Biomedical subjects

R A Mitchell

Publications and source records attributed to R A Mitchell.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vitro lymphocyte blastogenic responses and cytokine production in sickle cell disease patients with acute pneumonia.

BACKGROUND: Pulmonary infections continue to be a major cause of morbidity and mortality in patients with sickle cell disease (SCD). METHODS: In this study cell-mediated immunity in vitro was evaluated in 62 SCD patients (62 steady state and 16 with acute pneumonia) and compared with 44 normal controls (30 healthy and 14 with acute pneumonia). Lymphocyte blastogenic responses to phytohemagglutinin, tetanus toxoid and Candida albicans antigen were assessed in all subjects. In addition production of tumor necrosis factor, alpha- and gamma-interferon (IFN) were assayed. RESULTS: The results revealed comparable blastogenic responses to all three stimuli in all subjects except SCD patients with pneumonia. This group showed poor responses to all stimuli. The mean counts per minute were decreased 65 to 90% when compared with the other patients. Cytokine production of IFN-alpha and TNF was equivalent in all subjects. Conversely IFN-gamma production in both SCD groups, steady state (35 +/- 6 U/ml) and SCD with pneumonia (14 +/- 6 U/ml), was significantly decreased when compared with those in normal healthy controls (65 +/- 14 U/ml) and with pneumonia (48 +/- 17 U/ml). On analysis of individual titers 15 of 62 (24%) steady state and 10 of 16 (63%) SCD patients with pneumonia were deficient in IFN-gamma production in vitro. CONCLUSIONS: Acute pulmonary infections seem to have a profound effect on cell-mediated immunity in SCD. IFN-gamma deficiency, along with quantitative and qualitative T cell abnormalities, may represent significant factors to explain the frequent and severe infections seen in SCD.

Adolescent↗

PubMed1995

Hepatic metabolic response to injury and sepsis.

BACKGROUND: Experimental reports have indicated that hepatic oxidative and synthetic metabolism may become depressed in sepsis. Because the mechanism of infection-related liver dysfunction has not been established, further study of these functional alterations could contribute to the therapeutic management of septic organ failure syndromes. However, recently controversy has arisen over the existence of these derangements that must be reconciled before further progress in this field can be made. METHODS: Splanchnic balance studies for the measurement of glucose output and oxygen consumption were used to assess hepatic function in fasted normal volunteers (n = 18), injured patients (n = 10), and patients with sepsis (n = 18). The liver's contribution to splanchnic metabolism was estimated from a comparison of splanchnic oxygen utilization in response to increases in the liver-specific process of glucogenesis. In addition, in vivo liver albumin production was determined by using the [14C] carbonate technique. RESULTS: Glucose output after injury and sepsis was increased by 12.8% and 76.6%, respectively, compared with controls. On the basis of substrate balance studies, gluconeogenesis was estimated to account for 46%, 87%, and 93%, respectively, of splanchnic glucose output in each of the three groups. In patients with sepsis glucose output was also noted to be linearly related to regional oxygen consumption, indicating that these processes were coupled and increases in the respiratory activity of the splanchnic cellular mass could be accounted for by increases in new glucose output and gluconeogenic substrate clearance. The mean albumin synthetic rate increased during injury and sepsis by 22% and 29%, respectively, compared with normal volunteers. CONCLUSIONS: These studies cast doubt on the commonly held notion that tissue respiratory dysfunction may occur during sepsis. On the contrary, hepatic function is accelerated during hyperdynamic sepsis, and evidence indicating oxidative or synthetic functional depression is lacking.

Adult↗

PubMed1995

Serum interleukin-6 levels in the steady state of sickle cell disease.

In patients with childhood sickle cell disease (SCD) serum interleukin-6 (IL-6) levels were measured during the steady (healthy) state of disease. The corresponding measurements were made in comparable healthy normal controls. Serum IL-6 levels were assessed via ELISA in 27 SCD patients and 19 controls. Results revealed significantly higher circulating levels of IL-6 in the SCD patients (60 +/- 7 pg/ml) compared with the healthy controls (12 +/- 5 pg/ml). IL-6 is a multifunctional cytokine that plays a central role in host defense mechanisms. The impact of high circulating levels of IL-6 may be deleterious to humoral and cell-mediated immune functions in SCD, with resultant heightened risk for morbidity.

Adolescent↗

PubMed1994

Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF).

The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.

Amino Acid Sequence↗

PubMed1994

The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor.

For over 25 years, the cytokine known as macrophage migration inhibitory factor (MIF) has been considered to be a product of activated T lymphocytes. We recently identified the murine homolog of human MIF as a protein secreted by the pituitary in response to endotoxin administration. In the course of these studies, we also detected MIF in acute sera obtained from endotoxin-treated, T cell-deficient (nude), and hypophysectomized mice, suggesting that still more cell types produce MIF. Here, we report that cells of the monocyte/macrophage lineage are an important source of MIF in vitro and in vivo. We observed high levels of both preformed MIF protein and MIF mRNA in resting, nonstimulated cells. In the murine macrophage cell line RAW 264.7, MIF secretion was induced by as little as 10 pg/ml of lipopolysaccharide (LPS), peaked at 1 ng/ml, and was undetectable at LPS concentrations > 1 microgram/ml. A similar stimulation profile was observed in LPS-treated peritoneal macrophages; however, higher LPS concentrations were necessary to induce peak MIF production unless cells had been preincubated with interferon gamma (IFN-gamma). In RAW 264.7 macrophages, MIF secretion also was induced by tumor necrosis factor alpha (TNF-alpha) and IFN-gamma, but not by interleukins 1 beta or 6. Of note, MIF-stimulated macrophages were observed to secrete bioactive TNF-alpha. Although previously overlooked, the macrophage is both an important source and an important target of MIF in vivo. The activation of both central (pituitary) and peripheral (macrophage) sources of MIF production by inflammatory stimuli provides further evidence for the critical role of this cytokine in the systemic response to tissue invasion.

Animals↗

PubMed1993

MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia.

Cytokines are critical in the often fatal cascade of events that cause septic shock. One regulatory system that is likely to be important in controlling inflammatory responses is the neuroendocrine axis. The pituitary, for example, is ideally situated to integrate central and peripheral stimuli, and initiates the increase in systemic glucocorticoids that accompanies host stress responses. To assess further the contribution of the pituitary to systemic inflammatory processes, we examined the secretory profile of cultured pituitary cells and whole pituitaries in vivo after stimulation with bacterial lipopolysaccharide (LPS). Here we identify macrophage migration inhibitory factor (MIF) as a major secreted protein release by anterior pituitary cells in response to LPS stimulation. Serum analysis of control, hypophysectomized and T-cell-deficient (nude) mice suggests that pituitary-derived MIF contributes to circulating MIF present in the post-acute phase of endotoxaemia. Recombinant murine MIF greatly enhances lethality when co-injected with LPS and anti-MIF antibody confers full protection against lethal endotoxaemia. We conclude that MIF plays a central role in the toxic response to endotoxaemia and possibly septic shock.

Acute-Phase Reaction↗

PubMed1993

Contrast sensitivity in elderly subjects with a diagnosed ocular disease.

In a random sample of 3283 subjects, 1266 right eyes and 1334 left eyes were diagnosed as having one of the following: age-related macular degeneration (ARMD), cataract, or glaucoma. In this same sample 1369 right eyes and 1312 left eyes had no ocular disease. All eyes had their contrast sensitivity measured using the Arden-type gratings manufactured by The American Optical Corporation. This is a large sample survey and although prevalence rates for the diseases examined may be inferred, this must be carried out with caution. I found for those eyes where the disease interfered with the integrity of the retina (ARMD, glaucoma) the mean contrast sensitivity scores were higher, in the middle and upper frequencies, than for those eyes with lens opacities. For frequencies above 0.4 cpd contrast sensitivities associated with diseased eyes were significantly (p < 0.001) reduced when compared to the same measures obtained on nondiseased eyes.

Aged↗

PubMed1993

Prevalence of age related macular degeneration in persons aged 50 years and over resident in Australia.

STUDY OBJECTIVE: The aim was to determine, employing non-invasive procedures, the prevalence of age related macular degeneration in persons 50 years of age and over. DESIGN: A clinical investigation and a retrospective examination of ophthalmological records were employed in this study. SETTING: The study was conducted over the period 1988 to 1990 in the Western Metropolitan Health Region of New South Wales, Australia. PARTICIPANTS: A total sample of 3283 subjects stratified by local government area, age, and sex was obtained from the source population. It proved possible to confirm ophthalmological diagnoses in only 2522 of these subjects. MAIN RESULTS: All data were collected using accepted ophthalmological procedures and all diagnoses were confirmed through the use of independently derived ophthalmological records. A total of 428 subjects (13.0%) had a confirmed symmetrical diagnosis of age related macular degeneration. A total prevalence for diseased eyes of 14.9% was obtained. Prevalence of diseased eyes rose from 10.4% in those 50 to 64 years of age to 31.0% in those 85 years of age and over. CONCLUSIONS: There are several sources of error which can affect such a large sample study and are identified. Despite these, the prevalence rates obtained in this study provide normative rates for age related macular degeneration for persons 50 years of age and over.

Age Factors↗

PubMed1992

Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.

We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.

Base Sequence↗

PubMed1992

Topography of cat medullary ventral surface hypoxic acidification.

The topographic relationship between previously identified medullary ventral surface respiratory chemosensitive regions and brain surface extracellular fluid (ECF) acid production during acute hypoxia was explored in anesthetized, paralyzed, and artificially ventilated cats. Glass pH electrodes (0.8-mm diam, sheathed in stainless steel tubing) were mounted in mechanical contact with surfaces of medullary surface or adjacent pyramids, pons, spinal cord, or parietal cortex. Isocapnic hypoxia of 5 min [at arterial O2 saturation (SaO2) = 48 +/- 10%] reduced pH over rostral (Mitchell) and caudal (Loeschcke) areas by 0.12 +/- 0.09 and 0.07 +/- 0.04, respectively (n = 10, P < 0.05). Change in pH (delta pH) was proportional to desaturation with slopes 100 delta pH/delta SaO2 of 0.45 (rostral) and 0.20 (caudal) (R = 0.91 and 0.88, respectively). pH drop usually began within 3 min of hypoxia, became stable between 5 and 15 min, began to rise within 2 min of reoxygenation, and returned to control within 10 min. During equally hypoxic tests, intermediate area (Schläfke), pons, and spinal cord surfaces showed no significant acid shift. Parietal cortex ECF pH dropped more slowly but steadily by 0.079 +/- 0.034 during 20 min at SaO2 = 50% after a small but significant initial alkaline shift, and acidification of cortical surface continued for > 5 min after reoxygenation. We conclude that medullary ventral chemosensitive regions produce more lactic acid during hypoxia than neighboring brain surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The isolated perfused rat liver as an experimental model.

Intrinsic liver function may be specifically studied using the isolated perfused liver system. In this report, the metabolic characteristics of the perfused rat liver are described in response to lactate (5 mM) and ammonia (10 mM) loading. These conditions are analogous to the increased substrate availability that accompanies some acute disease states such as trauma. Glucose production (490 +/- 85 nm/gm liver/min) was constant during a 90-minute perfusion whereas albumin secretion exhibited a biphasic kinetic profile in the absence of exogenous amino acids. Liver oxygen consumption (VO2) was responsive to substrate loading as evidenced by a 20 to 60 per cent initial increase in VO2. The authors conclude that a large portion of hepatic VO2 may normally be directed toward substrate clearance. Additionally, although hepatic secretory protein synthesis persists despite the limitation of nutrient amino acid supply, the kinetics of secretion are complex.

Albumins↗

PubMed1991

Perceived health problems in subjects with varying cardiovascular diagnoses.

To study perceived health problems in subjects with differing cardiovascular status, the Nottingham Health Profile (NHP) was administered to 210 subjects 55 years of age and over. Subjects were categorized as being cardiovascular "Normals," being hypertensive, having isolated coronary artery disease, or both being hypertensive and having coronary artery disease. An analysis of variance between the four cardiovascular strata on each of the six subscales of the NHP yielded significant differences between the groups on the subscales Pain, Physical Mobility, Energy, and Social Isolation. Subsequent conservative post hoc analyses of the group means on each of these variables indicated that the group with isolated coronary artery disease differed significantly from both the hypertensives and the Normals in Physical Mobility. For the Pain subscale the subjects with isolated coronary artery disease differed significantly from those with hypertension. There were no differences among the four cardiovascular groups in perceived health problems on the subscales Emotional Reactions and Sleep.

Activities of Daily Living↗

PubMed1991

Australian caregivers of family members with dementia.

This study of Australian caregivers revealed that sufferers of dementia were men and women who were cared for mainly by their spouses. A small group of caregivers had little or no personal physical and emotional support from others. Changes in health status related to the caregiving role was reported by 83% of the caregivers. There is a clear need to provide physical and emotional support for caregivers generally and for those with little or no support in particular.

Aged↗

PubMed1991

Utility of femorofemoral bypass. Comparison of results with indications for operation.

Sixty-eight patients underwent femorofemoral bypass (FFB) at the University of Louisville Affiliated Hospitals over a 9-year period from 1980 to 1989. There were patients with acute ischemia requiring emergency operation, those with chronic ischemia following failed aortic reconstruction, good-risk patients with chronic ischemia who had FFB as the procedure of choice, and poor-risk patients who were unsuitable for other procedures. Patients operated on for acute ischemia had a significant number of infectious complications, with five of eight graft failures in this group due to infection. Primary patency rates were similar in all groups, with a 5-year primary patency of 42 per cent. Overall operative mortality was 4.4 per cent. Satisfactory limb salvage rates (75%) suggest that FFB is an acceptable option for providing inflow in patients with acute ischemia or for patients with a failed previous vascular prosthesis. FFB provides adequate inflow in poor-risk patients not suitable for aortic reconstruction, but patency rates do not warrant its use as a procedure of choice in good-risk patients.

Acute Disease↗

PubMed1990

Hepatic blood flow and splanchnic oxygen consumption measurements in clinical sepsis.

In an effort to characterize the hemodynamic response of the liver to sepsis, hepatic blood flow (HBF) was measured in 10 normal volunteers and compared with that of 9 patients with sepsis. Flow was determined according to two different indicators and three methods of analysis including indocyanine green dye clearance (HBFICG), galactose clearance (GC), and galactose clearance with splanchnic galactose gradient measurement (HBFGG). For normal subjects, these three analytic methods provided essentially identical results (HBFICG = 0.74 +/- 0.18, GC = 0.72 +/- 0.14, and HBFGG = 0.76 +/- 0.16 L/min-m2). With hepatic venous sampling, HBF in patients with sepsis was significantly higher than normal levels (HBFICG = 1.28 +/- 0.50 and HBFGG = 1.17 +/- 0.52 L/min-m2) (p less than 0.025), but HBF by the GC technique (0.89 +/- 0.41 L/min-m2), which uses peripheral venous sampling, was not significantly increased because of reduced splanchnic galactose extraction, which appears to be characteristic of sepsis. Thus HBF estimates based on peripheral venous sampling must be interpreted with caution in view of the reduced extraction fraction in sepsis. HBF in clinical sepsis tends to increase in response to this inflammatory stress.

Galactose↗

PubMed1988

Neurogenic inflammation in the rat trachea. II. Identity and distribution of nerves mediating the increase in vascular permeability.

This study addresses the question of whether increased vascular permeability, which is a prominent feature of neurogenic inflammation in the respiratory tract, is mediated by sensory axons that end near venules in the airway mucosa. In these experiments, neurogenic inflammation was produced in the tracheal and bronchial mucosa of atropine-treated Long-Evans rats by electrical stimulation of the left or right superior laryngeal nerve and/or cervical vagus nerve. The particulate tracer Monastral blue was injected intravenously to localize the sites of increased vascular permeability, and microspectrophotometry was used to measure the amount of extravasated Monastral blue in the trachea and thereby quantify the increase in vascular permeability. In some rats, selective denervations were made to locate the cell bodies of neurons that mediate the increase in vascular permeability; in others, fluorescence immunohistochemistry and quantitative electron microscopic methods were used to determine which structures in the tracheal mucosa are innervated by these neurons. The study revealed that the vagally mediated increase in vascular permeability was sudden, transient (half-life = 2.4 min) and restricted to venules. Stimulation of the left or right superior laryngeal nerve increased the permeability of venules in the extrathoracic trachea, whereas stimulation of either vagus nerve increased vascular permeability in the intrathoracic trachea and bronchi. All nerves had bilateral effects in the trachea, but the vagus nerves had largely unilateral effects in the bronchi. Neurons that mediated the increase in venular permeability had their cells bodies in the jugular (superior sensory) ganglion of the vagus nerve or rostral portion of the nodose (inferior sensory) ganglion. Preganglionic autonomic vagal neurons in the brain stem were not essential for this increase in venular permeability. Few nerves identifiable by substance P-immunohistochemistry or electron microscopy were located near the affected venules, and no nerves were within 1 micron of the walls of venules. However, the epithelium and arterioles of the airway mucosa were densely innervated. All intraepithelial nerves were within 0.1 micron of epithelial cells, and at least two-thirds of nerves near arterioles were within 1 micron of the vessel walls. We conclude that the increase in venular permeability associated with neurogenic inflammation in the trachea and bronchi of rats is mediated by sensory axons that travel in the vagus nerves and superior laryngeal nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

In vivo activity of tracheal parasympathetic ganglion cells innervating tracheal smooth muscle.

In vivo intracellular recording and intrasomal injection of Lucifer yellow revealed two populations of postganglionic parasympathetic neurons in the tracheal ganglia of cats. One consisted of large cells that had an inspiratory rhythm, had a significant post-spike afterhyperpolarization, and projected to the tracheal smooth muscle. The second consisted of small cells that fired with an expiratory rhythm, had no significant afterhyperpolarization, and projected to the intercartilaginous spaces.

Animals↗

PubMed1987

Insulin production following injury and sepsis.

It is known that alterations in insulin metabolism following injury and infection result in depression of insulin levels and the development of insulin resistance. In order to further study insulin during septic and traumatic stress, we estimated insulin production in control (Group 1, n = 6), postoperative (Group 2, n = 5), and postoperative-septic (Group 3, n = 8) human subjects by measuring the 24-hour urinary C-peptide excretion. In addition, basal and peak glucose and insulin levels in response to a standard (0.5 gm/kg) intravenous glucose stimulus were measured immediately thereafter to determine if insulin levels reflected insulin production. Basal insulin for Groups 1, 2, and 3 (16 +/- 8.4, 10 +/- 3.4, 9.5 +/- 4.4 microU/ml +/- SD, respectively) were not substantially different. Peak insulin response to glucose infusion declined from Groups 1 to 3 (51 +/- 14, 42.4 +/- 31, 20.4 +/- 6.8 microU/ml, respectively) with Group 3 exhibiting a significantly decreased mean peak level compared to the other groups. Corresponding C-peptide excretion rates increased from Groups 1 to 3 (28.3 +/- 15.3, 63.7 +/- 27.6, 121.3 +/- 95.2 micrograms/day, respectively) with Group 3 exhibiting a significantly (p less than 0.05) higher level than Group 1. These data suggest that low insulin levels which may be evident in injured or septic patients not in shock reflect increased clearance and not decreased production.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗