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Biomedical subjects

M H Gee

Publications and source records attributed to M H Gee.

At least 37 records · Page 2Linked to original sources
PubMed1990

Calcium is required for PMA induced superoxide release from human neutrophils.

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.

Calcium↗
PubMed1990

Physiology of aging related to outcome in the adult respiratory distress syndrome.

Thirty-nine patients with adult respiratory distress syndrome (ARDS) were enrolled in a study to identify potential age-related changes in organ system function that may help explain the apparent association between age and poor outcome in these patients. Criteria for enrollment included an arterial PO2-to-inspired O2 concentration ratio less than or equal to 200 in a clinical setting consistent with ARDS. Patients were excluded if they were less than 18 yr old, had clinical manifestations of congestive heart failure, were seropositive for the human immunodeficiency virus, or had stage II metastatic lung cancer. Patients were divided into two groups: those less than 60 yr old (mean 42 +/- 3 yr, n = 17) and those greater than or equal to 60 yr old (73 +/- 2 yr, n = 16). A group of six patients was analyzed as a separate subset based on a body temperature less than or equal to 97.5 degrees F at enrollment (hypothermic patients, 73 +/- 4 yr old). Sepsis was present in 67% of the nonhypothermic patients and in all the hypothermic patients. Mortality rates were 12% in the patients less than 60 yr and 69% in the nonhypothermic patients greater than or equal to 60 yr. All the hypothermic patients died. Sequential data obtained over 6 days were compared within and between groups. The following results were obtained. 1) The ratio of arterial PO2 to inspired O2 fraction was greater and the positive end-expiratory pressure used was significantly less in the patients greater than or equal to 60 yr old compared with the younger group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia.

In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion.

Alkaline Phosphatase↗
PubMed1989

Zymosan-activated plasma causes prolonged decreases in PMN superoxide release in sheep.

It is well established that activation of neutrophils within the pulmonary circulation produces acute lung injury in which adherence of neutrophils to endothelial cells is an obligatory step in the mechanism of injury. The effects of in vivo activation of neutrophils on the in vitro responses of these cells to stimulation have not been determined, although such information may be important in understanding how different etiological factors may interact to produce infection or acute respiratory failure. By using an assay to sequentially measure superoxide anion (O2-) release from adherent neutrophils stimulated with phorbol myristate acetate (PMA), we measured the in vitro activation response of peripheral blood neutrophils isolated before and 24 h after infusion of zymosan-activated plasma (ZAP; or untreated plasma as a control), air bubbles, or PMA in awake, instrumented sheep. Each of the three inflammatory agents produced an increase in lung microvascular permeability characteristic of acute lung injury; control plasma did not. For the in vivo ZAP experiments, stimulated O2- release in vitro by using PMA was approximately 50% lower (P less than 0.05) for neutrophils isolated 24 h after the in vivo infusion (4.3 +/- 0.8 nmol/500,000 cells) than before (8.1 +/- 0.2 nmol/500,000 cells). For the air emboli or PMA in vivo experiments, there were no changes in neutrophil activation responses in vitro. Similarly, infusion of control plasma did not result in reduced neutrophil O2- release. These results show that alterations in the inflammatory potential of neutrophils may occur in vivo and that such alterations appear to be dependent on the mechanism and agent by which lung injury is produced.

Animals↗
PubMed1989

Prolonged exposure of neutrophils to saline or bronchoalveolar lavage fluid does not alter superoxide anion generation.

The role played by neutrophils (PMNs) in the genesis of lung injury in diverse clinical situations, such as bronchial asthma, idiopathic pulmonary fibrosis, and the adult respiratory distress syndrome, is an area of intensive investigation. Functional studies of PMNs, particularly those obtained from the alveoli by bronchoalveolar lavage, should shed light on their contribution to lung injury. However, it has not been demonstrated whether procedures used to harvest cells from the lung (bronchoalveolar lavage), particularly the potentially prolonged exposure to saline, commonly used to perform lavage, and other components of lavage fluid, can alter the functional characteristics of PMNs. In this report we demonstrate that a 2- to 3-hour exposure of neutrophils to saline from both humans and sheep in vitro does not alter the functional characteristics of PMNs as determined by superoxide anion generation after activation with phorbol myristate acetate (PMA; 6.96 +/- 0.44 vs. 7.60 +/- 0.32 nmol O2-/250,000 PMNs for control and saline-treated human cells, respectively, after a 45-min incubation with 10(-7) M PMA, and 4.73 +/- 0.30 vs. 4.50 +/- 0.42 nmol O2-/250,000 PMNs for control and saline-treated sheep cells). In a second series of experiments, we studied the effect of exposure of human PMNs to bronchoalveolar lavage fluid supernatants obtained from normal volunteers on superoxide anion generation by neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Biochemical analysis of the activation of adherent neutrophils in vitro.

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.

Animals↗
PubMed1988

Morphological analysis of the activation of adherent neutrophils in vitro.

One approach to study the inflammatory potential of neutrophils involves in vitro methods, using either adherent or suspended cells. In order to do structure-function studies, traditional methods require that experiments be done in parallel: one experiment for structure and another for function. In this report new morphological methods for coordinated structure-function studies on the same cells are described. Isolation and biochemical analysis of sheep and human adherent neutrophils were done as described in the companion paper (Cerasoli et al., 1988). Then, at designated time points, the adherent cells were fixed and processed in the microtiter wells for high-resolution light microscopy and transmission electron microscopy. The principal obstacle to the morphological studies was chemical etching of the microtiter wells by the processing solutions and embedding media. Insertion of No. 3 BEEM capsule 'sleeves' (the cap and conical tip were removed) into the wells before processing eliminated the obstacle and provided standard-sized, polymerized blocks for microtomes. Adherent neutrophils activated in vitro with 10(-7) M phorbol myristate acetate developed prominent cytoplasmic vacuoles. Furthermore, the activated cells assumed irregular shapes and cytoplasmic processes. These changes in adherent cell morphology in vitro are similar to those seen in neutrophils which have been activated and fixed in vivo. Thus, the in vitro approach we devised retains the morphologic characteristics of cells in vivo and provides an efficient method to do integrated structure-function studies. Using these techniques, we have studied alveolar neutrophils obtained from a patient with acute respiratory failure. These cells contained conspicuous cytoplasmic vacuoles, few granules, and their border was ruffled. The same morphologic changes observed after activation of peripheral blood neutrophils with phorbol myristate acetate in vitro were seen in the alveolar neutrophils obtained from this patient. Therefore, these studies reveal that similar morphologic changes are seen in neutrophils stimulated in vitro as well as cells which have been activated pathophysiologically in vivo.

Animals↗
PubMed1987

Prostaglandin E1 prevents increased lung microvascular permeability during intravascular complement activation in sheep.

Prostaglandin E1 (PGE1) inhibits a variety of functions of activated neutrophils including respiratory burst, release of leukotriene B4, and adherence to endothelial cells. To determine if PGE, alters the pathophysiology of complement-induced lung vascular injury, experiments were conducted in anesthetized sheep with lung lymph fistulas given a 1-hour infusion of zymosan-activated plasma. PGE1 (30 ng/min/kg) or its saline vehicle was infused intravenously for 90 minutes beginning 30 minutes before the infusion of activated plasma. PGE1 had no effect on leukocyte count, the initial hypoxemia and thromboxane A2 release, or the development of acute pulmonary hypertension. However, PGE1 prevented steady-state increases in lung lymph flow that in vehicle-treated sheep signaled an increase in lung microvascular permeability. Furthermore, extraction of PGE1 by pulmonary endothelial cells was unaffected by the infusion of activated plasma. We propose that PGE1 prevented the increase in lung vascular permeability by inhibiting adherence of activated neutrophils to endothelial cells.

Alprostadil↗
PubMed1987

Resolution of increased permeability pulmonary edema in rats.

The rate and sequence of interstitial and alveolar fluid removal from the lung after the occurrence of pulmonary edema were examined. Rats were given intraperitoneal injections of 20 mg/kg alpha-naphthylthiourea (ANTU), resulting in an increased permeability edema with alveolar flooding. Animals were killed at intervals between 2 and 48 hours after ANTU for the gravimetric determination of extravascular lung water (Qwl/dQl) and histologic study of the lung. Interstitial fluid volume was quantified by a morphometric technique. The assumptions were made that edema fluid equaled the experimental Qwl/dQl minus the normal Qwl/dQl, and that the edema fluid volume equaled the sum of interstitial and alveolar fluid volume. It was found that between 2 and 4 hours after the induction of pulmonary edema, fluid was removed from the alveolar space faster than it was removed from the interstitial space. Between 4 and 48 hours after ANTU, the fluid removal rate from both compartments was much slower, and interstitial fluid was removed at a faster rate than alveolar fluid. It is hypothesized that the later phase of fluid removal from the lung is dependent on the removal of protein.

Animals↗
PubMed1986

The effects of cyclooxygenase inhibition on chemiluminescence and aggregation in sheep neutrophils.

Antiinflammatory actions of cyclooxygenase inhibitors may be related to inhibition of the synthesis and release of prostaglandins and thromboxane or to nonspecific actions of particular drugs. The role of cyclooxygenase products of arachidonic acid was studied in two leukocyte functions, free radical release and aggregation, after complement activation. Dose response curves were constructed after treatment with meclofenamate or ibuprofen. To differentiate between effects on free radical release from complement activated neutrophils and scavenging free radicals, additional experiments were made with a cell free system to generate free radicals. Both drugs inhibited complement initiated neutrophil chemiluminescence in a dose dependent manner. Meclofenamate acted primarily as a scavenger while ibuprofen inhibited free radical release. Neither drug had any inhibitory effects on complement induced leukocyte aggregation.

Animals↗
PubMed1986

Thromboxane as a mediator of pulmonary dysfunction during intravascular complement activation in sheep.

Intravascular complement activation results in thromboxane (TxA2) production, pulmonary hypertension, hypoxemia, and increased lung vascular permeability. The purpose of this study was to determine the role of TxA2 as a mediator of these responses. Experiments were made in anesthetized sheep subjected to intravenous injections of zymosan-activated plasma (ZAP) every 30 min for 4 h. Sheep were pretreated with dazoxiben, a TxA2 synthetase inhibitor, or SK and F 88046, a TxA2 end-organ antagonist, and the results were compared with those from untreated sheep. Dazoxiben, but not SK and F 88046, inhibited TxA2 release. The hypertensive response averaged 74 +/- 3 cm H2O after each injection of ZAP in untreated sheep. Neither drug altered this response. Pao2 decreased an average of 20 +/- 1 mmHg in untreated sheep, 3 +/- 1 mmHg in dazoxiben-treated sheep, and 11 +/- 1 mmHg in SK and F 88046-treated sheep. Increases in lung lymph flow and lymph protein clearance were unaffected by treatment. TxA2 appears to be an important mediator of hypoxemia during intravascular complement activation.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Dialysis-induced hypoxemia: membrane dependent and membrane independent causes.

Hypoxemia during hemodialysis may result from several differing processes. We initially studied patients undergoing standard acetate hemodialysis. At 15 minutes of dialysis, leukopenia (primarily neutropenia), a decline of platelet count, and hypoxemia occurred, but without a significant change in mean minute ventilation. Complement activation (V/A ratios of C5a greater than 1.0) persisted throughout dialysis. Leukocyte count returned to baseline by one hour. To separate the effects of solute and/or gas fluxes from those of blood-membrane interaction we studied changes in Po2, WBC, C5a, TxB2, and PGI2 during a period of blood membrane interaction without dialysis, and during subsequent acetate dialysis. Patients were studied with both polyacrylonitrile (PAN) and cuprophan membranes containing different priming solutions during membrane contact alone. Despite leukopenia and complement activation, hypoxemia failed to occur during membrane contact alone. At 15 minutes of subsequent acetate dialysis, significant hypoxemia occurred with both membranes. However, the degree of hypoxemia was twice as great with a cuprophan membrane primed with acetate (18.6 +/- 3.3 mm Hg) compared with air or bicarbonate (9.1 +/- 1.4 and 7.0 +/- 2.0 mm Hg, respectively), or compared with PAN (8 +/- 2.8 mm Hg). Changes in thromboxane B2, PGI2, and C5a did not correlate with changes in Po2. We conclude that there are two major components to dialysis related hypoxemia. One is membrane independent, and may relate to the metabolic effects of acetate or to dialyzer CO2 loss. The remaining portion is membrane dependent, occurring with cuprophan, but not with PAN, and is conditioned by an acetate dependent interaction between blood and membrane.

Adolescent↗
PubMed1985

Arachidonate cyclooxygenase metabolites as mediators of complement-initiated lung injury.

The pulmonary effects of intravascular complement activation can be divided into those that are initial events of activation and those that depend on the duration of intravascular activation. Pulmonary leukostasis is both an initial event and one that persists as long as intravascular complement activation is ongoing. This event is independent of synthesis and release of cyclooxygenase products of arachidonic acid. Initial events include synthesis and release of thromboxane and development of pulmonary hypertension and hypoxemia. Cyclooxygenase products are mediators of these initial pathophysiological events in part or in total. Complement-initiated increases in lung microvascular permeability are dependent on the duration of intravascular complement activation. Activation must be either prolonged or repeated to produce significant microvascular injury. This process is unrelated to synthesis and release of cyclooxygenase products, but is related to release of toxic oxygen metabolites.

Arachidonic Acids↗
PubMed1984

Aggregation and thromboxane synthesis and release in isolated sheep neutrophils and lymphocytes in response to complement stimulation.

In an attempt to determine possible cellular sources of thromboxane synthesis and release during intravascular complement activation, we prepared pure, viable fractions of neutrophils and lymphocytes from sheep blood. Lymphocytes were also isolated from thoracic duct lymph. Blood neutrophils and lymphocytes release thromboxane and the cells aggregate when challenged with zymosan-activated plasma. Blood lymphocytes release thromboxane in greater quantity than do neutrophils with the complement stimulus. Lymph lymphocytes, when stimulated with zymosan-activated plasma, did not release thromboxane or aggregate. These data suggest that both blood neutrophils and lymphocytes are likely sources of complement-initiated thromboxane synthesis and release in sheep.

Animals↗
PubMed1984

Role of interstitium in clearance of alveolar fluid in normal and injured lungs.

We investigated the contribution of the pulmonary interstitial space to the removal of alveolar fluid and solute. We prepared anesthetized sheep for the collection of lung lymph. A balloon-tipped catheter was advanced into a lower lung lobe, and 20 ml Ringer lactate solution (RL) were instilled in one group. Other groups received 20 ml RL with 4 mg/ml Evans blue dye (EB) or 10 micrograms/kg phorbol myristate acetate (PMA) or both. Instillation of 20 ml RL and EB resulted in an increase in lymph flow over RL alone, presumably by an osmotic mechanism. After 4 h, small perivascular fluid cuffs, which contained little EB, were present, and 1.9% of the instilled EB was removed by the lymphatics. An average of 9.2 ml of excess water remained in the lung. Instillation of RL, EB, and PMA resulted in an increase in lymph flow and large perivascular fluid cuffs, which contained large amounts of EB. Lymphatic removal of the instilled EB accounted for 1.2% of the total amount instilled. An average of 19.1 ml water was present in the lung after 4 h. We conclude that alveolar instillation of PMA results in epithelial and endothelial membrane injury and that when lung injury is present interstitial fluid reservoirs may be important sites of alveolar fluid accumulation and important routes of fluid removal from the air space.

Absorption↗