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Biomedical subjects

S D Reid

Publications and source records attributed to S D Reid.

33 records · Page 2Linked to original sources
PubMed1995

Differential expression of adhesion molecules during rat small bowel allograft rejection.

The adhesion molecules intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1) (alpha and beta chains), and very late activation antigen-4 (VLA-4) have an essential role in cell-cell interactions and the initiation of immune responses. This study used an indirect immunoperoxidase technique to investigate the expression of these molecules in the lamina propria of allografts and isografts after heterotopic rat small bowel transplantation. Normal untransplanted small bowel served as additional controls. Overall, ICAM-1 and LFA-1 alpha expression was significantly higher in allografts, although there was variable expression of these molecules in isografted animals. There were temporal differences in the expression of ICAM-1 and LFA-1 alpha in that increased ICAM-1 expression was more pronounced in the the early posttransplant period, whereas there was a progressive increase in LFA-1 alpha as rejection developed. In contrast, there was no difference between allograft and isograft expression of LFA-1 beta and VLA-4. This study has demonstrated a preferential increase in adhesion molecule expression with developing rat small bowel allograft rejection and suggests that adhesion molecules are involved in the development and progression of allograft rejection. Although the observed differences in antigen expression are not as marked as those previously reported in other organ transplants, appropriate adhesion molecules may present suitable targets for immunotherapeutic protocols after small bowel transplantation.

Animals↗
PubMed1995

Development of an enzyme immunoassay for rat soluble interleukin-2 receptors.

This study describes the development of a sandwich enzyme immunoassay (ELISA) for rat soluble IL-2 receptors (sIL-2R) using a combination of monoclonal antibodies reactive with different epitopes on the rat IL-2R. Coating plates with NDS61 and NDS64 monoclonal antibodies produced similar dose-response curves when incubated with a standard sIL-2R preparation followed by biotinylated OX39, streptavidin-alkaline phosphatase and the substrate. Although normal rat serum inhibited the assay, the effects were more profound when NDS64 was used as the capture antibody and subsequent development of the assay was performed using NDS61. The intra- and interassay variations were typically less than 5%. This assay will be valuable for monitoring immune activation status in a variety of experimental models.

Animals↗
PubMed1995

An enzyme immunoassay for rat soluble MHC class I molecules (RT1a) and the release of soluble class I from mitogenically stimulated mononuclear cells.

Soluble MHC class I antigens can be detected in the serum of humans and various animals and appear in the circulation shortly after liver transplantation. The precise role of these antigens is currently uncertain, but soluble MHC class I may be involved in immunomodulation. We have developed an enzyme linked immunosorbent assay for soluble rat MHC class I (RT1a) molecules and monitored the kinetics of antigen release following in vitro stimulation of splenic mononuclear cells. A 4 day DA splenocyte Con A supernatant provided a source of soluble class I antigens and was arbitrarily assigned a concentration of 1000 units/ml. Ninety six well plates were coated with a rat RT1a-specific mAb (MN4-91-6) and soluble class I binding was detected using a biotinylated mAb reactive with a monomorphic region of the rat MHC class I molecule (OX18) followed by a streptavidin-alkaline phosphatase conjugate and substrate. The intra- and interassay variations were typically less than 5% and 10% respectively, to give a working range for the assay of between 62.5 and 1000 units/ml. Mitogenic stimulation led to a progressive increase in soluble class I levels in culture supernatants. This assay will be valuable in differentiating recipient and graft responses following experimental organ transplantation.

Animals↗
PubMed1992

Rainbow trout hepatocyte beta-adrenoceptors, catecholamine responsiveness, and effects of cortisol.

Hepatocyte beta-adrenoceptors were characterized in trout with chronically raised plasma cortisol levels (132.1 +/- 14.8 ng/ml, n = 8, 11-12 days) and compared with shams (1.8 +/- 0.9 ng/ml, n = 8) using radioreceptor assay techniques. Hepatocytes prepared from sham trout possessed 1,696 +/- 179 beta-adrenoceptors/cell. These receptors were characterized as beta 2-adrenoceptors based on the potency order of specific inhibition of CGP binding. The number of putative surface beta 2-adrenoceptors significantly increased to 6,005 +/- 1,165 receptors/cell, in hepatocytes from trout exposed to the elevated plasma cortisol concentration. The physiological significance of the increase in hepatocyte surface receptors was assessed by the in vitro responsiveness of hepatocytes to a range of epinephrine concentrations (10-1,000 nmol/l). Both total glucose production and intracellular adenosine 3',5'-cyclic monophosphate content, indicators of epinephrine responsiveness, were enhanced as a result of chronically raised levels of plasma cortisol. We suggest that these cortisol-mediated alterations in the adrenergic responsiveness of trout hepatocytes may ultimately enhance the ability of the liver to supply glucose to the fish during acute stress after extended periods of chronic stress.

Animals↗
PubMed1992

Relationship between blood O2 content and catecholamine levels during hypoxia in rainbow trout and American eel.

Plasma catecholamine levels and arterial blood respiratory variables were monitored in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) acutely exposed (30 min) to graded levels of external hypoxia [water PO2 (PWO2) 20-90 Torr]. The experiments were designed to evaluate the factors controlling catecholamine mobilization in hypoxic fish and to elucidate the basis of marked interspecific differences. In trout, plasma catecholamine levels were unchanged when PWO2 remained above 50 Torr but increased markedly when PWO2 was lowered below this value; the predominant catecholamine released into the circulation was epinephrine. In eel, there was no such obvious PWO2 threshold for catecholamine release although plasma levels were consistently elevated above baseline only at PWO2 less than 35 Torr. The magnitude of the catecholamine release in eel was approximately an order of magnitude less than in trout. Unlike in trout, there was no increase in the plasma epinephrine-to-norepinephrine concentration ratio. During hypoxia, the relationship between arterial blood PO2 (PaO2) and PWO2 was similar in both species and thus could not explain the differences in the PWO2 thresholds for catecholamine release. In trout, the calculated PaO2 thresholds for catecholamine release were 25.3 (epinephrine) and 20.5 Torr (norepinephrine) whereas in eel the corresponding values were 12.5 and 11.6 Torr, respectively. These PaO2 thresholds were in good agreement with the in vivo values for PaO2 at half-maximal hemoglobin (Hb)-O2 saturation (P50) for trout and eel blood of 22.9 and 11.1 Torr, respectively. Thus both species displayed essentially equivalent catecholamine release thresholds when expressed in terms of arterial blood O2 content corresponding to approximately 45-60% Hb-O2 saturation.

Animals↗
PubMed1992

The relationship between beta-adrenoceptors and adrenergic responsiveness in trout (Oncorhynchus mykiss) and eel (Anguilla rostrata) erythrocytes.

Experiments were performed in vitro specifically to elucidate the underlying mechanism(s) of the attenuated adrenergic responses of eel (Anguilla rostrata) erythrocytes. This was achieved by comparing beta-adrenoceptor numbers and affinities in addition to (i) Na+/H+ exchange activity, (ii) cell swelling and (iii) cyclic AMP formation mediated by catecholamines in eel and trout (Oncorhynchus mykiss) erythrocytes under normoxic and hypoxic conditions. Under normoxic conditions, eel erythrocytes displayed a total absence of Na+/H+ exchange activity (as determined from measurements of extracellular pH) after addition of noradrenaline (50-1000 nmol l-1) in contrast to a pronounced dose-dependent response in trout. Incubation of the blood under hypoxic conditions, to achieve approximately 50% haemoglobin O2-saturation, further increased the extent of Na+/H+ exchange activation in trout and elicited a statistically significant, although physiologically small (10% of the response in trout), activation of H+ extrusion activity in eel. Catecholamine-mediated cell swelling, although obvious in trout, was absent in eel when estimated under hypoxic conditions. Eel erythrocytes possessed approximately 50% fewer surface beta-adrenoceptors than did trout erythrocytes, although the dissociation constants (KD) of these receptors did not differ between eel and trout. The numbers and affinities of the erythrocyte beta-adrenoceptors were not significantly affected by the hypoxic incubation. Both eel and trout erythrocytes displayed a dose-dependent elevation of cyclic AMP concentration in response to noradrenaline that was further increased by hypoxia. Surprisingly, eel erythrocytes produced larger quantities of cyclic AMP despite the lower numbers of surface beta-adrenoceptors. Thus, the absence of adrenergic swelling and the attenuated H+ extrusion response in eel erythrocytes cannot be attributed to insufficient numbers of beta-adrenoceptors or to functional uncoupling of these receptors from adenylate cyclase. Instead, the differences between trout and eel may reflect differing numbers of Na+/H+ exchangers or fundamental differences in the manner by which these exchangers are activated by cyclic AMP.

Anguilla↗
PubMed1991

Characterization of beta-adrenoreceptors of rainbow trout (Oncorhynchus mykiss) erythrocytes.

Although many studies have characterized these receptors according to pharmacological criteria, this work represents only the second direct characterization of the rainbow trout beta-adrenergic receptors. Radioligand binding assays using (+/-)-4-(3-t-butylamino-2-hydroxy-propoxy)-[5,7-3H]benzimidazol-2- one ([3H]CGP 12177) and 1-[4,6-propyl 3H]dihydroalprenolol ([3H]DHA) were conducted to determine equilibrium binding times, ligand-receptor dissociation constants (KD) and binding capacities (Bmax). Furthermore, we assessed the influence of erythrocyte handling, suspension medium and endogenous catecholamines on Bmax and KD. Maximal binding was obtained when erythrocytes were handled minimally and maintained suspended in plasma rather than physiological saline. Washing and resuspending the erythrocytes, as well as the transfer of the erythrocytes into saline, significantly impaired apparent radioligand affinity and receptor density. Endogenous catecholamines, at levels considered normal for non-stressed animals, did not interfere with the radioligand binding assays, and thus eliminated the need to wash and resuspend erythrocytes. Based on the binding characteristics after intentional lysis of erythrocytes, it is shown that the total receptor population of trout erythrocytes can be estimated by propranolol-displaceable DHA binding, the density of high-affinity surface receptors can best be determined by isoproterenol-displaceable CGP binding, and the number of receptors located within the erythrocytes can be calculated from the difference between the total receptor density and the number of isoproterenol-displacable DHA binding sites. Each of these components must be considered when performing radioreceptor assays using these radioligands and this has significant implications for the interpretation of erythrocyte beta-adrenorecptor localization and mobilization.

Animals↗
PubMed1991

The effects and physiological consequences of raised levels of cortisol on rainbow trout (Oncorhynchus mykiss) erythrocyte beta-adrenoreceptors.

We have investigated the influence of cortisol on the beta-adrenoreceptor population of rainbow trout [Oncorhynchus mykiss (Walbaum)] erythrocytes and determined what impact it has on the adrenergic responsiveness of erythrocytes in vitro to exogenous catecholamines. To do so, the erythrocyte beta-adrenoreceptors were characterized in fish with chronically elevated plasma cortisol levels (118 +/- 5.9 ng ml-1, greater than 10 days) and compared with shams, using radioreceptor assay techniques. The number of 'internalized', low-affinity receptors was increased when cortisol levels were raised, but the number of high-affinity, 'surface' receptors was not altered. The physiological significance of this response was ascertained by assessing the in vitro sensitivity (or responsiveness) of erythrocytes to adrenaline and noradrenaline (10-1000 nmol l-1) under normoxic (PO2 = 16.13 +/- 0.55 kPa, PCO2 = 0.41 +/- 0.01 kPa) or hypoxic (PO2 = 4.13 +/- 0.15 kPa, PCO2 = 0.43 +/- 0.01 kPa) conditions. Erythrocyte sensitivity to catecholamines, as determined by changes in both whole-blood pH (delta pHe) and intracellular cyclic AMP content, was greater in hypoxic than in normoxic blood. Although cortisol further enhanced the responsiveness of erythrocytes to catecholamines, this amplification in sensitivity was observed only during hypoxia. When the radioreceptor assay was conducted using erythrocytes from the catecholamine sensitivity experiments, results were consistent with initial receptor density data. An increase in surface receptor density was associated with hypoxia in vitro. This hypoxia-specific increase in surface beta-adrenoreceptors was significantly enhanced in the cortisol-treated erythrocytes, showing that cortisol had a significant impact on erythrocyte beta-adrenoreceptor dynamics in addition to the direct influence of hypoxia. This study has shown (1) that, by itself, in vitro hypoxia simultaneously initiates the movement of internal receptors to the cell surface, where they become physiologically active, and the replenishment of the internal receptor pool, (2) that cortisol increases receptor availability by increasing the internal pool of low-affinity receptors in the absence of any stimulus for receptor mobilization, and (3) that the sensitivity of erythrocytes to catecholamines is directly proportional to the number of high-affinity receptors present at the erythrocyte surface. Thus, we suggest that, under conditions of chronic stress, cortisol may pre-adapt the erythrocytes to receive additional physiological inputs that can ultimately enhance respiratory performance beyond that which would be possible in the absence of chronically elevated levels of cortisol.

Adrenergic beta-Antagonists↗
PubMedDate not supplied

Mutators and long-term molecular evolution of pathogenic Escherichia coli O157:H7.

It has been proposed that an increased mutation rate (indicated by the frequency of hypermutable isolates) has facilitated the emergence of Escherichia coli O157:H7. Analysis of the divergence of 12 genes shows no evidence that the pathogen has undergone an unusually high rate of mutation and molecular evolution.

Escherichia coli O157↗