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L G Mitchell

Publications and source records attributed to L G Mitchell.

At least 19 recordsLinked to original sources
PubMed2006

Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin generation in vitro.

BACKGROUND: Thrombin generation potential, a critical haemostatic measure, can be determined by continuous detection of total thrombin or direct subsampling. However, differences between methods exist in area under the curve or peak thrombin calculated. Also, impact of anticoagulants on thrombin generation may vary depending on mode of analysis. OBJECTIVE: We studied the effect of components on thrombin generation in the presence or absence of anticoagulants. METHODS: The continuous method was conducted with plasma +/- fibrin(ogen) +/- fibrin polymerization inhibitor. Plasma contained slow-reacting TG5134 substrate at 37 degrees C and reaction was started with dilute thromboplastin in CaCl(2)/Tris buffer. Absorbance (405 nm) was recorded over time and free thrombin calculated from total thrombin activity. For the subsampling method, similar plasma mixtures +/- TG5134 were reacted and free thrombin measured directly as the difference in activity against S2238 substrate of timed subsamples taken into EDTA or EDTA + antithrombin + heparin. RESULTS: Slow-reacting substrate in the continuous method acted as a competitor for thrombin, giving delayed but greater free thrombin than direct subsampling. These differences persisted to varying extents with all anticoagulants tested. In either method, presence of polymerization inhibitor increased the amount of free thrombin. Continuous method detection of alpha(2)macroglobulin complexes was hampered by sensitivity limits leading to inordinate free thrombin calculations. Especially with hirudin, although free thrombin remained at the end of the subsampling method, continuous method calculations assumed no residual free thrombin. CONCLUSION: In vitro plasma thrombin generation is delayed and increased by slow-acting substrate and fibrin polymerization inhibitor.

Adult↗
PubMed2005

Gene therapy progress and prospects: reprograming gene expression by trans-splicing.

The term 'trans-splicing' encompasses several platform technologies that combine two RNA or protein molecules to generate a new, chimeric product. RNA trans-splicing reprograms the sequences of endogenous messenger mRNA or pre-mRNA, converting them to a new, desired gene product. Trans-splicing has broad applications, depending on the nature of the sequences that are inserted or trans-spliced to the defined target. Trans-splicing RNA therapy offers significant advantages over conventional gene therapy: expression of the trans-spliced sequence is controlled by the endogenous regulation of the target pre-mRNA; reduction or elimination of undesirable ectopic expression; the ability to use smaller constructs that trans-splice only a portion of the gene to be replaced; and the conversion of dominant-negative mutations to wild-type gene products.

Animals↗
PubMed2004

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.

Animals↗
PubMed2004

Increased anticardiolipin antibody IgG titers do not predict recurrent stroke or TIA in children.

BACKGROUND: Increased anticardiolipin antibody (ACLA) immunoglobulin (Ig) G titers are commonly found in children with arterial ischemic stroke (AIS) or TIA (AIS/TIA). The associated risk of recurrent thromboembolism is unknown. OBJECTIVE: To determine the risk of recurrent thromboembolism associated with persistently increased ACLA titers of the IgG isotype in children with AIS/TIA. METHODS: The authors studied a cohort of children surviving first AIS/TIA tested by standardized ELISA for beta2-glycoprotein I-dependent ACLA of the IgG isotype. Children with ACLA titers >15 IgG phospholipid (GPL) units (per manufacturer's cutoff point) on more than two occasions > or =6 weeks apart were classified as ACLA-positive (ACLA+) and compared with ACLA-negative (ACLA-) children with respect to recurrent thromboembolic events (AIS/TIA, sinovenous thrombosis, and extracerebral thromboembolism). RESULTS: The authors recruited 34 ACLA+ children and 151 ACLA- children. Most ACLA+ children (30/34; 88%) had ACLA titers < or =40 GPL units. During the follow-up period (median duration, 2.8 years for ACLA+ children and 3.0 years for ACLA- children), AIS/TIA recurred in 26% of ACLA+ children and in 38% of ACLA- children; none developed sinovenous thrombosis or extracerebral thromboembolism. Based on survival analysis, this difference was nonsignificant (p = 0.54). Using binary partition evaluation, no titer criteria for ACLA positivity (range, 0 to 60 GPL units) predicted recurrent AIS/TIA. CONCLUSION: In children surviving arterial ischemic stroke/TIA, increased anticardiolipin antibody immunoglobulin G titers do not predict recurrent thromboembolism.

Antibodies, Anticardiolipin↗
PubMed2003

Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases.

Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.

Cell Line↗
PubMed2001

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

Cell Fractionation↗
PubMed2000

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.

Most messenger RNA precursors (pre-mRNA) undergo cis-splicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its pre-mRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conductance regulator (CFTR) mini-gene expressing mutant (deltaF508) pre-mRNA targets and tested this against a number of PTMs capable of binding to the CFTR target intron 9 and trans-splicing in the normal coding sequences for exons 10-24 (containing F508). When 293T cells were cotransfected with both constructs, they produced a trans-spliced mRNA in which normal exon 10-24 replaced mutant exon 10. To test whether SMaRT produced mature CFTR protein, proteins were immunoprecipitated from lysates of cotransfected cells and detected by Western blotting and PKA-phosphorylation. Tryptic phosphopeptide mapping confirmed the identity of CFTR. This proof-of-concept study demonstrates that exon replacement by SMaRT can repair an abnormal pre-mRNA associated with a genetic disease.

Blotting, Western↗
PubMed1999

Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

Animals↗
PubMed1999

Antithrombotic therapy in children.

Thromboembolic events are an increasingly common secondary complication in children who are successfully treated for serious, life-threatening primary diseases. In contrast to adults, thromboembolic events are rare enough in children to hinder clinical trials assessing optimal use of antithrombotic agents. Currently, pediatric patients are treated according to guidelines extrapolated from adults. However, optimal prevention and treatment of thromboembolic events in children likely differs from such treatment for adults. The following review summarizes the available information on commonly used antithrombotic agents in children, which include standard heparin, low molecular heparin, oral anticoagulants, thrombolytic therapy, antiplatelet agents, antithrombin concentrates, and protein C concentrates. The mechanisms, dosing, monitoring, therapeutic range, factors influencing dose-response relationship, and side effects are discussed.

Anticoagulants↗
PubMed1995

Fibrin clot lysis by tissue plasminogen activator (tPA) is impaired in plasma from pediatric patients undergoing orthotopic liver transplantation.

Large vessel thrombi can present life-threatening complications following orthotopic liver transplantation (OLT) in pediatric patients. We investigated the thrombolytic response to tissue plasminogen activator (tPA) of stored, pooled plasma (days 4-14 postoperatively) from 41 patients (mean age 4 years, 9 months) who underwent OLT at the Hospital for Sick Children, Toronto between 1986 and 1990. Trace-labeled fibrin clots were prepared by recalcifying 500-microliters aliquots of patient plasma spiked with 125I fibrinogen and then incubated at 37 degrees C in patient plasma in the presence or absence of tPA (0.1 or 0.3 mg/ml). At the end of the incubation period, the extent of clot lysis and concentrations of fibrinogen, plasminogen, and alpha 2 antiplasmin were determined. Pooled adult plasma was used as a control. Fibrin clot lysis in OLT plasma was significantly reduced compared with controls (P < 0.01). Initial concentrations of plasminogen were significantly reduced in OLT plasma. To determine if the low plasminogen levels limited the thrombolytic effect of tPA, we supplemented OLT plasma with purified plasminogen. Fibrin clots placed in OLT plasma containing adult levels of plasminogen showed a similar lytic response as adults. In summary, the reduced fibrinolytic response of OLT fibrin clots to tPA was due to low concentrations of plasminogen and corrected by plasminogen supplementation.

Adult↗
PubMed1995

Nephrotoxicity of xenobiotics.

Nephrotoxicity can be grouped by the xenobiotics place of action, by the clinical presentation or by the generic toxic effect. The latter can be dose related, indirect, idiosyncratic or allergic. Nephrotoxicity of lithium, demeclocycline, aminoglycosides, cyclosporine, mercuric ion, nonsteroidal anti-inflammatory drugs, methoxyflurane, ethylene glycol, D-penicillamine and methicillin is reviewed in light of all these three viewpoints, but emphasis is on toxic mechanisms.

Aminoglycosides↗
PubMed1995

Hemostasis in childhood acute lymphoblastic leukemia: coagulopathy induced by disease and treatment.

Thromboembolic events (TE) are serious complications of treatment for childhood acute lymphoblastic leukemia (ALL) that result in significant morbidity and occasionally mortality. These events are strongly associated with the administration of L'asparaginase (ASP). There have been many studies reporting TE and assessing the coagulopathy associated with treatment. The intention of these studies was to determine a potential mechanism for thrombosis. This article reviews the current literature in this area. First, data on thrombotic complications in terms of incidence, location, diagnosis, and timing of events are summarized. The second section discusses the coagulopathy associated with the disease and treatment. To minimize the effects of confounding treatments, the data are divided into sections covering pretreatment, after ASP only, after combination chemotherapy without ASP, and after combination chemotherapy with ASP. In addition, the effects of glucocorticoid steroids on the hemostatic system are discussed. As thrombin regulation is critically important to hemostasis, the next section of the review discusses the regulation of thrombin in children with ALL, both in vitro and in vivo, and the link between impaired thrombin regulation and TE in this population. Finally current hypothesis on mechanisms for TE and proposed preventative strategies are examined.

Anticoagulants↗
PubMed1994

Fresh frozen plasma has no beneficial effect on the hemostatic system in children receiving L-asparaginase.

L-Asparaginase (ASP), a chemotherapeutic agent used in the treatment of children with acute lymphoblastic leukaemia (ALL), is linked to thromboembolic complications secondary to an acquired deficiency of antithrombin III (ATIII). Fresh frozen plasma (FFP) is used to prevent and/or treat thrombotic complications in these children. However, the effect of FFP on plasma concentrations of ATIII and biochemical markers of activation of coagulation has never been tested. In this study, FFP (20 ml/kg) was administered to eight children with ALL receiving ASP in the consolidation phase of their treatment. Plasma samples were drawn pre-infusion, and following infusion at 1, 24, and 48 hr. Prior to the FFP infusions, plasma concentrations of prothrombin, fibrinogen, alpha 2-macroglobulin, heparin cofactor II, protein C, and protein S were similar to levels in healthy children. Only plasma concentrations of ATIII were significantly decreased (0.55 U/ml). Following FFP infusions, there was no statistical or clinically important increase in plasma concentrations of any coagulation protein at any time point. Pre-infusion plasma concentrations of markers of endogenous thrombin generation (thrombin-antithrombin III complexes (TAT)) and activation of the fibrinolytic system in response to activation of the coagulation system (D-dimer levels) were significantly increased. However, FFP had no statistical or clinically important effect on concentrations of these markers. We conclude that FFP administration for the prevention and treatment of acquired ATIII deficiency secondary to ASP has no demonstrable benefit on plasma levels of coagulation proteins and is unlikely to be of clinical benefit.

Adolescent↗
PubMed1994

Effect of disease and chemotherapy on hemostasis in children with acute lymphoid leukemia.

PURPOSE: We sought to determine the effect of disease and combination chemotherapy on the hemostatic system in children with acute lymphoid leukemia (ALL). PATIENTS AND METHODS: We conducted a prospective study of children newly diagnosed with ALL. Plasma samples were obtained at four time points: at diagnosis before therapy, 5 days after administration of L-asparaginase alone, after the remission induction program, and at completion of the consolidation phase. Plasma levels of 21 hemostatic proteins were measured. The amount of thrombin generated following activation with an APTT reagent was quantitated. RESULTS: At diagnosis there were significant elevations in factors VIII, IX, von Willebrand, alpha 2-macroglobulin and protein S. In contrast, there were significant reductions in protein C, prekallikrein, and factors XIIIA and XIIIS. L-asparaginase treatment alone decreased concentrations of 11 proteins, with antithrombin III being affected to the greatest extent. After multiagent chemotherapy, not including L-asparaginase, concentrations of most proteins increased to or above baseline. At completion of consolidation therapy, which included weekly L-asparaginase administration, concentrations of most proteins were decreased compared with baseline values. The amount of thrombin generated following activation with an APTT reagent was similar to adults. CONCLUSION: Plasma concentrations of coagulation proteins are affected by disease (ALL) alone and by combination chemotherapy with or without L-asparaginase. There is no impairment of in vitro capacity to generate thrombin. L-asparaginase alone caused a decrease in almost all proteins; however, ATIII was affected to the greatest extent.

Adolescent↗
PubMed1993

Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War.

Deoxyribonucleic acid (DNA) sequence analysis of the control region of the mitochondrial DNA (mtDNA) genome was used to identify human skeletal remains returned to the United States government by the Vietnamese government in 1984. The postmortem interval was thought to be 24 years at the time of testing, and the remains presumed to be an American service member. DNA typing methods using nuclear genomic DNA, HLA-DQ alpha and the variable number of tandem repeat (VNTR) locus D1S80, were unsuccessful using the polymerase chain reaction (PCR). Amplification of a portion of the mtDNA control region was performed, and the resulting PCR product subjected to DNA sequence analysis. The DNA sequence generated from the skeletal remains was identical to the maternal reference sequence, as well as the sequence generated from two siblings (sisters). The sequence was unique when compared to more than 650 DNA sequences found both in the literature and provided by personal communications. The individual sequence polymorphisms were present in only 23 of the more than 1300 nucleotide positions analyzed. These results support the observation that in cases where conventional DNA typing is unavailable, mtDNA sequencing can be used for human remains identification.

Anthropology, Physical↗
PubMed1992

Immunochemical characterization of a monoclonal antibody specific for Alzheimer's disease associated protein.

In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimer's disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.

Alzheimer Disease↗