by mechanical methods and exposed to Con A ferritin, the label was homogenously distributed on the cell surfaces of most cells. However, Con A binding was significantly higher on the surface of isolated cells than in the intact node. It is suggested that the increase in density of Con A binding sites on isolated cells may possibly be due to an unmasking of cell surface moieties in which additional Con A receptor sites become available as a result of the isolation procedure. The density of Con A ferritin binding sites was also significantly lower on the surface of isolated plasma cells than the lymphocyte and macrophage, suggesting that the density distribution of cell surface saccharides is different for various lymphoid cells.

Animals↗
PubMed1980

Lymphatic removal of fluids and particles in the mammalian lung.

The structure and distribution of pulmonary lymphatics and their permeability to fluids and particulate materials have been investigated in the lungs of rats following fixation by combined intratracheal and vascular perfusion. In such preparations, the lymphatics remain in a distended state, and a close relationship to other structural components of the pulmonary interstitium is maintained. They were identified in regions with an abundant amount of connective tissue, forming an eleborate plexus within the pleura, the interlobular septum, peribronchial and perivascular areas. Recent data have shown that water-soluble molecules and particulate matter are removed from the interstitium along the lymphatic capillary (initial lymphatics) segment. It is distinguished by attenuated endothelial cells with extensively overlapping cell margins which are easily separated. We have studied this segment of the lymphatic vascular system following intratracheal injections of colloidal particles (ferritin and carbon) to determine the structural features responsible for the transport of large molecules and particulate materials across the lymphatic endothelial wall in the lung. The results showed that the tracer particles cross the lymphatic endothelial wall via the clefts of intercellular junctions. While the tracer particles were observed within vesicles, the question of transport across the lymphatic endothelium via plasmalemmal vesicles is still not settled since the number and size of vesicles containing tracer particles also increased with time. Intravascular injected dextran was also localized within the clefts of intercellular junctions and plasmalemmal vesicles. The results obtained with intratracheal and intravascular injected tracer substances are consistent with those observed in lymphatic capillaries for other tissues.

Animals↗