Search PubMed⌕ Search

Biomedical subjects

G J Murphy

Publications and source records attributed to G J Murphy.

At least 19 recordsLinked to original sources
PubMed2005

Safety, efficacy, and cost of intraoperative cell salvage and autotransfusion after off-pump coronary artery bypass surgery: a randomized trial.

OBJECTIVE: We evaluated, in a randomized controlled trial, the safety and effectiveness of intraoperative cell salvage and autotransfusion of washed salvaged red blood cells after first-time coronary artery bypass grafting performed on the beating heart. METHODS: Sixty-one patients undergoing off-pump coronary artery bypass grafting surgery were prospectively randomized to autotransfusion (n = 30; receiving autotransfused washed blood from intraoperative cell salvage) or control (n = 31; receiving homologous blood only as blood-replacement therapy). Homologous blood was given according to unit protocols. RESULTS: The groups were well matched with respect to demographic and comorbid characteristics. Patients in the autotransfusion group had a significantly higher 24-hour postoperative hemoglobin concentration (11.9 g/dL; SD, 1.41 g/dL) than those in the control group (10.5 g/dL; SD, 1.37 g/dL) (mean difference, 1.02 g/dL; 95% confidence interval, 1.60-0.44 g/dL; P = .0007), as well as a 20% reduction in the frequency of homologous blood product use (11/31 vs 5/30; P = .095). Autotransfusion of washed red blood cells was not associated with any derangement of thromboelastograph values or laboratory measures of clotting pathway function (prothrombin time, activated partial thromboplastin time, and fibrinogen levels), increased postoperative bleeding, fluid requirements, or adverse clinical events. There was no statistical difference between groups in the total operation, hospitalization, and management costs per patient (median difference, USD 1015.90; 95% confidence interval, -USD 2260 to USD 206; P = .11). Conclusions Intraoperative cell salvage and autotransfusion was associated with higher postoperative hemoglobin concentrations, a modest reduction in transfusion requirements, no adverse clinical or coagulopathic effects, and no significant increase in cost compared with controls. This study supports its routine use in off-pump coronary artery bypass grafting surgery.

Adult↗
PubMed2004

Recognition and management of maternal cardiac disease in pregnancy.

Heart disease is a leading cause of maternal death. The aim of this study is to review the most common causes of cardiac disease, highlight factors that should be recognized by the clinician, and address recent advances in the anaesthetic management of these patients. Incipient cardiac disease, including peripartum cardiomyopathy, myocardial infarction and aortic dissection, accounts for approximately one in six maternal deaths. The keys to successful diagnosis and management of incipient disease are: a high index of suspicion, particularly in women with known risk factors for cardiovascular disease; a low threshold for radiological investigations; early cardiology input; and invasive monitoring during labour and delivery. Echocardiography is a safe, non-invasive test, under-used in pregnancy. Management of pregnant women with pre-existing cardiac problems should be undertaken by multidisciplinary teams in tertiary centres. In women with pre-existing cardiac disease wishing to proceed to term, cardiac status must be optimized preoperatively and planned elective delivery is preferable. Vaginal delivery is preferable, and with careful incremental regional anaesthesia is safe in most women with cardiac disease. The presence of adequate systems for early detection, appropriate referral to specialist centres, and timely delivery with multidisciplinary support can minimize the serious consequences of poorly controlled heart disease in pregnancy.

Anesthesia, Obstetrical↗
PubMed2003

Randomized clinical trial of the effect of microemulsion cyclosporin and tacrolimus on renal allograft fibrosis.

BACKGROUND: The aim of this study was to compare the effect of Neoral cyclosporin- and tacrolimus-based therapy on the development of renal allograft fibrosis (chronic allograft nephropathy; CAN) in a prospective randomized trial. METHODS: A total of 102 patients undergoing renal transplantation were randomized to immunosuppression with either microemulsion cyclosporin (Neoral; 15 mg per kg per day adjusted to whole-blood trough concentrations of 200-300 ng/ml) or tacrolimus (0.2 mg per kg per day adjusted to whole-blood trough levels of 8-15 ng/ml) in conjunction with steroids, or at a lower dose (7 mg per kg per day and 0.1 mg per kg per day respectively) with the addition of azathioprine for non-heart-beating renal transplant recipients. Renal transplant interstitial fibrosis was quantified using computerized histomorphometric measurement of picrosirius red-stained 1-year protocol renal transplant biopsies. Levels of interstitial fibrosis were compared in relation to observed efficacy and toxicity profiles of the two drugs. RESULTS: There was a significant increase in allograft interstitial fibrosis in the patients treated with Neoral compared with those given tacrolimus. There was no significant difference in the demographic characteristics between the patient groups or in the incidence of acute rejection (Neoral 36 per cent versus tacrolimus 35 per cent) or steroid-resistant rejection (both 10 per cent) between the two drugs. There was a higher incidence of insulin resistance in the tacrolimus group (post-transplant diabetes mellitus, glucose tolerance testing) but this was not statistically significant. Neoral was associated with a significant increase in total cholesterol (P = 0.030) and low-density lipoprotein (P = 0.021) levels, which persisted throughout the study period. CONCLUSION: Despite equivalent efficacy and pretransplantation risk factors for CAN, Neoral was associated with increased allograft fibrosis and significantly higher serum low-density lipoprotein cholesterol levels compared with tacrolimus.

Adult↗
PubMed2003

The effect of age on the outcome of surgical treatment for carcinoma of the oesophagus and gastric cardia.

OBJECTIVE: The aim of this study was to examine the effect of age on the outcome of surgical treatment for carcinoma of the oesophagus and gastric cardia. METHODS: From 1979 to 1999, 596 patients underwent gastro-oesophagectomy with two-field lymph node clearance for cancer under the care of a single surgeon. The clinicopathologic characteristics and survival of patients aged between 45 and 63 years (n=198, Group 1), 63 and 71 years (n=199, Group 2) and 71 and 89 years (n=199, Group 3) were compared. RESULTS: Thirty-day mortality for the first 300 patients (1979-1993) in this consecutive series was 5, 8 and 18% for Groups 1, 2 and 3, respectively, and 6, 6 and 6% for Groups 1, 2 and 3, respectively, in the second consecutive 296 patients (1993-1999, P=0.006, chi(2)). Tumours were poorly differentiated in 55.7, 59.1 and 53.4% of patients in Groups 1, 2 and 3, respectively, for 1979-1993 and 64.7, 53.2 and 40.2% of tumours in Groups 1, 2 and 3, respectively, for 1993-1999 (P=0.02, chi(2)). Adjuvant therapy was significantly more common in younger patients (P=0.006, chi(2)). Five-year survival in the first period was 22, 15 and 11% for Groups 1, 2 and 3, respectively, (P=0.02 log-rank) and 18, 16 and 14% for Groups 1, 2 and 3 in the second period (P=NS, log-rank). CONCLUSIONS: Elderly patients now have equivalent short and long-term outcomes compared to younger patients following gastro-oesophagectomy. Five-year survival, even in younger patients receiving adjuvant therapy remains poor, however, at approximately 20%. New therapeutic modalities are required to improve long-term survival following surgical treatment of gastro-oesophageal carcinoma.

Age Factors↗
PubMed2003

Rapamycin inhibits vascular remodeling in an experimental model of allograft vasculopathy and attenuates associated changes in fibrosis-associated gene expression.

BACKGROUND: Rapamycin inhibits extracellular matrix (ECM) accumulation (fibrosis) and vascular remodeling in experimental models of chronic allograft dysfunction (CAD) by poorly understood mechanisms. The aim of this study was to assess the effect of rapamycin on the expression of fibrosis-associated genes and correlate this with observed changes in ECM remodeling in an experimental of model allograft vasculopathy. METHODS: Vascular remodeling and ECM accumulation (picrosirius red) were measured by computerized histomorphometry of F344-to-Lewis rat aortic allograft sections harvested at serial timepoints. Expression of fibrosis associated genes was studied by means of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Rapamycin (0.5 mg/kg/day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodeling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intragraft gelatinase, collagen III and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA levels. At a lower dosage (0.25 mg/kg/day), rapamycin inhibited intimal hyperplasia and medial ECM accumulation, but there was a lesser effect on vascular remodeling. Lower dose allografts were also seen to have a more severe inflammatory infiltrate and larger amounts of intragraft matrix metalloproteinase 9 (MMP 9) mRNA than those treated with the higher dose. CONCLUSIONS: These data suggest that, in addition to the tissue response to injury, the alloimmune injury itself may contribute directly to the vascular remodeling that occurs in allograft vasculopathy. Rapamycin at higher but not lower doses inhibited both of these pathologic processes.

Animals↗
PubMed2003

Operative factors that contribute to post-operative atrial fibrillation: insights from a prospective randomized trial.

Atrial fibrillation (AF) is the most common complication following cardiac surgery and is associated with significant increases in postoperative morbidity, length of stay and cost of care. In a randomized study we assessed the impact of coronary artery bypass grafting (CABG) without cardiopulmonary bypass (CPB) (off-pump, n = 100), compared to conventional (on-pump, n = 100) CABG, on the frequency of postoperative AF. Arrhythmias were detected using an automated arrhythmia detector for the first 72 hours following surgery and by four hourly clinical observations thereafter. AF was defined as an irregular narrow complex rhythm (in the absence of bundle branch block) with absence of discrete P waves lasting greater then 10 minutes. There was a significant reduction in the incidence of postoperative AF in the off-pump group (11% versus 45%, P < 0.001) in addition to significant reductions in blood usage, postoperative pneumonia, inotrope requirements, and hospital and intensive care unit stay. Univariate analysis identified all these variables as risk factors for AF, however multivariate regression analysis identified CPB and cardioplegic arrest as the only independent predictor of postoperative AF (OR 7.4; 95% CI 3.4 to 17.9). This study therefore suggests that the inflammatory response to bypass, myocardial ischaemia and atrial cannulation are significant contributory factors to the development of AF following cardiac surgery. In the light of more recent trials it is apparent that this benefit may be most marked in patients with multiple perioperative risk factors for postoperative AF.

Adrenergic beta-Antagonists↗
PubMed2002

Microemulsion cyclosporin inhibits vascular remodelling and attenuates associated changes in profibrotic gene expression in an experimental model of allograft vasculopathy.

BACKGROUND: Chronic allograft dysfunction (CAD), the leading cause of solid organ transplant failure, is characterized by histological evidence of extracellular matrix (ECM) accumulation (fibrosis). The aim of this study was to characterize the changes in fibrosis-associated gene expression in an experimental model of CAD and to measure the effect of the immunosuppressant cyclosporin on these changes. METHODS: Lewis recipients of F344 rat thoracic to abdominal transplants were administered cyclosporin or no treatment. Vascular remodelling and ECM accumulation (picrosirius red) were measured using computerized histomorphometry. Fibrosis-associated gene expression was studied by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Cyclosporin inhibited medial ECM accumulation and vascular remodelling in allografts. This was associated with an attenuation of the graft inflammatory infiltrate and a reduction in intragraft matrix metalloproteinase (MMP) 2 and MMP-9 messenger RNA (mRNA) levels. There was a significant negative correlation between neoadventitial ECM density and MMP-9 expression, as well as with vessel circumference. Neoadventitial ECM density was significantly higher in the cyclosporin-treated group than in animals with untreated allografts, as were mRNA levels of collagen 3 and tissue inhibitor of metalloproteinase 1. CONCLUSION: The alloimmune injury itself may contribute directly to vascular remodelling and fibrosis in allograft vasculopathy. Cyclosporin attenuated this component of the pathophysiology of CAD effectively.

Animals↗
PubMed2002

Effects of the combination of rapamycin with tacrolimus or cyclosporin on experimental intimal hyperplasia.

BACKGROUND: Allograft vasculopathy remains the leading cause of late allograft failure following transplantation and can be inhibited by the antiproliferative drug rapamycin. This study assessed the efficacy of combining rapamycin therapy with calcineurin inhibition. METHODS: Male Sprague-Dawley rats received rapamycin 0.05 mg/kg daily and either tacrolimus 0.1 mg/kg or cyclosporin 5 mg/kg daily, and findings were compared with those in an untreated control group. Animals underwent left common carotid artery balloon angioplasty; the artery was explanted after 2 weeks. Morphometric analysis was performed on transverse sections and the intima : media ratio was calculated. Profibrotic gene expression was measured with competitive reverse transcriptase-polymerase chain reaction at 14 and 28 days. Proliferation was determined with proliferating cell nuclear antigen at 14 and 28 days. Extracellular matrix deposition was quantified with Sirius red. RESULTS: The combination of rapamycin and tacrolimus was associated with the greatest reduction in intimal thickening. Furthermore, treatment with rapamycin and tacrolimus significantly attenuated extracellular matrix deposition compared with rapamycin and cyclosporin (P < 0.02). CONCLUSION: The effects of rapamycin in combination with tacrolimus were better than those observed with rapamycin and cyclosporin.

Animals↗
PubMed2002

Pirfenidone inhibits early myointimal proliferation but has no effect on late lesion size in rats.

AIMS: intimal hyperplasia is mediated by smooth muscle cell proliferation, migration and deposition of extracellular matrix. The anti-fibrotic agent pirfenidone has been shown to inhibit pro-fibrotic growth factors in non-vascular inflammatory models. This study investigated the effect of the novel anti-fibrotic agent pirfenidone on the development of neointima. METHODS: male Sprague-Dawley rats received either standard diet or diet supplemented with pirfenidone (250, 500, 1000 mg/kg/day). Animals underwent left common carotid balloon angioplasty and were explanted at 4, 8, 14 and 28 days and analysed for intimal thickening, pro-fibrotic gene expression, extracellular matrix deposition and metalloproteinase activity. RESULTS: neointimal thickness was significantly reduced in a dose-dependent manner at 14 days; pirfenidone 250 mg/kg (p<0.005), pirfenidone 500 mg/kg (p<0.001), pirfenidone 1 g/kg ( p<0.001). There were no significant differences in intimal thickening at 28 days. Expression of MMP-2, MMP-9, TIMP-1, collagen III and TGF-beta were all significantly inhibited at 14 days. Both collagen III expression and ECM deposition were reduced at 28 days ( p<0.05 and <0.002 respectively). CONCLUSION: pirfenidone reduces expression of MMPs governing smooth muscle cell proliferation and migration (MMP-2 and 9), and genes favouring ECM accumulation (TIMP-1 and collagen III). This study shows that inhibition of MMP activity is not sufficient to inhibit late lesion size.

Angioplasty, Balloon↗
PubMed2002

Autogeneous elbow fistulas: the effect of diabetes mellitus on maturation, patency, and complication rates.

OBJECTIVES: diabetes mellitus is an increasingly common cause of end stage renal failure (ESRF) and the establishment of adequate permanent vascular access for dialysis is a major cause of morbidity and mortality in these patients. The aim of this study was to compare the availability of suitable vein, maturation rates, patency and complication rates of autogeneous elbow fistulas in diabetics and non-diabetics at a single centre where an autogeneous vein only policy is employed. DESIGN: retrospective series. PATIENTS AND METHODS: two hundred and ninety-three elbow fistulas were attempted in 232 patients over a seven year period, [median age 60 years (range 14-94 years)], of which 210 were in non-diabetic and 83 were in diabetic patients. The diabetic group had a significantly higher proportion of male patients (p < 0.05), peripheral vascular disease and established ESRF. RESULTS: there was a trend towards a higher technical success rate in the non-diabetic group (98% versus 93% p = 0.057). There was no significant difference between the primary failure rate, fistula maturation rate, revision rate or incidence of complications between the two groups. Diabetes had no effect on cumulative secondary fistula patency even when stratified for Type 1/Type 2 diabetes, female sex, old age or primary versus subsequent procedures. CONCLUSION: diabetes mellitus has no significant detrimental effect on outcome following formation of autogeneous elbow fistulas for haemodialysis.

Adolescent↗
PubMed2002

An aggressive policy of bilateral saphenous vein harvest for infragenicular revascularisation in the era of multidrug resistant bacteria.

BACKGROUND: The success of infragenicular revascularisation for lower limb ischaemia is limited by the high proportion of patients without ipsilateral long saphenous vein (LSV) of adequate length or quality. The aim of this study was to report the results of an autogenous vein only policy for infragenicular revascularisation utilising contralateral LSV when ipsilateral LSV is inadequate. The treatment and outcome of infection of autogenous grafts with methicillin resistant Staphylococcus aureus (MRSA) is also reported. PATIENTS AND METHODS: The vascular audit database and patient case notes were reviewed retrospectively for patients with arterial occlusive disease requiring infragenicular reconstruction. There were 68 critically ischaemic legs in 65 patients of whom 48 were male: median age (range) 74 years (41-94), over a three year period. RESULTS: Thirty six patients (53%) underwent revascularisation (eight infragenicular femoropopliteal bypass, 28 femorodistal), 24 (35%) underwent primary amputation and a further eight (12%) were found to have unsuitable distal vessels for revascularisation after tibial vessel exploration and intraoperative angiography. Thirty three grafts (92%) utilised LSV and three (8%) were polytetrafluoroethylene grafts. Thirteen patients (39%) lacked adequate ipsilateral LSV of whom 12 had the contralateral leg explored providing suitable LSV in 10/12 (83%). Contralateral LSV was used as a single length conduit in two cases and as a venovenous composite graft in eight cases. Primary, primary assisted, and secondary patency rates at two years were 38%, 77%, and 81% respectively. Actuarial limb survival and patient survival rates at two years were 86% and 61% respectively. Eleven patients developed ipsilateral wound complications (30%) including seven (21%) who developed MRSA infection of the ipsilateral leg wound. MRSA wound infection was treated successfully in all cases by antibiotic therapy (intravenous vancomycin). No patient subsequently required saphenous vein harvesting for a secondary reconstruction or coronary artery bypass graft. CONCLUSION: Excellent long term results can be achieved using autogenous vein for infragenicular revascularisation and the contralateral LSV is an excellent alternative in the absence of suitable ipsilateral LSV. Autogenous vein may confer some protection against severe complications observed with MRSA infection seen in vascular patients and therefore its use is recommended.

Adult↗
PubMed2002

Elbow fistulas using autogeneous vein: patency rates and results of revision.

BACKGROUND: The provision and maintenance of vascular access remains a major cost to end stage renal failure programmes. There are few reports regarding the surgical revision of the failing native elbow arteriovenous fistula (AVF). PATIENTS AND METHODS: A retrospective case note review was performed on all patients identified from the hospital vascular access database as having undergone construction of an autogeneous vein elbow AVF. Over a seven year period 282 autogeneous vein AVFs were fashioned in 232 patients using the brachial artery as the in-flow conduit. Of these 208 were brachiocephalic fistulas, or a variant thereof, and 74 were fashioned using the transposed autologous basilic vein (136 male: 96 female; median age 60 years, range 14-94 years). RESULTS: Of 282 elbow fistulas 197 were successfully used for dialysis (70%). Cumulative primary patency of elbow fistulas using autogeneous vein in this series was 68%, 54%, and 44% at one, two, and three years respectively. A further 34 revision procedures were performed on 28 fistulas to maintain fistula function, and cumulative secondary patency after surgical revision was 75%, 60%, and 46% at one, two, and three years. Overall 21 out of 34 procedures (62%) successfully restored fistula function and cumulative primary patency of the revised fistulas was 56% at one year. Eighteen AVFs (brachiocephalic, n=12; autologous basilar vein, n=6) required revision for access dysfunction secondary to a short stenoses within 4 cm of the arteriovenous anastomoses. Of these 18 AVFs eight were revised by excision of the stenosed segment and either primary anastomoses of the two cut ends of arterialised vein or reanastomoses of the proximal venous limb proximally on the brachial artery. In another nine fistulas the excised segment was replaced with a short interposition graft (polytetrafluoroethylene, n=7; native basilic vein, n=1; bovine carotid artery, n=1). One fistula with postanastomotic stenoses and a more proximal needle site stenoses was revised using two vein patches. Overall 100% were patent at 24 hours, 13 provided successful dialysis (72%), and cumulative primary patency was 67% and 50% at six months and one year respectively. CONCLUSIONS: Successful surgical revision of failing native elbow fistulas can restore patency and improve cumulative secondary patency with potential benefits in terms of patient morbidity and mortality. These results compare favourably to published patency rates after fistula salvage using interventional radiological techniques.

Adolescent↗
PubMed2001

Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases.

GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

Amino Acid Sequence↗
PubMed2001

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels.

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.

Animals↗
PubMed2001

A case-control comparison of the results of renal transplantation from heart-beating and non-heart-beating donors.

INTRODUCTION: The decline in heart-beating brainstem dead organ donors has necessitated the search for other organ sources. In the field of renal transplantation one alternative source currently available, but little used, is that of kidneys from non-heart-beating donors (NHBD). Reticence to use NHBD kidneys is in part due to concerns over the effect that warm ischemic may have subsequent graft function. Presented here are the results of the NHBD renal transplants at the Leicester transplant unit, and compared with matched heart-beating donor transplants as a case control analysis. METHODS: In order to analyze any differences in graft performance between the two organ sources, the confounding effect of other variables known to influence the outcome of renal transplantation was minimized by matching NHBD and HBD transplants for the following criteria: donor age and sex, first or re-transplant, anastomosis and cold times, tissue match and PRA sensitisation. Transplant performance was assessed primarily by graft survival, the statistical evaluation of which was by log rank analysis of Kaplan-Meier curves. RESULTS: 72 NHBD and 192 HBD transplants were performed over an eight year period. Of the 192 HBD transplants, 105 matched one or more of the NHBD by the criteria outlined above, and thus constituted the control group for comparison. There was no significant difference in overall graft survival between the two groups. The 5 year survival for the NHBD was 73% compared with 65% for HBD kidneys. When death with a functioning graft is treated as censored data, then these figures become 75% and 81% respectively, again without statistical significance. CONCLUSION: NHBD kidneys are a valuable additional source of organs for transplantation, with long-term survival, comparable to transplants from HBD.

Acute Disease↗
PubMed2001

Characterization of Mpl mutants using primary megakaryocyte-lineage cells from mpl(-/-) mice: a new system for Mpl structure-function studies.

Mpl is the thrombopoietin (TPO) receptor. The current molecular understanding of how Mpl activation stimulates proliferation of megakaryocyte-lineage cells is based largely on the engineered expression of Mpl in nonmegakaryocyte-lineage cell lines. However, the relevance of these findings to Mpl signaling in primary megakaryocyte-lineage cells remains largely unknown. Therefore, a system was developed to study Mpl function in primary mpl(-/-) megakaryocyte-lineage cells. Expressing avian retroviral receptors on the surfaces of mammalian cells overcomes their natural block to avian retroviral infection; 815 bp of human GPIIb regulatory sequence was used to generate transgenic mice with megakaryocyte-lineage expression of the subgroup A avian leukosis virus receptor, TVA. Avian retroviral infection of unfractionated bone marrow from these mice is restricted to megakaryocyte-lineage cells. The transgenic mice were crossed to an mpl(-/-) background generating GPIIb-tva+mpl(-/-) mice. By using avian retroviruses to express wild-type or mutant Mpl on the surfaces of primary megakaryocyte-lineage cells, it was demonstrated that (1) the 10 membrane-proximal, cytoplasmic amino acids of Mpl are required for TPO-induced proliferation; (2) Y582F mutation confers a proliferative advantage over wild-type Mpl and imparts a constitutive anti-apoptotic signal; (3) truncating the 50 C-terminal Mpl amino acids reduces but does not eliminate TPO-induced mitogen-activated protein kinase activation, yet it does not alter the synergistic effect of stem cell factor on TPO-induced proliferation; and (4) TPO-induced proliferation of early, primary megakaryocyte-lineage cells does not require Stat-5 phosphorylation. The system reported provides an improved approach for Mpl structure-function studies, and the method can be applied to any hematopoietic lineage.

Amino Acid Sequence↗