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Biomedical subjects

R C Hunt

Publications and source records attributed to R C Hunt.

At least 37 records · Page 2Linked to original sources
PubMed1995

A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.

PURPOSE: A spontaneously arising, apparently transformed, cell line has been cloned from a primary culture of human retinal pigment epithelial (RPE) cells and has been subcultured more than 200 times. The similarities of these cells to human RPE cells in vivo have been determined. METHODS: The structure of the transformed cells has been determined by light and electron microscopy and by immunocytochemistry using antibodies that detect cytoskeletal and other proteins. The ability of the cell line to bind and phagocytose photoreceptor material has also been assessed by fluorescence and electron microscopy. The metabolism of all-trans-retinol has been investigated by incubation of the cells with 3H-all-trans-retinol and analysis of the metabolic products by high-performance liquid chromatography. RESULTS: The transformed cells possess an epithelial cobblestone morphology with intercellular junctional complexes containing N-cadherin. The cytoskeleton of these cells comprises cytokeratins that are characteristic of epithelial cells, together with actin, spectrin, and vimentin. The keratins expressed are those typical of RPE cells. The cells also express cellular retinaldehyde binding protein and retinol dehydrogenase activity but do not express retinoid isomerase or lecithin retinol acyl transferase activities. These cells also exhibit phagocytic activity. CONCLUSIONS: This cell line retains many of the metabolic and morphologic characteristics of RPE cells in vivo although there are some differences, particularly the loss of some enzymatic activities and cytoskeletal polarization. These cells should be useful in further studies of RPE cell metabolism and other functions.

Cell Line, Transformed↗
PubMed1994

Adverse effect of helicopter flight on the ability to palpate carotid pulses.

STUDY OBJECTIVE: To determine if the air medical helicopter environment compromises the ability to palpate carotid pulses. DESIGN: Using a carotid pulse model, flight nurses were tested for their ability to palpate the simulated carotid pulse at normal (120/80 mm Hg) and low (80/60 mm Hg) blood pressures on the ground and during helicopter flight. SETTING: Palpation tests were performed during flight in an MBB BO-105 twin-turbine engine, single-rotor air medical helicopter; control palpation tests were performed on the ground. TYPE OF PARTICIPANTS: Ten flight nurses. MEASUREMENTS AND MAIN RESULTS: Tracings of pulsatile pressure from the carotid pulse model verified its ability to simulate a wide range of arterial pressures. Analyses of variance for repeated measures, including polynomial contrasts, were performed to compare the number of correct detections of the presence or absence of pulse pressures in flight with the number of correct detections in the two control conditions for both carotid pulse pressures. The mean in-flight number of correct detections was lower than both the preflight and postflight control tests, which were themselves nearly equal, at each simulated carotid pulse pressure. The quadratic terms for both the 120/80 mm Hg trial (F1,14 = 9.28; P = .0087) and the 80/60 mm Hg trial (F1,18 = 5.69; P = .0283) were statistically significant. CONCLUSION: Factors associated with transport of patients in an MBB BO-105 helicopter impair the ability of flight nurses to detect carotid pulses in a simulated physiologic model.

Aircraft↗
PubMed1994

Effect of an in-flight helicopter environment on the performance of ALS interventions.

INTRODUCTION: Patients transported by air ambulance are often critically ill and, thus, have a relatively high likelihood of requiring advanced life support (ALS) measures during transport. Despite this fact, the ability of the air medical crew to perform ALS interventions efficiently while in flight has not been investigated. SETTING: The study was conducted with BO 105 and BK 117 helicopters and a ground transport unit at a university hospital-based air ambulance service. METHODS: Ten flight nurses were timed performing various ALS tasks while in flight in each aircraft and in the ground ambulance. Mean times for performance of each task in the aircraft and in the ground unit were analyzed using Dunnett's method for analysis of multiple means; alpha was set at 0.01. RESULTS: Times required to perform all ALS tasks were significantly increased in the BO 105 setting compared to the setting of the BK 117 or the ground ambulance (p < 0.01). Times for performance of interventions in the BK 117 did not differ significantly from corresponding times in the ground setting. CONCLUSION: It appears that the environment of the BO 105 may impose limitations on the timely performance of certain ALS interventions.

Air Ambulances↗
PubMed1994

Interhospital transfer of cardiac patients: does air transport make a difference?

INTRODUCTION: The purpose of this study was to compare the outcome of interhospital transported cardiac patients for whom bias in selecting transport mode was removed due to helicopter unavailability. METHODS: Patients with the diagnosis of unstable angina or myocardial infarction who underwent transport by ground only because helicopter transport was not available, were compared to patients transported by helicopter. Patients were matched by gender and referring hospital. Ninety-six patients were studied and both groups were comparable in age, diagnosis, Killip classification, treatment with thrombolytics and post-transport procedures. RESULTS: There were no statistically significant differences between the groups for mean ICU days (5.3 air vs. 3.5 ground) and mean hospital days (9.9 vs. 8.2, respectively). No differences were detected in the proportions of deaths within 72 hours of arriving at the receiving institution (1/48 air vs. 0/48 ground), but air transportation was associated with more total deaths (9/48 vs. 1/48, respectively). CONCLUSION: It appears that the interhospital transport of cardiac patients by air offers no outcome advantage over ground transport.

Air Ambulances↗
PubMed1994

Improved flight following through continuous quality improvement.

INTRODUCTION: Flight following is a key component of an air medical transport service's safety program. National standards require conscientious adherence to flight following. EastCare monitored its compliance with internal flight-following requirements for three years. SETTING: EastCare is a single-helicopter, hospital-based air medical transport service located in a rural region of the southeastern United States. It has a full-time staff of flight nurses, pilots and communications specialists. METHODS: A continuous quality improvement (CQI) process was initiated to delineate specific areas requiring improvement. These areas were discussed at the air medical service's monthly CQI meetings. RESULTS: In 1989, the communications specialists achieved flight following intervals of < or = 15 minutes in 98% of attempts. In 1990 and 1991, the frequency of flight-following intervals of < or = 15 minutes were 98.8% and 99.6%, respectively. The CQI process pointed out educational requirements, technological problems and other areas for improvement. CONCLUSION: The use of a structured CQI process for this service directly contributed to consistently strong compliance in the frequency of flight following.

Air Ambulances↗
PubMed1994

Interleukin-1-beta changes the expression of metalloproteinases in the vitreous humor and induces membrane formation in eyes containing preexisting retinal holes.

PURPOSE: Proliferative vitreoretinopathy occurs when cells migrate into the vitreous humor, where they proliferate and produce a membrane composed of extracellular matrix. Interleukin-1-beta (IL-1-beta) may be involved in these processes because it is chemotactic and mitogenic, and it stimulates metalloproteinase production. In the present study, the effects of intravitreally injected IL-1-beta on retinal membrane formation and the associated changes in metalloproteinase content of vitreous humor were examined. METHODS: Rabbit eyes were injected with IL-1-beta in a buffer, with or without the prior creation of retinal holes. Control eyes received the buffer alone or no injection, with or without retinal holes. Animals were examined by slit lamp biomicroscopy and indirect ophthalmoscopy for 1 month. Zymography was performed on a portion of vitreous humor to assess collagenase content, and the remaining tissue was subjected to histologic analysis. RESULTS: Intraocular IL-1-beta induced perilimbal vessel engorgement, keratic precipitates, synechiae, flare, lens deposits, optic disk hyperemia, and granulomatous formations that gradually subsided during the first week. Intravitreal injection of IL-1-beta in eyes with preexisting retinal holes additionally induced membrane formation. Zymographic analysis of vitreous humor from animals sacrificed 24 hours after IL-1-beta injection showed a 100-kd and a 65-kd gelatinase, whereas control vitreous humor contained predominantly a single gelatinase species of approximately 65 kd. Retinal holes did not affect IL-1-beta induction of the 100-kd gelatinase. CONCLUSIONS: IL-1-beta induces a 100-kd gelatinase in the vitreous humor and epiretinal membrane formation in eyes containing preexisting retinal holes. The presence of retinal holes and abnormal production of cytokines may lead to a cascade of events, including aberrant extracellular matrix remodeling, that result in proliferative diseases of the eye.

Animals↗
PubMed1994

Cytokines and serum cause alpha 2 beta 1 integrin-mediated contraction of collagen gels by cultured retinal pigment epithelial cells.

PURPOSE: Integrins, heterodimeric cell surface glycoproteins, are involved in cell-substratum and cell-cell interactions. The role of these molecules in cytokine-mediated contraction of extracellular matrix by retinal pigment epithelial cells has been investigated in a model system that may mimic epiretinal membrane contraction during retinal detachment in proliferative vitreoretinopathy. METHODS: Retinal pigment epithelial cells were cultured on three-dimensional collagen gels that they cause to contract. The involvement of new protein synthesis and C-kinase-mediated signal transduction were studied using specific inhibitors. Cell surface integrins synthesized by pigment epithelial cells were identified using immunofluorescence, and the same antibodies were used in gel contraction assays to identify the integrin species responsible for force transduction. RESULTS: Contraction of collagen type I gels was small without cytokines but was greatly enhanced in the presence of serum or IL1 plus TGF beta. It was dependent on new protein synthesis. Contraction induced by the combination of cytokines was dependent on active protein kinases, whereas that induced by serum was not because only the former was inhibited by staurosporine, a C-kinase inhibitor. Pigment epithelial cells were found to produce beta 1, alpha 2, and alpha 5 integrins, but only alpha 2 and beta 1 appeared to participate in the contraction process because only antibodies against these integrins inhibited contraction. CONCLUSIONS: Retinal pigment epithelial cells contract an extracellular matrix in vitro in a manner similar to epiretinal membrane contraction in vivo. This contraction is mediated via cell surface glycoproteins of the integrin family that bind directly to extracellular matrix molecules.

Cell Communication↗
PubMed1993

Role of oligosaccharides in the processing and function of human transferrin receptors. Effect of the loss of the three N-glycosyl oligosaccharides individually or together.

When the coding sequence for human transferrin receptors was expressed in a Chinese hamster ovary cell line lacking endogenous transferrin receptors, 86-kDa molecules containing three N-glycosidically linked oligosaccharides were synthesized. These rapidly dimerized to form 172-kDa molecules which increased in size to 190 kDa. After site-directed mutagenesis of all three N-glycosylation sites, 80-kDa receptors were synthesized and only a few dimers were formed. 84-kDa monomers were synthesized in the absence of the oligosaccharide attached to Asn727 or Asn317. Dimerization and maturation through the Golgi body of the Asn727 mutant receptors were much slower than the wild type whereas the Asn317 mutant receptors behaved more similarly to the wild type. Lack of the oligosaccharide at Asn251 gave rise to 73-kDa monomers because of proteolytic processing (Hoe, M. H., and Hunt, R. C. (1992) J. Biol. Chem. 267, 4916-4923), but a second mutation at a potential cleavage site allowed the formation of 84-kDa receptors. These also dimerized at a similar rate to wild type receptors. The three-site mutant receptors were degraded in the endoplasmic reticulum but all three 84-kDa single site mutant receptor species migrated to the cell surface. However, receptors lacking the oligosaccharide at Asn727 bound and internalized little transferrin as a result of reduced affinity.

Animals↗
PubMed1993

Transferrin is made and bound by photoreceptor cells.

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, control the access of blood-borne components such as diferric transferrin to the neural retina. It has recently been shown that RPE cells remove iron from diferric transferrin in a low pH compartment and subsequently release it in a low molecular weight form that can be chelated by apo-transferrin (Hunt and Davis: J. Cell Physiol. 152:102-110, 1992). It is now shown that photoreceptor cells can bind diferric transferrin to receptors on their inner segments. Moreover, polymerase chain reaction and in situ hybridization show that cells of the neural retina, particularly photoreceptors, make apo-transferrin.

Base Sequence↗
PubMed1993

Comparison of motor vehicle damage documentation in emergency medical services run reports compared with photographic documentation.

STUDY OBJECTIVE: To determine whether emergency medical services (EMS) run reports adequately document vehicle damage when compared with vehicle photographs by using a traffic accident scoring system. DESIGN: A prospective study consisting of three phases: photographing motor vehicle collisions and collecting their respective EMS run reports, traffic accident damage score development, and comparison of photographs to the run reports by emergency medical technicians using the traffic accident damage score. SETTING: Data were collected in North Carolina and Ohio from motor vehicle crashes to which nine different EMS squads responded during a three-year period. TYPE OF PARTICIPANTS: EMS squads ranged from basic to paramedic levels of training. MEASUREMENTS AND MAIN RESULTS: Three emergency medical technicians were unable to determine the area of vehicle damage in 48% and the severity of damage in 61% of the EMS run reports. In contrast, there were no instances in which all three emergency medical technicians were unable to determine both area and severity of damage from the photographs. CONCLUSION: Most EMS run reports do not document vehicle damage adequately.

Accidents, Traffic↗
PubMed1993

Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels.

PURPOSE: Because retinal pigment epithelial cells in epiretinal membranes remodel and contract their surrounding extracellular matrix, investigations were performed to determine if these cells can produce matrix metalloproteinases and contract collagen gels in vitro in the presence of serum or cytokines. METHODS: Cells were grown on collagen gels and their production of metalloproteinases was measured using zymography. RESULTS: Cells grown in a three-dimensional collagen gel culture system produce several latent metalloproteinases that are secreted into the gel and the surrounding medium. These include molecules of 49, 56, 66, and 100 kD. In addition, an enzyme that is probably the active form of the 66 kD enzyme is present. When interleukin 1 beta is added to the cultures, latent 49 kD and 100 kD gelatinase production is greatly stimulated and an active form of both enzymes is also observed in the medium. In contrast, transforming growth factor beta has no stimulatory effect. The cells contract the collagen gel but this is small without cytokines; however, contraction is greatly enhanced in the presence of serum or interleukin 1 beta plus transforming growth factor beta. Contraction is unlikely to be the result of metalloproteinase action on the underlying extracellular matrix because complete inhibition of these enzymes has little effect. CONCLUSIONS: These results show that cytokines can cause cultured retinal pigment epithelial cells to produce metalloproteinases that can, when activated, degrade most collagens and other structural molecules in extracellular matrix. In addition, they can stimulate the contraction of extracellular matrix constituents but there is not a simple causal relationship between matrix remodeling and contraction.

Adult↗
PubMed1992

Loss of one asparagine-linked oligosaccharide from human transferrin receptors results in specific cleavage and association with the endoplasmic reticulum.

The coding sequence for the human transferrin receptor gene has been mutated in order to abolish the attachment of the oligosaccharide closest to the transmembrane sequence. Expression of the mutant receptor in Chinese hamster ovary cells resulted, after analysis under nonreducing conditions, in an 85- and a 73-kDa receptor species. After reduction, the 85-kDa receptors were mostly converted to 73 kDa, although with short labeling periods some 85-kDa receptor remained suggesting that the mutated gene was capable of coding for the entire polypeptide which was then proteolytically processed. This was supported by in vitro translation of mRNA from either wild type or mutant cells which in both cases yielded an 80-kDa protein, the full size of the nonglycosylated protein. The solubility characteristics of the mutant receptor suggest that it contains the COOH-terminal extracellular domain but not the transmembrane sequence. This is supported by the endoglycosidase H sensitivity of the 73-kDa protein which is compatible with the retention of two of the original three high mannose oligosaccharides. The receptor that lacks one oligosaccharide is unable to form intermonomer disulfide bridges or to migrate to the cell surface and is located in the endoplasmic reticulum where it is further degraded after a lag period of about 30 min.

Animals↗
PubMed1992

Release of iron by human retinal pigment epithelial cells.

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, take up iron in association with transferrin by a typical receptor-mediated mechanism (Hunt et al., 1989. J. Cell Sci. 92:655-666). This iron is dissociated from transferrin in a low pH environment and uptake is sensitive to agents that inhibit endosomal acidification. The dissociated iron enters the cytoplasm as a low molecular weight (less than 10 kD) component and subsequently binds to ferritin. No evidence for recycling of iron in association with transferrin was found. Nevertheless, much of the iron that is taken up is recycled to the extracellular medium, primarily from the low molecular weight pool. This release of iron is not sensitive to inhibitors of energy production or of vesicular acidification but is increased up to a maximum of about 40% of the total 55Fe incorporated when cells are incubated with serum or the medium is changed. When a short loading time for 55Fe from 55Fe-transferrin is used (i.e., when the low molecular weight pool is proportionately larger), a much larger fraction of the cell-associated radiolabel is released than when longer loading times are used. The data suggest that a releasable intracellular iron pool is in equilibrium with the externalized material. The released iron may be separated into a high and a low molecular weight component. The former is similar on polyacrylamide gel electrophoresis to ferritin although it cannot be immune precipitated by anti-ferritin antibodies. The low molecular weight 55Fe which is heterogeneous in nature can be bound by external apo-transferrin and may represent a form that can be taken up by cells beyond the blood-retinal barrier.

Biological Transport↗
PubMed1991

Inability to assess breath sounds during air medical transport by helicopter.

This study assessed the capabilities of a traditional and an amplified stethoscope used by flight nurses to assess breath sound during air medical transport in an MBB BO-105 helicopter. We developed a normal breath sound model using a prerecorded tape of breath sounds interspersed with segments without breath sounds; the recorder had been placed in the chest wall of a resuscitation training manikin. Flight nurses completed control listening sessions in a quiet environment and experimental sessions during flight using a traditional stethoscope for half of the sessions and an amplified stethoscope for the remaining half. In the quiet environment, flight nurses accurately reported the presence or absence of breath sounds in 110 (92%) of 120 trials. During helicopter flight, none of the flight nurses heard breath sounds during any of the recorded segments with either the traditional stethoscope or the amplified stethoscope. We conclude that flight nurses are unable to hear normal breath sounds using a traditional or amplified stethoscope during flight in a medically configured MBB BO-105 helicopter. Improved stethoscopes, innovative methods of listening, and reduction of aircraft noise are potential solutions to the problems of breath sound assessment during air medical transport.

Aircraft↗
PubMed1990

Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro.

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.

Antibodies, Monoclonal↗
PubMed1990

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems.

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.

Animals↗
PubMed1990

Selective inhibition of growing pigment epithelial cells by a receptor-directed immunotoxin.

An immunotoxin conjugate of a murine monoclonal antibody against human transferrin receptors and the A chain of ricin was examined for its potential to inhibit selectively the growth of retinal pigment epithelial (RPE) cells which grow in an uncontrolled manner in proliferative vitreoretinopathy. The probable efficacy of such an agent in vivo stems from the observation that actively proliferating cells possess many more transferrin receptors than normal quiescent cells. The authors showed in vitro that the immunoconjugate (454A12 MAB-rRA) inhibits protein synthesis in actively dividing RPE cells but has a smaller or no effect on protein synthesis by confluent, nondividing RPE cells. The effect was specific in that neither the free ricin A chain (rRA) nor the monoclonal antibody (454A12 MAB) alone has any inhibitory effect. Furthermore, the antibody competes with the immunotoxin and suppresses the latter's toxicity. This immunotoxin has applications for therapy in conditions in which the pathologic proliferation of RPE cells occurs.

Antibodies, Monoclonal↗