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S Metcalfe

Publications and source records attributed to S Metcalfe.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mutational biosynthesis of novel rapamycins by a strain of Streptomyces hygroscopicus NRRL 5491 disrupted in rapL, encoding a putative lysine cyclodeaminase.

The gene rapL lies within the region of the Streptomyces hygroscopicus chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into rapL by phiC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this rapL mutant on media containing added L-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that rapL encodes a specific L-lysine cyclodeaminase important for the production of the L-pipecolate precursor. In the presence of added proline derivatives, rapL mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.

Ammonia-Lyases↗
PubMed1996

Amplification of natural regulatory immune mechanisms for transplantation tolerance.

There is a need to derive donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and side effects of continual immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it were "self." Here we show that, once generated, this state of operational tolerance becomes self-sustaining, imposing itself on new cohorts of lymphocytes as they arise. These new cohorts retain specificity for the tolerizing antigen and can be selectively amplified to tolerate new antigens that have linked expression with the original tolerogen. Regulation is critically dependent upon the continuous presence of tolerizing antigen and is mediated by the CD4+ lymphocyte population. We propose that such natural mechanisms of immune regulation may eventually be exploited for transplantation tolerance, even in fully immune-competent recipients.

Animals↗
PubMed1995

Effect of dietary fat on the pharmacokinetics and pharmacodynamics of cyclosporine in kidney transplant recipients.

OBJECTIVE: To investigate the effect of dietary fat on the pharmacokinetics and pharmacodynamics of cyclosporine. METHODS: Sixteen stable kidney transplants recipients (mean age, 50.4 years; age range, 19 to 63 years; six women) who were maintained on oral cyclosporine therapy were randomized to receive a high- or low-fat diet for periods of 7 days in a balanced crossover study. The crossover was separated by a 7-day washout period, when the usual diet was followed. Oral cyclosporine was taken once daily with breakfast. Twenty-four-hour pharmacokinetic studies were conducted during each dietary period on day 6 after oral cyclosporine and on day 7 after a 3-hour intravenous cyclosporine infusion (30% of oral dose). Sequential blood samples were also taken after the oral dose on day 6 for lymphocyte transformation studies. RESULTS: The mean breakfast fat intake and total daily fat intake were 6.5 and 5.5 times higher, respectively, during the high-fat diet than during the low-fat diet. The bioavailability and clearance of cyclosporine were found to be significantly higher during the high-fat diet (p = 0.02 and p = 0.01, respectively). As a consequence, the area under the blood concentration-time curve (AUC) after the oral dose was not significantly different between the two diets. There were no significant differences in concanavalin A-stimulated proliferation of peripheral blood lymphocytes between the high- and low-fat diets. CONCLUSIONS: An increased fat content of food significantly increases cyclosporine bioavailability and clearance. However, this is unlikely to be of clinical importance during oral administration because the AUC and pharmacodynamics of cyclosporine are not affected significantly.

Adult↗
PubMed1994

FK506 and cyclosporin A each inhibit antigen-specific signaling in the T cell line 171 in the absence of a calcium signal.

Antigen-specific signal transduction leading to IL2 induction and secretion in the T cell line 171 is augmented by association of p56lck with CD4. Although no change in cytoplasmic calcium level ([Ca2+]i) was detectable during antigen-specific signal transduction of 171-CD4+ cells, IL2 induction was inhibited by FK506 and CsA. Since these drugs are thought to act selectively by inhibiting calcineurin, a calcium-calmodulin-dependent protein phosphatase associated with activation of the IL2 promoter, we considered the possibility that calcineurin is constitutively active in 171 cells. However, we found no evidence for this because PMA failed to supplement any putatively active calcineurin to induce IL2 secretion. We suggest that IL2 secretion induced by antigen presentation to TCR/CD4/p56lck requires an FK506 and cyclosporin A-sensitive step which may be independent of calcium signaling. Rapamycin did not inhibit IL2 secretion induced by TCR/CD4/p56lck, emphasizing the specific action of FK506 and cyclosporin A.

Amino Acid Sequence↗
PubMed1994

Rat liver blood flow after ischemia and reperfusion. The effects of the platelet-activating factor antagonist WEB-2170 and of removing circulating leukocytes.

The inflammatory response to trauma induces release of platelet activating factor (PAF), which promotes leukocyte adherence to the vascular endothelium. Ischemia and reperfusion induces inflammatory reactions that play a role in reperfusion injury, and here we investigate the role of both PAF and of leukocytes in damage to reperfused rat liver. The experimental procedure consisted of the temporary interruption of blood flow to the left lateral and medial lobes of the rat liver in vivo, and subsequent reperfusion after defined periods. Rats were pretreated either with the PAF-antagonist WEB-2170 or with vinblastine to induce leukopenia, and compared with controls. The postischemic liver blood flow and liver oxyhemoglobin saturation were recorded using an He-Ne Laser doppler flowmeter and photometer. Reperfusion after 30 and 45 min of ischemia was associated with partial recovery to normal values and was inversely proportional to the duration of ischemia. In the WEB-2170-treated group, liver flow and hemoglobin saturation upon reperfusion did not show significant differences when compared with the untreated control groups, suggesting that inhibition of PAF activity did not protect against the microcirculatory disturbance induced by ischemia and reperfusion in the liver. In contrast, rats made leukopenic by treatment with vinblastine showed significantly better recovery of blood flow and hemoglobin saturation than the control group after 45 min of ischemia. Thus, we found that although PAF alone did not appear to have a pivotal role in the cascade of reperfusion injury, the effect of leukocytes is critical.

Animals↗
PubMed1994

Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW).

A panel of 127 monoclonal antibodies against canine leukocyte antigens, including controls, was distributed to 29 laboratories that performed a variety of experiments to identify groups of antibodies against the canine equivalents of some of the human CD antigens. Cluster analysis was performed centrally, using the submitted antibody binding data from immunofluorescence, ELISA and immuno-histology experiments. Immunoprecipitation for molecular weight determination was also performed centrally with T-cell blasts and a B-cell line as the sources of antigen. Clusters of three or more antibodies were found that defined the canine equivalents of the CD5, CD4, CD8 and Thy-1 antigens, and these could be used to label T-cell subsets from the peripheral blood. Other groups of monoclonal antibodies recognized the canine homologues of the CD11/18 group of antigens, CD44 and the CD45/CD45R antigen family: these should be useful in isolating functional subsets of CD4+ helper T cells. There was a cluster of four antibodies that bound strongly to platelets (probably CD41 antigen), three antibodies that were specific to B cells (including CD21) and two antibodies against a granulocyte antigen (possibly CD15). A number of reagents were found against canine MHC-II and immunoglobulin, with some of the latter able to distinguish between Ig subclasses. Properties of each of the canine antigens defined by these monoclonal antibodies are discussed and compared with other species. The availability of such a panel of reagents should allow rapid improvements in the immunological diagnosis of canine disease, and there might now be a potential for testing novel therapeutic strategies in a clinical veterinary setting.

Animals↗
PubMed1994

FK 506 and cyclosporin each block antigen-induced T cell receptor signalling that is dependent on CD4 co-receptor and operates in the absence of detectable cytoplasmic calcium fluxes.

The T cell hybridoma "171", which lacks CD4 but expresses T cell receptor (TCR) for hen egg white lysozyme, requires introduction of wild-type CD4 for antigen-mediated induction and secretion of interleukin-2 (IL-2). Mutant CD4, which fails to associate with the tyrosine kinase p56lck does not support IL-2 secretion, suggesting that a role of CD4 is to bring cytoplasmic p56lck into alignment for signal transduction to the IL-2 promotor. Using 171, 171-CD4 (wild-type) and 171-CD4 (mutant), we found that IL-2 secretion was inhibited by FK 506 and cyclosporin but not by rapamycin. However, this inhibition was not associated with calcium fluxes since no change in cytoplasmic free calcium levels ([Ca]i; resting level 80 nM) was detectable during antigen stimulation of the 171 or 171-CD4 cells. Thus, although FK 506 and cyclosporin inhibited calcium-dependent signalling to the IL-2 promoter via inhibition of the protein phosphatase calcineurin, it is possible that IL-2 induction via TCR/CD4 requires an FK 506 (and cyclosporin) sensitive step which is independent of cytoplasmic calcium changes.

Animals↗
PubMed1994

Serum PAF-acetylhydrolase in severe renal or hepatic disease in man: relationship to circulating levels of PAF and effects of nephrectomy or transplantation.

(1) PAF-acetylhydrolases form a major pathway for the degradation of platelet-activating factor (PAF). Here we investigate the role of the kidney and the liver in the control of PAF-acetylhydrolase levels by comparing normal subjects to patients with abnormal liver or kidney function. These patients had either severe chronic liver disease, chronic renal failure or were anephric. In a few cases PAF was also measured. (2) In those patients where PAF was measured there was no evidence that circulating PAF levels determined PAF-acetylhydrolase release. (3) In anephric patients serum PAF-acetylhydrolase levels were normal or even raised. Therefore the kidney is unlikely to be the usual major source of serum PAF-acetylhydrolase in man. (4) Liver patients with chronic cholestasis had elevated serum PAF-acetylhydrolase especially in stage III or IV primary biliary cirrhosis, as well as in a patient with secondary biliary cirrhosis and one with cholangiocarcinoma. Since normalisation of liver function following liver transplantation was accompanied by a reduction to normal or near normal PAF-acetylhydrolase levels, it is likely that the liver can play an important role in regulating levels of this enzyme in serum.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1993

Prodigiosin 25C: effect in in vitro models for T cell activation and T cell cycling and in vivo for rat heart allografts.

Prodigiosin 25C inhibited both lymphocyte activation (concanavalin-A-activated mouse spleen cells) and constitutive cell cycling of T cells and fibroblasts. This suggests that prodigiosin 25C is not selective for activation of T lymphocytes as has been previously reported. Use of prodigiosin 25C to suppress allograft rejection of rat hearts (DA to PVG) showed that 1 mg/kg/day i.p. had no effect whilst 10 mg/kg/day i.p. was toxic as manifest by weight loss and diarrhoea at 3 days. Although this toxicity was reversible upon reversion to 1 mg/kg/day, no major prolongation in allograft survival was seen. Our data suggests that prodigiosin 25C has a low therapeutic index and may not be selective for T cell activation.

Animals↗
PubMed1992

Tolerance in the mouse to major histocompatibility complex-mismatched heart allografts, and to rat heart xenografts, using monoclonal antibodies to CD4 and CD8.

Mice given a single short course of anti-CD4 and anti-CD8 monoclonal antibodies (mAb) became tolerant of major histocompatibility complex (MHC)-incompatible vascularized heart allografts in a donor- and organ-specific manner. T cell depletion was not important, as blocking antibodies were equally effective. Anti-CD4 antibody therapy alone was sufficient to establish tolerance. Anti-CD8 mAb therapy alone was associated with poor recipient survival, although survivors were rendered tolerant. A second donor-type heart allograft to the neck was always accepted in recipients which had carried the first abdominal heart allograft for over 120 days, with the first heart also continuing to function. However, donor-type skin grafted at 100 days was sometimes rejected, albeit chronically. Those recipients that accepted the donor-type skin were able to reject third-party skin grafts mismatched at the MHC, or over multiple minor differences alone. Thus, the tolerance induced appeared to be donor and tissue specific, although spreading in some cases to tolerance of skin. Similarly donor-specific tolerance was found when xenogeneic PVG rat hearts were grafted into CBA/Ca mice using the same protocols of anti-CD4 plus anti-CD8 mAb. Resolution of the mechanisms underlying these forms of tolerance may permit the design of improved immunosuppressive protocols for human organ transplantation.

Animals↗
PubMed1992

FK506 and rapamycin: differential sensitivity of human, baboon, cynomolgus monkey, dog and pig lymphocytes.

It has recently been suggested that there are species differences in the sensitivity of T lymphocytes to the immunosuppressive effect of FK506. We explore this phenomenon further and compare FK506 with rapamycin in lymphocytes from dog, pig, baboon, cynomolgus monkey and man. We found that the relative sensitivity of T cells to FK506 did not necessarily correlate with their sensitivity to rapamycin, further emphasising the different mode of action of each drug. The serum may alter results of dose-response curves, and thus autologous serum may introduce an uncontrolled variable when different species are being compared in vitro. We conclude that the differences in sensitivity of lymphocytes from various species to FK506 and rapamycin are independent of each other, and these differences are likely to reflect variation in the overall interactive pathways involved in T-cell mitogenesis.

Animals↗