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Biomedical subjects

R A Clark

Publications and source records attributed to R A Clark.

At least 217 records · Page 12Linked to original sources
PubMed1990

Comparison of the efficacy and systemic effects of 4 mg and 8 mg formulations of salbutamol controlled release in patients with asthma.

The purpose of the present study was to compare the efficacy and systemic effects of 4 mg and 8 mg doses of salbutamol controlled release (SCR) after single dosing and at steady state in patients with asthma. Fifteen asthmatic patients (Age 36 y, FEV1 85% predicted) were given SCR 4 mg and 8 mg twice daily for 7 days in a randomised double-blind cross-over design, with at least 7 days washout between treatments. There were no differences between the bronchodilator effects of 4 mg and 8 mg doses. There was no evidence of tolerance to the bronchodilator effects after chronic dosing. Morning and evening PEFR measurements also showed improvements during treatment with SCR 4 mg and SCR 8 mg, although there were no differences between the two formulations. Both doses of SCR caused significant objective tremor responses which were maintained after chronic dosing. The 8 mg dose produced a larger tremor response after single dosing, but not at steady-state. Subjective tremor occurred in 7 patients with SCR 8 mg, and in 2 patients with SCR 4 mg. There were no cardiac arrhythmias on Holter ECG monitoring. These results suggest that the 8 mg dose of SCR was no more effective than the 4 mg formulation, and was associated with more systemic adverse effects.

Adult↗
PubMed1990

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction.

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.

Actins↗
PubMed1990

Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA.

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.

Cell Adhesion↗
PubMed1990

Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro.

Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

Cells, Cultured↗
PubMed1990

Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin.

During cutaneous tissue organization, numerous critical interactions occur between cells and the extracellular matrix (ECM). Cell-matrix interactions depend on the presence of ECM receptors. Many ECM receptors, known as integrins, are heterodimeric glycoproteins consisting of one alpha and one beta chain. Integrins containing beta 1 or beta 3 chains are ECM receptors, whereas those containing beta 2 chains are leukocyte cell-cell receptors. We have used porcine cutaneous wounds as a paradigm for tissue organization and probed healing wounds and adjacent normal skin with polyclonal antibodies to fibronectin and fibronectin (alpha 5 beta 1) receptor. During re-epithelialization, the epidermis transits over a provisional matrix containing fibronectin. Migrating epidermal cells expressed fibronectin receptors in a bright linear peripheral pattern. At 10 days, when reepithelialization was complete and the basement membrane was re-established, the fibronectin matrix was markedly reduced and fibronectin-receptor expression was limited to the basolateral aspect of basal cells, as observed in normal epidermis. Beneath the migrating epidermis in 5-d wounds, granulation tissue had filled 80% of the wound space. Day-5 wound fibroblasts did not express fibronectin nor other beta 1 integrin receptors, were randomly oriented, and contained no actin bundles. Fibronectin fibrils were assembled on the surfaces of day-5 wound fibroblasts but formed few linkages between cells. Day-7 wound fibroblasts expressed fibronectin receptors, contained peripheral cytoplasmic actin bundles consistent with a contractile fibroblast phenotype, and were coaligned across the wound in parallel array with interconnecting fibronectin fibrils. The wounds contracted between 7 and 10 days. Thus the migrating epidermis consistently expressed fibronectin receptors. Fibronectin receptors were expressed by fibroblasts just prior to wound contraction.

Animals↗
PubMed1990

Optimization of gradient-echo imaging parameters for intracaval filters and trapped thromboemboli.

Flow-phantom magnetic resonance (MR) gradient-echo (GRE) imaging at 1.5 T was performed on a titanium Greenfield filter containing trapped blood clots with a high concentration of either deoxyhemoglobin (DHb) or methemoglobin (MHb), simulating acute and older thromboemboli, respectively. Flip angle, repetition time (TR), and echo time (TE) were varied, and a contrast-to-noise ratio between trapped clots and flowing fluid (clot-flow contrast) was determined for each set of imaging parameters. Use of very low flip angles (less than or equal to 10 degrees) rendered MHb clots indistinguishable from flowing fluid. In general, DHb clots displayed greater clot-flow contrast than MHb clots regardless of flip angle. With increasing TE values, T2* effect was observed with MHb clots, and magnetic susceptibility artifacts increased. Overall, optimum clot-flow contrast for imaging of both DHb and MHb clots was achieved with a flip angle of 45 degrees-60 degrees, a TR of 50 msec, and the shortest TE possible. Using GRE parameters similar to the optimum parameters determined in vitro, the authors imaged four patients with nickel-titanium Simon filters and one dog with a titanium Greenfield filter. MR imaging was successful in demonstrating filter location, caval patency, and the presence and extent of intraluminal thrombus.

Adult↗
PubMed1990

Acute hematomas: effects of deoxygenation, hematocrit, and fibrin-clot formation and retraction on T2 shortening.

Acute hematomas can appear hypointense on T2-weighted magnetic resonance (MR) images at field strengths as low as 0.35 T. Using Raman spectroscopy to measure blood oxygenation and taking T2 measurements at 2.1 and 9.4 T, the authors examined the relaxation mechanisms acting during deoxygenation, increases in hematocrit, and fibrin-clot formation and retraction. Individual contributions to overall T2 from deoxyhemoglobin and the interactions of water with protein hydration layers in hemoglobin, plasma proteins, and fibrin were measured. Overall T2 values estimated by summing individual relaxation rates were in reasonable agreement with the T2 values of clotted blood. Results suggest that deoxygenation may be most important in T2 shortening, followed by increased hematocrit. T2 shortening from fibrin polymerization was minimal at the field strengths used. Effects of deoxygenation and increasing hematocrit are more sensitive to field strength than fibrin T2 shortening. Effects of fibrin may be more significant at middle and low field strengths.

Acute Disease↗
PubMed1990

Role of microfilaments in maintenance of proximal tubule structural and functional integrity.

To determine the selective effect of microfilament disruption on both cellular structure and function, microfilament-specific doses of cytochalasin D (10 microM) were used in an isolated perfused kidney system. Structurally, cytochalasin D resulted in extensive disruption of the apical surface with blebbing, vacuolization, and patchy loss and fusion of microvilli. Functionally, cytochalasin D resulted in an initial decrease in glomerular filtration rate (300.8 +/- 29.9 vs. 541.6 +/- 51 microliters.min-1.g-1, P less than 0.05) with subsequent stabilization throughout the duration of the perfusion. In contrast, the tubular reabsorption of sodium decreased significantly in a linear fashion from 97.1 +/- 0.7 to 64.3 +/- 7.0% over the duration of the perfusion. Similarly, the tubular reabsorption of lithium decreased linearly from 74.8 +/- 2.6%, before the addition of cytochalasin, to 33.6 +/- 6.8% by the end of the perfusion. Correlation of the decrements in percent tubular reabsorption of sodium and lithium for individual kidneys was 0.87 (P less than 0.01), suggesting the effect of microfilament disruption on tubular reabsorption of sodium was localized primarily to the proximal tubule. Because ischemic injury is characterized by time-dependent structural alterations in the apical membrane of proximal tubule cells, we set out to determine whether microfilament disruption occurs during ischemic acute renal failure. Utilizing indirect immunofluorescence with an anti-actin antibody, control kidneys demonstrated intact circumferential apical immunofluorescence representing brush-border and terminal web actin staining. Fifteen minutes of ischemia resulted in multiple large gaps in the terminal web, and 50 min of ischemia caused diffuse redistribution of actin immunofluorescence throughout the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1990

Comparison of the effects of prolonged treatment with low and high doses of inhaled terbutaline on beta-adrenoceptor responsiveness in patients with chronic obstructive pulmonary disease.

Eleven patients with chronic obstructive pulmonary disease (age, 61 +/- 2 yr; FEV1, 1.36 +/- 0.24 L, 46 +/- 7% predicted) were given 4 wk of treatment with either a conventional low dose of inhaled terbutaline (LDT), 500 micrograms four times a day, or a high dose of inhaled terbutaline (HDT), 2,000 micrograms four times a day, delivered by a spacer. A randomized double-blind crossover design was used with 2-wk run-in and washout periods, when ipratropium bromide was substituted for inhaled beta-agonists. Dose response curves (DRC) to cumulative doubling doses of inhaled terbutaline (125 to 4,000 micrograms) were constructed after each treatment period, and baseline spirometry, finger tremor (Tr), plasma potassium (K), plasma cAMP, and ECG (HR and T wave) were measured at each dose step of the DRC. Daily PEFR measurements (A.M. and P.M.) and Holter ECG were performed during run-in and treatment periods. Baseline values for FEV1 were not significantly different during run-in, treatment, or washout periods. There were dose-related increases in FEV1 (p less than 0.0001) with no significant differences between DRC after treatment with HDT compared with those with LDT: delta FEV1 max, 0.46 +/- 0.14 L, 15.5 +/- 3.7% predicted (HDT); 0.50 +/- 0.11 L, 16.0 +/- 3.1% predicted (LDT). There were also no differences between DRC for delta FVC: 1.08 +/- 0.22 L, 31.1 +/- 5.4% predicted (HDT); 0.99 +/- 0.14 L, 28.5 +/- 3.8% predicted (LDT). There were no significant changes in K or HR in response to cumulative doses of terbutaline after either treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1990

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

The superoxide-forming respiratory burst oxidase of human neutrophils is composed of membrane-associated catalytic components and cytosolic constituents required for oxidase activation. This study concerns the hypothesis that cytosolic oxidase components translocate to a membrane fraction when neutrophils are stimulated and the oxidase is activated. A polyclonal antiserum that recognizes two discrete cytosolic oxidase components of 47 and 67 kD was used to probe transfer blots of electrophoresed membrane and cytosol fractions of resting and stimulated neutrophils. In contrast to their strictly cytosolic localization in unstimulated cells, both proteins were detected in membrane fractions of neutrophils activated by phorbol esters and other stimuli. This translocation event was a function of stimulus concentration as well as time and temperature of exposure to the stimulus. It was inhibited by concentrations of N-ethylmaleimide that blocked superoxide formation but was unaffected by 2-deoxyglucose. There was a correlation between translocation of the cytosolic proteins and activation of the oxidase as determined by superoxide formation. Quantitative analyses suggested that approximately 10% of total cellular p47 and p67 became membrane-associated during phorbol ester activation of the oxidase. Analysis of Percoll density gradient fractions indicated that the target membrane for translocation of both proteins was the plasma membrane rather than membranes of either specific or azurophilic granules. In the cell-free oxidase system arachidonate-dependent but membrane-independent precipitation of the cytosolic oxidase proteins was demonstrated. The data show that activation of the respiratory burst oxidase in stimulated human neutrophils is closely associated with translocation of the 47- and 67-kD cytosolic oxidase components to the plasma membrane. We suggest that this translocation event is important in oxidase activation.

Biological Transport↗
PubMed1990

Fibronectin fragments containing the RGDS cell-binding domain mediate monocyte migration into the rabbit lung. A potential mechanism for C5 fragment-induced monocyte lung accumulation.

Many inflammatory processes are characterized by an early phase of neutrophil migration and a later phase of monocyte migration into the inflammatory site. Mechanisms that govern the transition between phases are the subject of these investigations. Acute lung inflammation induced by C5 fragments in the rabbit leads to an initial neutrophil influx and plasma leakage into the alveolar space, followed by monocyte influx that we have previously shown to be dependent on prior emigration of neutrophils. Neutrophil enzymes are known to cleave intact fibronectin into fragments that are monocyte chemotaxins in vitro. Accordingly, generation of appropriate fibronectin fragments in situ by proteolytic enzymes from infiltrating neutrophils might represent a potential mechanism for attraction of monocytes into the lung. The studies reported herein demonstrate that a 120-kD fragment of fibronectin containing the RGDS fibroblast cell-binding domain induced monocyte migration into the rabbit lung in vivo. Intact fibronectin was inactive. A significant proportion of the monocyte migration was neutrophil independent. Intact fibronectin was present in bronchoalveolar lavage fluid from C5 fragment-treated animals rendered neutropenic, but absent in lavage from normal C5 fragment-treated animals. Fibronectin fragments were present in bronchoalveolar lavage fluid from both C5 fragment-treated and control rabbits. In addition, the amount of fibronectin was significantly increased in lavage of C5 fragment-treated normal but not neutropenic animals. Monoclonal antibodies directed against an epitope of fibronectin containing the RGDS cell-binding domain significantly inhibited the C5 fragment-induced monocyte migration, but not neutrophil migration. These studies suggest that chemotactic fibronectin fragments may in part be responsible for the recruitment of monocytes into areas of acute lung inflammation.

Animals↗
PubMed1990

Total body irradiation as an alternative to systemic chemotherapy in small-cell anaplastic lung cancer.

The effectiveness of total body irradiation (TBI) plus local radiotherapy in the treatment of small-cell lung cancer was studied in 13 patients, using 4,000 cGy in 15 fractions over three weeks to the local site and 150 cGy in ten fractions over two weeks to the whole body. The mean survival for 12 patients was 31 weeks, with a median survival of 32 weeks. One patient received six courses of combination chemotherapy for recurrent disease four months after TBI without marrow depression and survived 72 weeks, the longest survivor in this series. Brain metastases occurred in only one patient, the most common site of metastases being the liver. All patients tolerated TBI well without nausea, vomiting or hair loss. When bone marrow suppression occurred it was asymptomatic, requiring no treatment and resolving within eight weeks.

Aged↗
PubMed1990

Cryptococcus neoformans pulmonary infection in HIV-1-infected patients.

Cryptococcus neoformans (Cn) is a frequent pathogen in patients infected with the human immunodeficiency virus (HIV-1). We review the initial presentation and clinical course of 18 HIV-1-infected (HIV+) patients with a Cn pulmonary infection. Simultaneous positive cerebrospinal fluid (CSF) cultures were found in 10 (63%) of 16 examined. The most frequent presenting symptoms were fever (87%) and pulmonary complaints (60%). Although the most common chest radiographic finding was bilateral diffuse interstitial infiltrates, nodules and cavitary lesions were also seen. Nine (50%) of the 18 patients died within 6 weeks of diagnosis. Of six patients with an isolated Cn pulmonary infection, five have subsequently died. Three of these five patients did not receive maintenance therapy and had confirmed or probable relapse. Patients initially presenting with an isolated Cn pulmonary infection may later show disseminated disease, suggesting that such patients should receive both acute and maintenance therapy.

Adult↗
PubMed1989

Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils.

Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.

Actin Cytoskeleton↗
PubMed1989

Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system.

Chronic granulomatous disease, a syndrome of recurrent infections and failure of oxidative microbicidal activity in phagocytes, results from defects in the gene for one of several components of an oxidase system that can undergo activation. To determine the relative prevalence of certain of the genetic variants of this disorder, we used immunoblotting to detect two specific neutrophil cytosolic proteins of 47 and 67 kd recently shown to be required for oxidase activation. Chronic granulomatous disease is usually an X-linked disorder associated with the absence of membrane cytochrome b558. Of our 94 patients with chronic granulomatous disease, however, 36 had a phenotype characterized by autosomal inheritance, normal membrane oxidase components (including cytochrome b558), and functionally defective cytosolic activity in a cell-free oxidase system. We studied 25 of these 36 patients and found that 22 lacked the 47-kd cytosolic protein, and the remaining 3 were missing the 67-kd component. Patients with chronic granulomatous disease whose functional defect was localized to the neutrophil membrane (classic X-linked cytochrome b-negative type and two other rare variants) had normal amounts of both cytosolic components. We estimate that approximately 33 percent of all patients with chronic granulomatous disease are missing the 47-kd cytosolic oxidase component and about 5 percent of patients are missing the 67-kd component. Chronic granulomatous disease caused by a defect in any cytosolic factors other than the 47-kd and 67-kd proteins, if it exists, is apparently rare.

Adolescent↗