Extraction and purification of RNA from plant tissue enriched in polysaccharides.
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Carbon Dioxide↗
Biomedical subjects
S H Cheng
Publications and source records attributed to S H Cheng.
Preoperative concurrent chemotherapy and radiotherapy in rectal cancer patients.
The management of rectal cancer has changed significantly in recent years. The key end-point is no longer survival but rather preservation of sphincter function with improved quality of life. Preoperative radiation can not only render a low-lying rectal tumor amenable to sphincter-preserving surgery but has also been reported to give better local control and lower toxicity than postoperative radiotherapy. From October 1991 through July 1996, 46 patients with local advanced or low-lying rectal cancer were treated with preoperative high-dose radiotherapy and concurrent chemotherapy. All patients underwent pelvic radiotherapy with 5,000 to 5,400 cGy in 25 to 27 fractions. Chemotherapy was given concomitantly and consisted of two courses of 5-fluorouracil (5-FU) at 1,000 mg/m2 for 4 days in week 1 and week 5 plus mitomycin C 10 mg/m2 single bolus on day 1 of week 1. In 30 patients, postoperative adjuvant chemotherapy with 5-FU and levamisole weekly was also given, for a total of 12 months. The most common acute toxicity was grade 1 to 2 diarrhea and tenesmus during radiation or soon afterward. Only five of the 46 patients experienced symptomatic grade 3 acute toxicity. Forty-two patients underwent subsequent surgery 6 to 8 weeks after concurrent chemoradiotherapy. Pathologic examination disclosed complete tumor regression in eight patients and microscopic residual disease in 13 patients after preoperative chemoradiation. Of the 42 patients who completed the intended treatments, only one had local recurrence. The sphincter was preserved in 21 of the 26 patients in whom the tumor was located within 5 cm above the anal verge. Twelve of the 16 evaluable patients had good to excellent sphincter function. The 2-year overall survival rate was 93% and the disease-free survival was 81%. Our findings indicate that preoperative concurrent chemoradiotherapy not only allows low-lying rectal tumors to be resected while preserving sphincter function but also results in good local control and acceptable toxicity.
Adenocarcinoma↗
Aeromonas hydrophila sepsis presenting as meningitis and necrotizing fasciitis in a man with alcoholic liver cirrhosis.
Aeromonas hydrophila is rarely reported as a causative organism of meningitis in humans. A 39-year-old man with alcoholic liver cirrhosis was admitted with a 2-day history of fever, chills, and confusion. Laboratory data revealed leukocytosis with granulocytosis, marked impairment of renal and liver function, and an elevated serum ammonia level. A. hydrophila was isolated from both blood and cerebrospinal fluid samples. Skin and soft-tissue lesions, consisting of bullae and necrotizing fasciitis, were found in the lower left leg 2 days after admission. Cultures of the bullous fluid, subcutaneous tissue, and fascia all yielded A. hydrophila. Pathologic examination revealed extensive necrosis. Although the patient was appropriately managed with antibiotics, debridement, and fasciotomies, his clinical status rapidly deteriorated, resulting in death 3 days after admission.
Adult↗
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↗
The effect of universal health insurance on health care utilization in Taiwan. Results from a natural experiment.
CONTEXT: The government of Taiwan introduced universal health insurance to cover all citizens in 1995. This national health insurance program was proposed to assure the accessibility to health care at reasonable cost. Evaluation of the consequences, including health care utilization and expenditure, is crucial for policy adjustment. OBJECTIVES: To evaluate the effect of Taiwan's national health insurance on health care utilization. DESIGN: Cohort survey conducted before and after the implementation of the national health insurance program. PARTICIPANTS: A total of 1021 randomly selected Taiwanese adults. MAIN OUTCOME MEASURES: Physician visits in the 2 weeks prior to the survey and hospital admissions and emergency department visits in the immediate past year. RESULTS: After the introduction of universal health insurance, the newly insured consumed more than twice the amount of outpatient physician visits (0.21 vs 0.48, P<.05) and hospital admissions (0.04 vs 0.11, P<.05) than before universal health insurance was implemented, bringing them to the same amount of health care contacts as the previously insured group. The newly insured also experienced an insignificant increase in emergency department visits. In contrast, the previously insured group had a small but statistically significant increase in outpatient visits (0.48 vs 0.59, P<.05) and insignificant changes in hospital admissions and emergency department visits. CONCLUSION: The universal health insurance removed some barriers to health care for those newly insured. The copayment design in the insurance scheme seemed to have an insignificant effect on curbing medical care utilization. Taiwanese health policy analysts should seriously consider the growth of health care expenditures since the implementation of universal health insurance.
Adult↗
Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.
Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.
Adenoviridae↗
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↗
A concentrated and stable aerosol formulation of cationic lipid:DNA complexes giving high-level gene expression in mouse lung.
Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.
Administration, Inhalation↗
Biophysical characterization of cationic lipid: DNA complexes.
To better understand the structures formed by the interaction of cationic lipids with DNA, we undertook a systematic analysis to determine the biophysical characteristics of cationic lipid:DNA complexes. Four model cationic lipids with different net cationic charge were found to interact in similar ways with DNA when that interaction was compared in terms of the apparent molar charge ratio of lipid to DNA. When DNA was present in charge excess over the cationic lipid, the complex carried a net negative charge as determined by zeta potential measurements. Under these conditions, some DNA was accessible to ethidium bromide, and free DNA was observed in agarose gels and in dextran density gradients. Between a lipid:DNA charge ratio of 1.25 and 1.5:1, all the DNA became complexed to cationic lipid, as evidenced by its inaccessibility to EtBr and its complete association with lipid upon agarose gel electrophoresis and density gradient separations. These complexes carried a net positive charge. The transition between negatively and positively charged complexes a occurred over a very small range of lipid to DNA ratios. Employing a fluorescent lipid probe, the addition of DNA was shown to induce lipid mixing between cationic lipid-containing vesicles. The extent of DNA-induced lipid mixing reached a maximum at a charge ratio of about 1.5:1, the point at which all the DNA was involved in a complex and the complex became positively charged. Together with freeze-fracture electron micrographs of the complexes, these biophysical data have been interpreted in light of the existing models of cationic lipid:DNA complexes.
Cations↗
Nomenclature for synthetic gene delivery systems.
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Gene Transfer Techniques↗
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↗
Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes.
We have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mM NaCL as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitro aerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (1-3 microns). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.
Administration, Intranasal↗
Association between migraine and stroke in a large-scale epidemiological study of the United States.
OBJECTIVE: To examine the association between stroke and migraine in an epidemiological study. DATA SOURCES AND DESIGN: The National Health and Nutrition Examination Survey baseline and first follow-up data were used to investigate cross-sectional and longitudinal associations between headache/migraine and stroke. SETTING: Study participants from a national probability sample of the civilian noninstitutionalized population of the United States. MAIN OUTCOME MEASURE: Self-reported physician diagnosis of stroke. RESULTS: After controlling for established risk factors for stroke (hypertension, diabetes, heart disease, and gender), both migraine and severe nonspecific headache were associated with a significantly increased risk for stroke reported at follow-up. The risk for stroke associated with migraine decreased as the age at stroke increased. CONCLUSIONS: Our results strengthen previous evidence regarding a nonrandom association of both headache and migraine with stroke, particularly among young women. To our knowledge, this is the first systematic examination in a large-scale prospective epidemiological study of men and women with sufficient statistical power to test the association between migraine and stroke in women. Severe headache and migraine should be considered as risk factors for the development of stroke, particularly in the absence of other well-established stroke risk factors. Further investigation is required to identify the putative mechanisms underlying comorbidity of migraine and stroke.
Adult↗
Safety of a single aerosol administration of escalating doses of the cationic lipid GL-67/DOPE/DMPE-PEG5000 formulation to the lungs of normal volunteers.
Several groups are assessing the use of cationic lipids for respiratory gene therapy. To date no human data are available regarding the safety of intra-pulmonary cationic lipid delivery. In preparation for a trial of pulmonary delivery of the CFTR gene, we have assessed the safety of nebulised lipid GL-67/DOPE/DMPE-PEG5000 (GL-67A), the cationic lipid formulation to be used in this study. Fifteen healthy volunteers were given incremental doses of GL-67A via a Pari LC Jet nebuliser; three volunteers in each of five dosing cohorts with a week interval between cohorts. Markers of safety included clinical assessment, measurement of lung function, chest CT scan, serological testing and analysis of induced sputum. Measurements were taken before administration and at intervals up to 21 days thereafter. No adverse clinical events were seen or any statistically significant changes in spirometry or gas transfer. There were no clinically significant changes in any of the blood parameters and no CT changes were seen. Comparisons of the cellular subpopulations (neutrophils, eosinophils, lymphocytes and macrophages) in induced sputum showed no significant alterations following administration of the GL-67A. This study suggests that a single application of aerosol formulation of GL-67A does not result in clinically detectable changes when given by nebulisation into the lungs of normal volunteers and provides an indication of a lipid dose tolerated in man.
Aerosols↗
Clostridium difficile--associated diseases: comparison of symptomatic infection versus carriage on the basis of risk factors, toxin production, and genotyping results.
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Adult↗
Diagnosis of late onset neonatal sepsis with cytokines, adhesion molecule, and C-reactive protein in preterm very low birthweight infants.
AIMS: To evaluate the commonly used markers--namely IL-6, TNF alpha, IL-1 beta, C-reactive protein and E-selection for identification of late onset neonatal sepsis; to define the optimal cutoff value for each marker in preterm neonates; to assess whether these markers could assist in early discontinuation of antibiotics in non-infected cases; and to delineate the profile of these markers during systemic infection and in relation to successful treatment. METHODS: Very low birthweight infants in whom clinical sepsis was suspected when they were > 72 hours of age were eligible for study. A full sepsis screen was performed in each episode. Cytokines, C-reactive protein, and E-selectin were serially measured on days 0 (at the time of sepsis evaluation), 1, 2, 4 and 7. The optimal cutoff value for each marker was calculated after minimising the number of misclassified episodes over all possible cutoff values for days 0 and 1. The sensitivity, specificity, positive and negative predictive values for each test and combination of tests for predicting systemic infection were also determined. RESULTS: One hundred and one episodes of suspected clinical sepsis were investigated in 68 infants. Forty five episodes were proved to be infections. The optimal cutoff values were IL-6 31 pg/ml, TNF alpha 17 pg/ml, IL-1 beta 1 pg/ml, C reactive protein 12 mg/l and E-selectin 174 ng/ml. IL-6 had the highest sensitivity (89%) and negative predictive value (91%) for detecting late onset infection on day 0. However, between 24 and 48 hours of onset, C-reactive protein was the best single marker, with an overall sensitivity and specificity of 84% and 96%, respectively. The use of serial and multiple markers in the first 48 hours further enhanced the sensitivity and specificity of these tests. Performing IL-6 and C-reactive protein on day 0, together with either TNF alpha on day 1 or C-reactive protein on day 2, showed the best overall sensitivity (98%) and specificity (91%) for the diagnosis of late onset infection. CONCLUSIONS: Optimal cutoff values for these markers in detecting late onset systemic infection in very low birthweight infants have been defined. Withholding antibiotic treatment at the onset of infection could be fatal and is not recommended, but the concomitant use of IL-6 and C-reactive protein or TNF alpha should allow antimicrobial treatment to be discontinued at 48 hours without waiting for microbiological results, provided that the infants are in good clinical condition.
Biomarkers↗
Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.
The correct patterning of vertebrate skeletal elements is controlled by inductive interactions. Two vertebrate hedgehog proteins, Sonic hedgehog and Indian hedgehog, have been implicated in skeletal development. During somite differentiation and limb development, Sonic hedgehog functions as an inductive signal from the notochord, floor plate and zone of polarizing activity. Later in skeletogenesis, Indian hedgehog functions as a regulator of chondrogenesis during endochondral ossification. The vertebrate Gli zinc finger proteins are putative transcription factors that respond to Hedgehog signaling. In Drosophila, the Gli homolog cubitus interruptus is required for the activation of hedgehog targets and also functions as a repressor of hedgehog expression. We show here that Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 (C.-c. Hui and A. L. Joyner (1993). Nature Genet. 3, 241-246) mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development. The role of Gli2 and Gli3 in Hedgehog signaling during skeletal development is discussed.
Animals↗
A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.
Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.
Administration, Intranasal↗