Biomedical subjects
Publications and source records attributed to K Shannon.
Explore the source record for details and available documents.
Many agents are being considered for treatment of Parkinson disease (PD). Given the large number of agents and the limited resources to evaluate new agents, it is essential to reduce the likelihood of advancing ineffective agents into large, long-term Phase III trials. Futility design methodology addresses this goal. The authors describe how a single-arm Phase II futility study uses a short-term outcome to compare a treatment group response to a predetermined hypothesized or historically based control response. The authors present advantages and limitations of futility designs along with examples derived from the data archive of a large Phase III efficacy study of treatments to delay PD progression, the Deprenyl And Tocopherol Antioxidative Therapy Of Parkinsonism (DATATOP) trial. Using the same control progression rate and treatment effect assumptions used to power the original DATATOP trial, the authors calculated the number of subjects needed to conduct two 12-month futility studies. DATATOP was designed to enroll 800 patients. Using data on 124 consecutive subjects randomized into each of the DATATOP treatment groups, the authors identified tocopherol as futile and deprenyl as worthy of further study. Using Phase II information, DATATOP could have been simplified from a 2 x 2 factorial design to a comparison of deprenyl vs placebo. While not testing efficacy, futility designs provide a strategy for discarding treatments unlikely to be effective in Phase III. A limitation is the dependence on historical data or hypothesized outcomes for untreated controls. Futility studies may decrease the time to identify treatments unworthy of further pursuit and reduce subjects' exposure to futile treatments.
Severe congenital neutropenia (CN) is characterized by a maturation arrest of myelopoiesis at the promyelocyte stage. Treatment with pharmacological doses of recombinant human granulocyte colony-stimulating factor (rh-G-CSF) stimulates neutrophil production and decreases the risk of major infectious complications. However, approximately 15% of CN patients develop myeloid malignancies that have been associated with somatic mutations in the G-CSF receptor (G-CSFR) and RAS genes as well as with acquired monosomy 7. We report a CN patient with chronic myelomonocytic leukemia (CMML) who never received rh-G-CSF. Molecular analysis demonstrated a somatic G-CSFR mutation (C2390T), which led to expression of a truncated G-CSFR protein in the CMML. Normal G-CSFR expression was unexpectedly absent in primary and cultured CMML. In addition, CMML cells showed monosomy 7 and an oncogenic NRAS mutation. In vitro culture revealed a G-CSF-dependent proliferation of CMML cells, which subsequently differentiated along the monocytic/macrophage lineage. Our results provide direct evidence for the in vivo expression of a truncated G-CSFR in leukemic cells, which emerged in the absence of rh-G-CSF treatment and transduces proliferative signals.
BACKGROUND: Frequent use of emergency rooms by injection drug users (IDUs) has been attributed to a lack of access to primary care and barriers to health services. Using a community-based sample of IDUs, we examined rates of primary care and emergency room use among IDUs and identified correlates of frequent emergency department use. METHODS: From January to November 2003, we enrolled IDUs into a prospective cohort study involving a baseline questionnaire, comprehensive retrospective and prospective health record linkages. We examined rates of primary care and emergency department utilization, and diagnoses upon arrival in the emergency room. Logistic regression was used to determine factors independently associated with frequent emergency room use. RESULTS: Of the 883 IDUs included in this analysis, 687 (78 per cent) accessed a primary care clinic in the previous year, while 528 (60 per cent) participants accessed the emergency room (ER) during the years 2002 and 2003. Abscesses, cellulitis and other skin infections accounted for the greatest proportion of ER use. Factors independently associated with frequent ER use included: frequent crystal methamphetamine injection (AOR = 2.4, 95 per cent CI: 1.0-5.6); non-fatal overdose (AOR = 2.1, 95 per cent CI: 1.4-3.3); HIV-positive status (AOR = 1.5, 95 per cent CI: 1.1-2.1), having been physically assaulted (AOR = 1.5, 95 per cent CI: 1.1-2.1); and primary care utilization (AOR = 1.5, 95 per cent CI: 1.0-2.1). DISCUSSION: high rates of ER use were observed among IDUs, despite high rates of primary care use among this same population. ER use was due primarily to preventable injection-related complications that are less amenable to primary care interventions, and therefore educational and prevention efforts that encourage and enable sterile injection practices should be promoted.
OBJECTIVE: We studied the ability of patients with Parkinson's disease to improve their performance in a motor task requiring both speed and accuracy in the execution of elbow flexion movements. Our goal was to investigate the changes in electromyographic activity associated with the changes in movement performance. METHODS: Eleven patients on anti-Parkinsonian medication were tested. The patients were selected for being bradykinetic, having little or no resting tremor or dyskinesias, and being in stages II or III of the Hoehn and Yahr rating scale. RESULTS: The untrained patients displayed multiple bursts of agonist activity, characteristic of Parkinsonian EMG recordings. All patients improved their performance by increasing peak velocity while maintaining movement accuracy within strict boundaries. With practice, the patients' performance changed in a manner similar to that which has been previously observed for performance curves in neurologically normal subjects. As movement duration decreased (i.e. peak velocity increased), we observed a slight decrease in the number of agonist bursts and an increase in the average burst duration. However, the patients continued to generate a fractionated, multi-burst agonist pattern. CONCLUSIONS: We conclude that Parkinsonian patients benefit from practice by improving their performance but remain fundamentally impaired in the generation of muscle activation patterns. This study has shown that the generation of fractionated, multiple short bursts of EMG activity that is characteristic of movements made by Parkinsonian patients is not normalized by practice.
Klebsiella aerogenes K41, resistant to third generation cephalosporins and aminoglycosides, was isolated from clinical samples of 153 in-patients. Blood cultures accounted for 24 (15.7%) of isolates. The MIC(90) of ceftazidime for the isolates of 84 patients was >512 mg/l and was reduced to 2.0 by 4 mg/l of clavulanic acid, but only to 64 by 4 mg/l of sulbactam. Isolates of K. aerogenes K41 produced extended-spectrum beta-lactamase (ESBL) SHV-5 and TEM-1, identified by isoelectric focusing. Plasmid profiles showed that co-dissemination of cephalosporin and aminoglycoside resistance, plus ESBL production, coincided with the acquisition of a 116-kb plasmid. This plasmid was transferable in vitro from K. aerogenes K41 to other serotypes and genera of the Enterobacteriaceae.
In the emergence of the evidence based practice movement, critical care nurses have struggled to identify scientific evidence on which to base their clinical practice. While the lack of critical care nursing research is a major concern, other important issues have significantly stalled the implementation of evidence even when it is available. A descriptive study of 274 critical care nurses was undertaken to examine nursing research activity in Victorian critical care units. The study aimed to identify critical care nurses' research skills, the barriers encountered in participation and implementation and the current availability of resources. Results revealed that 42 per cent of the nurses who participated in the study believed that they were not prepared adequately to evaluate research, and less than a third believed they were sufficiently skilled to conduct valid scientific studies. An association was found between nurses' ability to confidently perform research activities and higher academic qualifications. The study found that there is a lack of organisational support and management commitment for the development of evidence based nursing. In order to facilitate the implementation of evidence based practice, clinicians must be made aware of the available resources, be educated and mentored when carrying out and using clinical research, and be supported in professional initiatives that promote evidence based practice. It is argued that this will have positive implications for patient outcomes in the critical care environment.
Explore the source record for details and available documents.
The NF1 tumor-suppressor gene is frequently inactivated in juvenile myelomonocytic leukemia, and Nf1 mutant mice model this myeloproliferative disorder (MPD). Competitive repopulation assays were performed to quantify the proliferative advantage of Nf1(-/-) hematopoietic cells in vivo. Nf1 mutant stem cells demonstrated a growth advantage that was greatest in myeloid lineage cells and least pronounced in T lymphocytes. Surprisingly, although low numbers of Nf1-deficient cells consistently outcompeted wild-type cells, levels of chimerism were stable over months of observation, and MPD was not observed unless threshold numbers of mutant cells were injected. These data showing that normal competitor cells can strongly modulate the growth of mutant populations in vivo have general implications for modeling cancer in the mouse. In particular, strains in which cancer-associated mutations are expressed in fields of target cells may not accurately model early events in tumorigenesis because they eliminate the requirement for a mutant clone to outcompete resident normal cells.
Amiodarone (AD) is gaining support as a first-line antiarrhythmic drug despite its potentially fatal pulmonary toxicity involving inflammation and fibrosis. The goals of this study were to characterize a rat model of AD-induced pulmonary toxicity (AIPT) and identify a serum biomarker to aid in the diagnosis of the onset of pulmonary toxicity. Male F344 rats were instilled intratracheally with AD (6.25 mg/kg with a 3.125 mg/ml solution) in sterile water or the sterile water vehicle on days 0 and 2, a protocol that led to the development of pulmonary fibrosis on day 28 in the AD-treated animals. Animals were killed on days 3, 5, 6, 7, or 10 and bronchoalveolar lavage (BAL) was performed. Recovery of alveolar macrophages and eosinophils was increased on days 3 and 5, while neutrophil recovery and albumin levels in the first BAL fraction were significantly elevated only on day 3. BAL cells recovered from AD-treated rats at day 3 produced more phorbol myristate acetate-stimulated luminol-dependent chemiluminescence (LDCL) over 20 min than BAL cells from control rats. Experiments using specific inhibitors implicated superoxide and nitric oxide in at least part of the LDCL response. Serum levels of surfactant protein-D (SP-D), a surfactant-associated protein, were increased concurrently with the inflammatory response in the lungs. These findings indicate that this model exhibits transient pulmonary inflammation and damage, with the potential for elevated oxidant production in the lungs and subsequent pulmonary fibrosis. Also, SP-D is proposed as a specific biomarker to monitor the onset of AIPT in this model.
Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.
Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.
The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for members of the p21(ras) (Ras) family, which negatively regulates Ras output by accelerating the conversion of active Ras. GTP to inactive Ras.GDP. Analysis of tumors from patients with neurofibromatosis type 1 (NF1) has shown biochemical evidence of hyperactive Ras as well as frequent loss of the normal NF1 allele, consistent with its role as a tumor suppressor gene. Taken together, these data suggest that novel therapeutics directed against components of the Ras signaling cascade might provide effective treatments for certain pathological complications of NF1. Here we summarize data that support a role for hyperactive Ras in NF1 disease, including Ras processing, activation, and down-regulation. We review targets for rational drug design, provide preliminary results, and discuss implications for future studies. Am. J. Med. Genet. (Semin. Med. Genet.) 89:14-22, 1999.
Explore the source record for details and available documents.
Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.
Between July and September 1997 a ceftazidime- and aminoglycoside-resistant strain of Klebsiella pneumoniae infected or colonized seven patients on three paediatric wards at Guy's Hospital in London. The patients were mostly neonates or infants recovering from cardiac surgery for congenital defects. The organism was probably introduced by an asymptomatic patient from Greece and the subsequent outbreak could largely be explained by person-to-person spread on individual wards and frequent transfers of patients between wards. The outbreak was controlled by patient isolation and attention to handwashing, and there were no fatalities. The organisms were non-typeable by serology but had a characteristic RAPD profile. They produced the extended spectrum beta-lactamase SHV-5 and the aminoglycoside-modifying enzymes AAC(6') + probably AAC(3)II, encoded on a conjugative plasmid of approximately 160 kb. Two other patients had multi-resistant klebsiellas, one of them an SHV-5 producer and one a TEM-5 producer, but these could be distinguished from each other and from the outbreak strain by serological and RAPD typing and by the genetics and mechanisms of their resistances. Three other multi-resistant enterobacteria were isolated during the outbreak: an Escherichia coli that had acquired the 160 kb resistance plasmid from the epidemic klebsiella, a Citrobacter isolated from one of the patients with the klebsiella but which did not produce SHV-5, and a TEM-5-producing Enterobacter. This outbreak illustrates the importance of screening patients from high-risk areas for multiply-resistant organisms on admission, and the value of bacterial typing and analysis of resistance mechanisms to define the epidemiology of hospital infection.
Treatment failures with vancomycin prompted us to investigate the phenomenon of tolerance to glycopeptides in recent clinical isolates of Staphylococcus aureus. We used both MBC/MIC determinations and time-kill measurements to study tolerance to vancomycin and teicoplanin in 35 blood or heart valve isolates of S. aureus from patients with endocarditis or bacteraemia. There was generally good agreement between vancomycin tolerance indicated by an MBC:MIC ratio of > or =32 and by < or =90% kill after 6 h incubation in the presence of 20 mg/L vancomycin. However, two isolates were tolerant according to their MBC:MIC ratios but non-tolerant as judged by time-kill measurements. Seven of 15 methicillin-resistant S. aureus (MRSA) isolates but only two of 20 methicillin-susceptible ones were tolerant as judged by time-kill experiments (chi2 = 4.27 with Yates' correction, P = 0.04). Seven of the 16 isolates from patients with endocarditis were tolerant, compared with only two of the 19 isolates from patients with other conditions (chi2 = 3.43 with Yates' correction, P = 0.06). Within the endocarditis and non-endocarditis subgroups, tolerance was associated more frequently with methicillin resistance than with susceptibility, but the numbers were too small for the differences to be statistically significant. Most of the vancomycin-tolerant isolates were also tolerant to teicoplanin. We conclude that glycopeptide tolerance is a real phenomenon in S. aureus, particularly amongst MRSA isolates, and can be reliably determined by our method of time-kill analysis. Tolerance may compromise glycopeptide therapy of serious S. aureus infection and should be taken into account when deciding treatment.
Explore the source record for details and available documents.