Biomedical subjects
S Murphy
Publications and source records attributed to S Murphy.
Mismatched platelet transfusions to alloimmunized patients.
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Outbreak of staphylococcal dermatitis in housed lactating Suffolk ewes.
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Differential in vivo activation of the class II and class III snRNA genes by the POU-specific domain of Oct-1.
Many snRNA genes contain binding sites for the ubiquitous transcription factor Oct-1. In vitro studies have shown that this factor potentiates binding of an essential transcription factor (PTF) to the proximal sequence element (PSE) of snRNA genes, and activates transcription. Using Gal4 fusion proteins, I show here that the POU-specific region of the DNA-binding domain of Oct-1 is sufficient both to potentiate PTF binding in vitro and to transactivate pol II- and pol III-dependent snRNA genes in vivo . A single amino acid change in this domain abrogates both activation and interaction with PTF. The N- and C-terminal regions of Oct-1 also activate transcription of both classes of snRNA genes. Wild-type levels of Pol II-dependent U2 expression require all activation domains, whereas efficient activation of the pol III-dependent 7SK and U6 genes is effected by the POU-specific domain alone. These results indicate that contacts between PTF and amino acids in the POU-specific domain of Oct-1 are critical for efficient transactivation of snRNA genes in vivo. The POU-specific domain of Oct-2A also activates these genes, but the N- and C-terminal domains are relatively inactive.
HIV-induced T cell syncytia are self-perpetuating and the primary cause of T cell death in culture.
Prior studies suggested that induced fusion in T cell cultures infected with syncytium-inducing HIV was not the primary cause of T cell death. However, these studies failed to assess the contribution of fusion in terms of syncytium volume, rather than syncytium frequency. This question has been reassessed by monitoring the frequency, volume, rate of growth, longevity, p24 production, viral budding, and self-propagating ability of syncytia in HIV-infected SUP-T1 cell cultures and individually isolated syncytia seeded in uninfected SUP-T1 cell cultures. The results demonstrate that in these cultures syncytium formation is the principal mechanism of T cell death, syncytia are the main source of virus production, and both virus production and syncytium longevity are independent of syncytium size, but dependent on syncytium age, suggesting that both are programmed events.
Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients.
We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1alpha. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- +/- 5-fold expansion of CD34+/CD61+ cells, a 17- +/- 5-fold expansion of colony-forming units-megakaryocytes, and a 58- +/- 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 x 10(5)/kg CD61 cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.
Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappaB binding to DNA.
Treatment of astroglial cells with interleukin 1beta and interferon gamma transcriptionally activates the nitric oxide synthase (NOS)-2 gene. The duration of mRNA expression is brief because of transcript instability. In addition, NO donors reduce the expression of NOS-2 mRNA dramatically by reducing the rate of transcription. In this study we observed that the NO donor, spermine NONOate did not inhibit the activation and translocation of NF-kappaB, a key transcription factor in the induction of NOS-2, but inhibited formation of the NF-kappaB-DNA complex. This effect was reversed by methaemoglobin (acting as an NO trap) and by the reducing agent dithiothreitol. Formation of the interferon-regulatory factor-DNA complex was unaffected by NO. These results suggest that NO can modulate its own production by interfering with NF-kappaB interaction with the promoter region of the NOS gene, a negative feedback effect that may be important for limiting NO production in vivo.
Serum erythropoietin levels in children with leukemia.
OBJECTIVE: Our aim was to test the hypothesis that, in leukemic children, serum erythropoietin (EPO) levels vary inversely with hemoglobin. DESIGN: Twenty-four children (15 males, nine females) with an age range of 1-16 years (mean, 7.7 years) diagnosed with acute leukemia (22 acute lymphocytic, two acute myeloid) were studied over 4 months. Serum EPO and hemoglobin were measured simultaneously at multiple time points in the course of their disease, and a multiple regression analysis was performed to describe the EPO-hemoglobin relationship. RESULTS: In a model adjusted for individual subject, there was a significant correlation between hemoglobin and logEPO in these leukemic children (r = -0.55, P < .01, n = 100). When measurements at hemoglobins less than 10.0 were analyzed the correlation increased significantly (r = -0.88, P < .01, n = 21). However, approximately 20% of the observations fell into one of two groups: an inappropriately low EPO for hemoglobin or an inappropriately elevated EPO for hemoglobin. The clinical characteristics of the children at each of these determinations were not different in any manner from the determinations which fell within the 95% confidence intervals for predicted mean EPO value: each of the outlying points came from a patient who at other times had an appropriate EPO for hemoglobin. CONCLUSIONS: There existed a significant inverse relationship between hemoglobin and EPO, suggesting that the feedback mechanism for EPO is intact. Reasons for inappropriately high or low EPO, for level of hemoglobin, are not clear and may be reflective of other aspects of bone marrow or EPO metabolism.
Activated astrocytes induce nitric oxide synthase-2 in cerebral endothelium via tumor necrosis factor alpha.
Astrocytes under pathological conditions become activated and produce a variety of cytokines and low molecular weight signal molecules. Previously we demonstrated that activated astrocytes release nitric oxide which can downregulate the expression of nitric oxide synthase (NOS)-2 in co-cultured cerebral endothelium, and also release a transcriptionally regulated factor that can induce NOS-2 expression in endothelium (Borgerding and Murphy: J Neurochem 65:1342, 1995). The activity of this NOS-2-inducing factor was impeded by inhibitors of tyrosine kinases and NF-kappaB activation. Tumor necrosis factor (TNF alpha) alone, or in combination with IL-6, induced NOS-2 expression in endothelial cells. A neutralizing antibody against TNF alpha attenuated the NOS-2 expression in endothelial cells exposed to activated astrocytes. These results imply that cytokine-activated astrocytes release TNF alpha which can induce NOS-2 expression in endothelium and suggest that activated astrocytes within the CNS may induce expression of NOS-2 in cells of the adjacent microvasculature. The ensuing alterations in blood-brain barrier properties may be either beneficial or detrimental.
Gene therapy of Duchenne muscular dystrophy.
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Orthodromic activation of peptidergic cells in the medial amygdala.
Electrical recordings from vasopressin-containing cells in the medial amygdala were obtained. Electrical stimulation of one major afferent structure, the accessory olfactory bulb, invariably elicited single unit discharge in the peptidergic cells and set up a field potential indicating widespread excitation in the structure. Pheromonal stimuli, normally borne into the brain by the accessory olfactory bulb, were ineffective in activating the medial amygdala. These results in combination with preexisting research suggest that the accessory olfactory bulb is an important influence, but not the only influence, on the activity of the peptidergic cells.
Thalamic NMDA receptors modulate inflammation-produced hyperalgesia in the rat.
The effects of inhibition of thalamic NMDA receptor function and synthesis on thermal and mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan in the rat were studied in the 'acute' phase (within 3-5 h) and the 'subacute' phase (24 h) after carrageenan administration. Blockade of NMDA receptors was produced by intrathalamic injection of D,2-amino-5-phosphonovaleric acid (D-APV) and NMDA receptor synthesis was decreased (or not) by pretreatment of rats with intrathalamic (hindlimb representation area) injections of antisense, sense or missense oligodeoxynucleotides (ODNs) directed against the NR1 subunit of the NMDA receptor complex. Treatment with D-APV, but not saline, in the contralateral (but not ipsilateral) thalamus significantly reduced both the acute thermal and mechanical hyperalgesia in the injected paw; these same rats demonstrated significantly less thermal and mechanical hyperalgesia in the sub-acute phase than rats that had received saline or D-APV in the ipsilateral thalamus. None of the treatments had any effect on withdrawal responses of the uninjected hindpaw. Rats pretreated with NR1 sense or missense ODNs developed both thermal and mechanical hyperalgesia that was equivalent in magnitude and duration to rats that received intrathalamic saline injections. In contrast, rats pretreated with NR1 antisense ODN did not develop either acute or subacute thermal hyperalgesia; they developed less mechanical hyperalgesia than saline, sense or missense ODN-treated rats. Antisense ODN-treated rats also displayed a decrease in the number of thalamic NMDA receptors as determined by receptor binding assay. These results suggest an involvement of thalamic NMDA receptors in the development and maintenance of hyperalgesia associated with neurogenic inflammation in a model of tonic pain.
Paramyotonia congenita and hyperkalemic periodic paralysis associated with a Met 1592 Val substitution in the skeletal muscle sodium channel alpha subunit--a large kindred with a novel phenotype.
Paramyotonia congenita (PC) and Hyperkalemic periodic paralysis (HyperPP) are caused by amino acid substitutions in the alpha subunit of the human skeletal muscle sodium channel. One such substitution, methionine for valine at position 1592, has been associated with HyperPP with myotonia and cold sensitivity. We report clinical, electromyographic (EMG), genetic and pathological features of a large kindred with the Met1592Val substitution. Affected members were phenotypically heterogenous and had episodic potassium-sensitive paralysis, and stiffness and weakness induced by exercise and cold, which was confirmed by EMG studies. These features indicate a combined PC-HyperPP phenotype not previously described with this mutation.
Health locus of control and value for health in smokers and nonsmokers.
A representative sample of 11,401 persons completed a questionnaire, including measures of health locus of control, value for health, and smoking frequency. Smokers held stronger internal, chance, and powerful others beliefs than never smokers. Ex-smokers had lower scores on internal and chance dimensions and placed a higher value on their health than smokers. The interaction between chance health locus of control and value for health was a significant predictor of smoking status, suggesting that health value may moderate the relationship between health locus of control and smoking status. Within smokers, the health locus of control dimensions and value for health explained less than 1% of the variance in smoking frequency, with only the chance dimension emerging as a significant predictor.
The hyper-CVAD regimen improves outcome in relapsed acute lymphoblastic leukemia.
Sixty-six adults with refractory acute lymphocytic leukemia received salvage therapy with the 'hyper-CVAD' regimen, consisting of eight courses of alternating intensive chemotherapy with growth factor support, followed by oral maintenance chemotherapy. Their outcome was compared with 63 prognostically similar historical control patients treated with high-dose Ara-C plus mitoxantrone with or without GM-CSF. Overall, the complete response rates were similar in the treatment and control groups (29 of 66 (44%) vs 24 of 63 (38%)). There were more patients in the current study with primary resistant disease (10 of 66 (15%) vs one of 63 (2%), P = 0.006), and conversely fewer patients with secondary resistance (19 of 66 (29%) vs 28 of 63 (44%), P = 0.06). Recovery of granulocyte counts above 500/microl was significantly faster in the current study when compared to high-dose Ara-C-treated patients who were given GM-CSF (20 vs 25 days, P = 0.04). Survival was prolonged in the hyper-CVAD-treated patients, with most of the benefit seen in first salvage patients (42 vs 20 weeks, P = 0.016). When only first salvage patients were considered, there was a significant difference in disease-free survival in favor of hyper-CVAD (52 vs 20 weeks, P = 0.008). The hyper-CVAD regimen is a more effective and less toxic salvage regimen for relapsed acute lymphocytic leukemia than high-dose Ara-C-based regimens.
Regulation of astrocyte nitric oxide synthase type II expression by ATP and glutamate involves loss of transcription factor binding to DNA.
Pretreatment of astrocytes with either glutamate or ATP, acting via specific receptors, suppresses subsequent cytokine-induced expression of type II nitric oxide synthase (NOS). This effect is downstream from the activation and translocation of nuclear factor (NF)-kappaB, as western blotting revealed no difference in accumulation of the p50 subunit in nuclear extracts from agonist-treated vs. nontreated cells. However, evidence from gel-shift assay suggests that the binding of nuclear protein from agonist-treated cells to NF-kappaB and activator protein-1 consensus oligonucleotides is markedly reduced. Selective inhibitors of protein kinases A and C could not restore either binding of transcription factors to DNA or type II NOS mRNA expression in agonist-pretreated cells. The modulation of proinflammatory cytokine-evoked type II NOS expression by ATP and glutamate may play an important role in CNS pathologies associated with stroke and trauma.
Fat embolism with the use of intraosseous infusion during cardiopulmonary resuscitation.
The objective of this prospective study was to assess the incidence and magnitude of fat emboli after cardiopulmonary resuscitation and intraosseous infusions. An animal laboratory at a university center was used to study 33 mixed-breed piglets. The piglets underwent hypoxic cardiac arrest followed by chest compressions and mechanical ventilation for a minimum of 30 minutes. The animals were divided in groups: group 1 (n = 5), which had no intraosseous cannulas, group 2 (n = 6), which had intraosseous cannulas with infusion, groups 3 (n = 6), 4 (n = 6), and 5 (n = 8), which had intraosseous cannulas with infusion of epinephrine, normal saline, and sodium bicarbonate respectively, and group 6 (n = 2), which was a sham group with no intraosseous cannulas and no cardiopulmonary resuscitation. At cessation of cardiopulmonary resuscitation, representative lung samples were collected from upper and lower lobes of each lung and observed for fat globules and bone marrow elements. Fat globules were seen in the peribronchial blood vessels and intravascular areas throughout all lung fields of groups 1 through 5. There was no difference in appearance or distribution of fat globules among the 5 treatment groups. Analysis of variance showed no statistical significance (P < 0.05) within or among groups 1 through 5. The use of the intraosseous cannula for infusion of emergency drugs and fluids did not increase the magnitude of fat embolization over cardiopulmonary resuscitation alone in this animal model. The benefits of using this procedure in critically ill children as a means of rapid vascular access for resuscitation is well established. However, the risk of fat embolism in this population needs further study.
Intraosseous and central venous blood acid-base relationship during cardiopulmonary resuscitation.
OBJECTIVE: The objectives of this study were: 1) to determine whether obtaining intraosseous (IO) blood samples was practical during cardiopulmonary resuscitation (CPR), and 2) to compare the acid-base status (pH and partial pressure of CO2 (PCO2) of venous and IO blood during CPR. DESIGN: A prospective repeated measure study. SETTING: An animal laboratory at a university medical center. INTERVENTIONS: Nine mixed breed piglets (mean weight 43 kg) were anesthetized, tracheotomized, and placed on a ventilator (Siemens 900C Elema, Sweden). Placement of a pulmonary artery catheter was done via a surgical incision in the neck. An IO cannula was then placed in the tibial marrow cavity. The animals were positioned under a mechanical thumper (Thumper, Michigan Instruments, Grand Rapids, MI) for chest compressions. Blood gases were analyzed during steady state (baseline) after five minutes of ventricular fibrillation and during CPR at seven, nine, 11, 13, 15 and 18 minutes. MAIN RESULTS: Blood samples for acid-base analysis were easily obtained from the IO sites during all sampling times. Mixed venous blood was slightly more acidic than IO blood, especially at 13, 15, and 18 minutes. However, there were no significant differences in pH and Pco2 values between IO and central venous (CV) gases at all time intervals except the PCO2. At nine minutes, a significant difference (P < 0.006) was found in PCO2 (59 +/- 4 vs 47 +/- 5 torr) for the CV versus IO sample, respectively. As the duration of CPR progressed, the differences in PCO2 between IO and CV sites were clinically relevant (though not statistically significant). CONCLUSION: Obtaining blood from the IO site is practical during CPR. The divergence in values as CPR progresses suggests that, during longer periods of CPR, IO blood may reflect local acidosis and yield lower PCO2 and higher pH values that CV blood. This finding may limit the usefulness of IO blood to judge acid base status as CPR progresses.