New drug treatments for alcohol problems. Response to Moncrieff & Drummond.
Explore the source record for details and available documents.
Alcohol Deterrents↗
Biomedical subjects
J Marshall
Publications and source records attributed to J Marshall.
In response to The ethics of organ transplantation.
Explore the source record for details and available documents.
Alcoholism↗
Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.
Cationic lipids show promise as vectors for transfer of CFTR cDNA to airway epithelia of patients with cystic fibrosis (CF). However, previous studies have not compared the effect of DNA-lipid to DNA alone. Recently, we developed a formulation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) that generated greater gene transfer in mouse lung than previously described DNA-lipid vectors. Therefore, we tested the hypothesis that DNA-lipid complexes were more effective than DNA alone at transferring CFTR cDNA to airway epithelia in vivo. We administered complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study. Electrophysiologic measurements showed that DNA-lipid complexes partially corrected the Cl- transport defect. Importantly, the pCF1-CFTR plasmid alone was at least as effective as complexes of DNA with lipid. Measurements of vector-specific CFTR transcripts also showed gene transfer with both DNA-lipid and DNA alone. These results indicate that nonviral vectors can transfer CFTR cDNA to airway epithelia and at least partially restore the Cl- transport defect characteristic of CF. However, improvements in the overall efficacy of gene transfer are required to develop a treatment for CF.
Adolescent↗
Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression.
Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.
Animals↗
Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.
Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.
Administration, Intranasal↗
Optimization of plasmid vectors for high-level expression in lung epithelial cells.
Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.
Animals↗
Subtractive hybridization of cDNA from small amounts of plant tissue.
A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.
DNA, Complementary↗
Excimer laser photorefractive keratectomy for the correction of hyperopia using an erodible mask and axicon system.
PURPOSE: The purpose of the study is to evaluate photorefractive keratectomy for the correction of hyperopia using the erodible mask and Axicon system. METHODS: Forty-three patients (43 eyes) with a mean refraction (spherical equivalent) of +4.54 diopter (D) (range, +1.75 to +7.50 D) were treated using a Summit Technology "Apex Plus" excimer laser. This system uses an erodible mask to create a 6.50-mm diameter hyperopic correction over the axial cornea. An Axicon then is used to fashion a 1.50-mm "blend zone" around the correction. On the basis of preoperative refractions, patients were assigned to 3 groups: 2 groups of 14 patients underwent either "+2.00 D" or "+3.00 D" corrections and 15 patients had "+4.00 D" corrections. RESULTS: All patients had a reduction in their hyperopia with an overcorrection, especially in the first month after surgery and some stability in the refractive change at 3 to 6 months. The mean manifest refraction (n = 43) at 6 months was -0.17 D (range, +4.50 D to -3.125 D). Patient satisfaction was high. At 6 months, all eyes had an improvement in unaided near visual acuity. Unaided distance acuity was improved in 37 eyes (86%). A ring of haze 6.5 mm in diameter appeared in all eyes 1 month after surgery. Night halo measurements at 6 months showed no differences from preoperative levels. Flicker contrast sensitivity and forward light scatter (glare) measurements showed no differences after surgery. CONCLUSIONS: In this short-term study, photorefractive keratectomy for hyperopia using the erodible mask and Axicon system appeared to be a promising procedure. Visual performance, in terms of flicker contrast sensitivity, forward light scatter, and night halos, was not compromised. There was an overcorrection based on the manufacturer's algorithms. Manipulation of the treatment algorithms should improve future predictability.
Adult↗
IGF-1 modulates N and L calcium channels in a PI 3-kinase-dependent manner.
Receptor tyrosine kinases (RTKs) have long been associated with proliferation in non-neural cells, although they are also expressed in postmitotic neurons. We demonstrate that insulin-like growth factor-1 (IGF-1) induces within seconds a large, tyrosine-kinase-dependent increase in calcium channel currents in cerebellar granule neurons. Separation of channel subtypes reveals that, while P, Q, and R channels are unaffected, N and L channel activities are strongly potentiated at specific membrane voltages: N currents triple at depolarized potentials, while L currents rapidly increase 4-fold at hyperpolarized potentials. Moreover, transient expression of dominant-negative and wild-type phosphatidylinositol 3-OH kinase (PI 3-kinase) subunits, as well as application of specific inhibitors, demonstrates that PI 3-kinase is an essential and rate-limiting messenger in this signaling pathway. Our results indicate that N and L calcium channels are downstream targets of neuronal RTKs and suggest that RTK modulation may control calcium-dependent processes, such as neurotransmitter release and IGF-1-dependent differentiation or survival.
Animals↗
Choroidal abnormalities in birdshot chorioretinopathy: an indocyanine green angiography study.
Birdshot chorioretinopathy is a rare inflammatory disorder with an insidious onset that can slowly progress to severe visual loss. The pathogenesis is unknown. This study used indocyanine green (ICG) angiography to investigate the degree of choroidal vascular involvement with progression of disease and to determine the nature of the birdshot lesions. Seven patients with birdshot chorioretinopathy had ICG angiography performed with a scanning laser ophthalmoscope at various stages of clinical disease. Results were compared with fluorescein fundal angiography (FFA). All large choroidal vessels appeared normal. The birdshot lesions were demonstrated with ICG but not with FFA and were represented by dark areas on ICG angiography. Typically these areas were bordered by large or medium-sized choroidal vessels and their appearance suggested small choroidal vessel hypoperfusion. In disease of recent onset, some lesions masked fluorescence from large underlying choroidal vessels possibly due to inflammatory choroidal infiltrates. In long-standing disease, the choroidal angioarchitecture was relatively normal within the birdshot lesions. This study of birdshot chorioretinopathy demonstrates abnormalities in the small choroidal vessels within the birdshot lesions. ICG angiography detects the birdshot lesions more readily than FFA and may be of benefit in assessing disease activity.
Adult↗
Effect of co-lipids in enhancing cationic lipid-mediated gene transfer in vitro and in vivo.
Complexes of DNA and cationic lipids are promising vectors for gene transfer. Most cationic lipid formulations contain both a cationic component and a neutral co-lipid. We found that the co-lipid could influence DNA uptake in COS-1 cells, but processes subsequent to uptake were even more important in determining gene expression. We compared dioleoylphosphatidylethanolamine (DOPE) and structural analogs of DOPE combined with cationic lipids and found that DNA uptake and transgene expression did not always correlate. Transgene expression was dependent on DNA uptake into the cell, on entry of DNA into the cytoplasm, and on release of DNA from the lipid complex. We found that some co-lipids had a greater effect on DNA uptake, whereas others had a greater effect on steps subsequent to entry. Based on those results, we tested the hypothesis that co-lipids conferring different properties could be combined to enhance gene transfer. The results showed that a combination of co-lipids had a synergistic effect on expression. We also found that structural analogs of DOPE were more effective than DOPE in enhancing gene transfer to mature human airway epithelia studied in vitro and to mouse lung studied in vivo. These data provide insight into the mechanism by which co-lipids influence cationic lipid-mediated gene transfer and show that optimization of the effects of co-lipids can enhance gene transfer both in vitro and in vivo.
Animals↗
Both the disposition and the means of cure: "Severe SIRS," "sterile shock," and the ongoing challenge of description.
Explore the source record for details and available documents.
APACHE↗
Confocal microscopy using oblique sections for measurement of corneal epithelial thickness in conscious humans.
PURPOSE: Thickness measurements by confocal microscopy in conscious human subjects may be liable to error as a result of instability of the eye or instrument. Our aim was to evaluate a technique which was expected to be less sensitive to such problems. METHOD: Thickness of corneal epithelium was determined from oblique confocal sections through cornea. A contact lens of known thickness worn by subjects was used to calibrate images. RESULTS: There were two layers in images which could have corresponded to the stromal/epithelial interface. The mean result in each subject ranged from 38 to 53 microm using the more superficial layer and 46 to 60 microm using the deeper one. The smaller values gave the distance between the epithelial surface and the sub-epithelial nerve plexus and thus seemed to correspond to epithelial thickness. CONCLUSIONS: Measurements of epithelial thickness by our new method are comparable with results of earlier studies.
Adult↗
Effect of beam variables on corneal sensitivity after excimer laser photorefractive keratectomy.
AIM: To investigate changes in corneal touch sensitivity following excimer laser photorefractive keratectomy (PRK) using different beam configurations. METHODS: 20 subjects were given a unilateral -3.00 D correction with either a 5 mm (26 micrograms, n = 10) or 6 mm (42 micrograms, n = 10) beam diameter. Thirty subjects underwent a unilateral -6.00 D correction with 5 mm (62 micrograms, n = 10), 6 mm (78 micrograms, n = 10), or multizone (62 micrograms, n = 10) treatments. The multizone treatment was 6 mm in diameter with the depth of the 5 mm treatment. Corneal sensitivity was measured using a slit-lamp mounted Cochet-Bonnet aesthesiometer before and at 1, 3, 6, and 12 months after PRK. Stimulus locations included points lying within the ablated zone (central) and outside (peripheral). These were compared with the equivalent locations in control (untreated) eyes. RESULTS: There was a significant reduction in corneal sensitivity within the central (ablated) zone in all treatment groups after PRK. In most groups a return to full sensitivity was achieved by 6 months with the exception of the multizone treatment group which showed significant corneal hypoaesthesia at 12 months. Peripheral corneal sensitivity was also reduced in this group up to 3 months after the procedure. A comparison between the -3.00 D and -6.00 D treatment groups showed no significant difference. However, combining data from all treatment groups, a significant correlation was found between the interocular difference in central corneal sensitivity and postoperative haze at 3 and 6 months. CONCLUSIONS: For corrections up to -6.00 D ablation depth and treatment zone diameter do not appear to be clinically important determinants of corneal hypoaesthesia. In contrast, postoperative corneal haze appears to correlate with sensitivity loss.
Adult↗
Administration of medicines by emergency nurse practitioners according to protocols in an accident and emergency department.
OBJECTIVE: To present the legal and professional issues related to nurse administration of drugs according to protocols, and describe the implementation and initial audit findings of such a scheme. SETTING: Accident and emergency (A&E) department of a district general hospital. METHODS: Analysis of legal and professional opinion. Protocols acceptable to the medical, nursing, and pharmacy professions were developed across a wide range of drugs appropriate for administration by accident and emergency nurse practitioners (ENPs). The first six months of the scheme were audited. Audit initially addressed general compliance with protocols and later the specific areas of tetanus immunisation and emergency contraception. RESULTS: ENPs assessed 2925 patients in six months (10.9% of all new patients); 455 patients (15.5% of the ENP patients) were given drugs according to protocols. There were no breaches of the protocols. Subsequent audit of tetanus immunisation showed 94-100% compliance with protocol standards and 71-100% compliance for emergency contraception. CONCLUSIONS: There are no legal or professional obstacles to the development of protocols for the administration of drugs to patients by nurses without reference to a doctor, providing the protocols meet all the requirements of the UKCC and have the support of consultant medical staff. Such a system must be subject to regular audit to promote a dynamic approach to protocols and training. The system safely enhanced the quality of care of patients treated by ENPs in A&E.
Clinical Protocols↗
Does transfusion practice affect mortality in critically ill patients? Transfusion Requirements in Critical Care (TRICC) Investigators and the Canadian Critical Care Trials Group.
In 4,470 critically ill patients, we examined the impact of transfusion practice on mortality rates. As compared with survivors, patients who died in intensive care units (ICU) had lower hemoglobin values (95 +/- 26 versus 104 +/- 23 g/L, p < 0.0001) and were transfused red cells more frequently (42.6% versus 28.0%, p < 0.0001). In patients with cardiac disease, there was a trend toward an increased mortality when hemoglobin values were < 95 g/L (55% versus 42%, p = 0.09) as compared with anemic patients with other diagnoses. Patients with anemia, a high APACHE II score (> 20), and a cardiac diagnosis had a significantly lower mortality rate when given 1 to 3 or 4 to 6 units of allogeneic red cells (55% [no transfusions] versus 35% [1 to 3 units] or 32% [4 to 6 units], respectively, p = 0.01). Adjusted odds ratio (OR) predicting survival were 0.61 (95% CI; 0.37 to 1.00, p = 0.026) after 1 to 3 units and 0.49 (95% CI; 0.23 to 1.03, p = 0.03) after 4 to 6 units compared with nontransfused anemic patients. In the subgroup with cardiac disease, increasing hemoglobin values in anemic patients was associated with improved survival (OR = 0.80 for each 10 g/L increase, p = 0.012). We conclude that anemia increases the risk of death in critically ill patients with cardiac disease. Blood transfusions appear to decrease this risk.
APACHE↗