Impact of reduced resuscitation fluid on outcomes of children with 10-20% body surface area scalds.

'Permissive hypovolaemia' fluid regimes in adult burn care are suggested to improve outcomes. Effects in paediatric burn care are less well understood. In a retrospective audit, outcomes of children from the South West Children's Burn Centre (SWCBC) less than 16 years of age with scalds of 10-20% burn surface area (BSA) managed with a reduced volume fluid resuscitation regime (post-2007) were compared to (a) an historical local protocol (pre-2007) and (b) current regimes in burn services across England and Wales (E&W). Outcomes included length of stay per percent burn surface area (LOS/%BSA), skin graft requirement and re-admission rates. 92 SWCBC patients and 475 patients treated in 15 other E&W burn services were included. Median LOS/%BSA for patients managed with the reduced fluid regime was 0.27 days: significantly less than pre-2007 and other E&W burn services (0.54 days, 0.50 days, p<0.001). Skin grafting to achieve healing reduced post-2007 compared to pre-2007 and remains comparable with other E&W services. Re-admission rates were comparable between all groups. A reduced fluid regime has significantly shortened LOS/%BSA without compromising burn depth as measured by skin grafting to achieve healing. A prospective trial comparing permissive hypovolaemia to current regimes for moderate paediatric scald injuries would help clarify.

Activation of latent precursors in the hippocampus is dependent on long-term potentiation.

The recent discovery of a large latent population of precursor cells in the dentate gyrus of adult mice led us to investigate whether activation of this population is regulated by synaptic activity, thereby explaining the observation that environmental signals can affect neurogenesis. Using a variety of stimulation protocols, we found that only a long-term potentiation (LTP)-inducing protocol activated the latent precursor pool, leading to increased neurogenesis in the dentate gyrus. LTP induced by high-frequency stimulation (HFS) of the perforant pathway in vivo produced a two-fold increase in the number of neurospheres cultured from the stimulated hippocampus, compared with the unstimulated hippocampus. No increase in neurosphere number or neurogenesis was observed when the HFS failed to induce LTP. These results show that LTP can activate latent neural precursor cells in the adult mouse dentate gyrus, thereby providing a direct mechanism for regulating activity-driven neurogenesis. In the future, it may be possible to utilize such learning- or stimulation-induced neurogenesis to overcome disorders characterized by neuronal loss.

Characterisation of the Dunaliella tertiolecta RbcS genes and their promoter activity in Chlamydomonas reinhardtii.

The availability of highly active homologous promoters and terminators is critical in the development of a transformation system for the unicellular microalga Dunaliella tertiolecta. To facilitate transformation of this species, we isolated and characterised two native ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes (RbcS) including flanking sequences. The two non-allelic cDNA sequences share approximately 80% identity and have approximately 60% identity to the RbcS genes of Chlamydomonas reinhardtii. The D. tertiolecta RbcS promoter and 3' untranslated regions were shown to drive expression of the bleomycin resistance gene (ble) in C. reinhardtii. This is the first demonstration of a heterologous algal promoter being used to drive transgene expression in C. reinhardtii. In addition, promoter deletions were shown to further increase transformation efficiency.

Enhanced in vivo tumour response from combination of carboplatin and low-dose c-myc antisense oligonucleotides.

BACKGROUND: Phosphorothioate oligonucleotides ([S]ODNs) contain a modified phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used to varying success in vivo. Carboplatin is a commonly used chemotherapeutic and is associated with chemoresistance in some human tumours. The potential for combined antisense [S]ODNs and carboplatin chemotherapy has only recently been explored in vivo. MATERIALS AND METHODS: This study examines the effect of c-myc antisense oligomers delivered in isolation as naked DNA and in combination with carboplatin upon the growth kinetics of an in vivo transplantable adenocarcinoma using rodents. RESULTS: Tumours treated with a combination of 600 microg of 15-mer c-myc phosphorothioate antisense oligodeoxyribonucleotide and an intravenous administration of carboplatin (3 mg/kg), demonstrated a significant (p<0.05) retardation in tumour growth kinetics relative to a control. Two mismatch antisense controls did not significantly inhibit tumour growth. C-myc protein studies in tumour sections failed to show significant differences in c-myc expression in any of the treated tumours. CONCLUSION: This study demonstrates that carboplatin affects the relative abundance of c-myc and that combination treatment of carboplatin and c-myc phosphorothioate antisense oligonucleotides in vivo results in synergistic tumour retardation.

Varus derotation osteotomy for the treatment of hip subluxation and dislocation in cerebral palsy: statistical analysis in 73 hips.

The purpose of this study is to review the results of varus osteotomy in patients with cerebral palsy and to determine factors that influence the rates of residual hip displacement. A retrospective chart review of 65 patients who underwent 79 varus osteotomies was performed. Preoperative, postoperative and follow-up radiographs were analyzed for routine radiographic measurements, pelvic obliquity, osteonecrosis (avascular necrosis), joint incongruity or degenerative joint disease. The average follow-up was 5.2 years (range, 1.1-18.4 years). At follow-up, 3 hips were dislocated, 19 were subluxated and 57 were stable (72%). Age at surgery and the degree of preoperative hip displacement had significant effects on outcome. The average age at surgery for initially subluxated hips, which were located at follow-up, was 7.2 years. This was significantly younger (P = 0.008) than initially subluxated hips, which were displaced (10 years of age). Subluxated hips at surgery were also more likely to be located at follow-up than dislocated hips.

Effect of surgery on the nontreated hip in severe cerebral palsy.

We studied the fate of the nonoperated hip in 35 patients with cerebral palsy who underwent surgical stabilization for unilateral hip subluxation (24 patients) or dislocation (11 patients). Review of medical records and radiographs was performed and analysis was accomplished on the effect of preoperative and radiographic variables on the radiographic outcome of the nonoperated hip. The average age at surgery was 5.5 years and at follow-up was 9.7 years, with an average follow-up of 4.2 years. Before subsequent surgery (in 15 nonoperated hips) or at follow-up, 10 of the nonoperated hips were dislocated and 16 hips were subluxated. Hips were stable and less likely to have surgery if they had a lower initial migration index and higher center edge angles. We conclude that there are few indications for unilateral hip surgery in patients with diplegia or quadriplegia undergoing initial hip stabilization surgery, especially if any degree of dysplasia is present.

A microsphere-liposome (microplex) vector for targeted gene therapy of cancer. II. In vivo biodistribution study in a solid tumor model.

Cationic liposomes are commonly used for transfection of plasmids into mammalian cells, while microspheres have been traditionally used for selective delivery of anticancer agents into tumor vasculature. We have developed a novel vector, comprised of cationic liposomes electrostatically bound to ion-exchange microspheres (termed 'microplex') for targeted gene therapy of solid tumors. The delivery modes tested in a rat solid tumor model were free plasmids, plasmids bound to microspheres, to liposomes, or to the combination vector. The greatest amount of chloramphenicol acetyltransferase (CAT) reporter gene expression in tumors was achieved using the microplex vector; 3.4-fold compared with free, and 1.8-fold compared with both microspherical and liposomal deliveries (p < 0.01). Tumor-to-normal kidney tissue CAT expression ratios were as follows: free 1.9:1; microspherical 3.7:1; liposomal 1.4:1 and microplexical 2.7:1. Expression between the two types of tissues was significantly different (p < 0.01) for all delivery modes. Microspheres targeted the plasmids to the tumors, while the action of cationic liposomes on cellular membranes allowed more plasmids to breach the cell membrane. This study has proven that the novel microplex vector is capable of selective delivery of genes to tumors and has the potential to target genes in clinical trials.

Comparison of nickel-titanium and stainless-steel spreader penetration and accessory cone fit in curved canals.

The purpose of this study was to compare nickel-titanium and stainless-steel spreader penetration in curved canals. Twenty prepared plastic blocks with a 30 degrees curvature were used for each part of the study. In part 1, the force required to insert each spreader to within 1 mm of working length in an empty canal was measured. In part 2, the force required to insert each spreader to within 3 mm of working length was measured in a canal containing a master cone. In part 3, the depth of penetration of each spreader with a master cone in place using a 1.5 kg force was measured. Additionally, in part 3, the depth of penetration of the first accessory cone was measured. The results from part 1 showed that a nickel-titanium spreader required significantly less force than a stainless-steel spreader (0.30 kg vs. 0.59 kg). In part 2, a nickel-titanium spreader required significantly less force than a stainless-steel spreader (1.56 kg vs. 2.42 kg). In part 3, a nickel-titanium spreader penetrated significantly deeper than a stainless-steel spreader (15.0 mm vs. 14.0 mm). There was no significant difference in the depth of penetration of the first accessory cone used after either spreader (0.8 mm vs. 0.7 mm). Therefore, the potential for vertical root fracture in curved canals during lateral condensation may be minimized by using nickel-titanium spreaders.

A Comparison of Three Cytotoxicity Endpoints in the Corneal HCE-T Model.

The prediction of ocular irritation potential from in vitro assays still presents a problem, despite a number of validation trials. A study with coded cosmetic formulations, for which historic in vivo data were available, has been conducted with a human corneal multi-layered model system. This corneal model, the HCE-T model, was developed by using HCE-T cells, a transfected human corneal epithelial cell line. The relative effectiveness of three endpoints that provide a measure of cytotoxicity in the HCE-T model was evaluated. Cell viability immediately after exposure to the test materials was determined by using the MTT and Alamar Blue™ (AB) assays, and, 24 hours later, by using the MTT, AB and lactate assays. Viability measurements with the MTT, AB and lactate assays gave similar dose-response curves at the 24-hour endpoint. One formulation (an anti-dandruff shampoo) caused a less severe drop in viability in assays conducted immediately after the exposure than at the 24-hour time-point. There was little deterioration in viability with the other test materials. The ranking of the test formulations on the basis of relative loss of viability and release of lactate resulted in the same order as for the Modified Maximum Average Draize Test Score. Comparison of the HCE-T model cytotoxicity assay results with historic in vitro data from two different cytotoxicity assays, conducted by using fibroblast monolayer cultures and the same materials, indicated that the multi-layered corneal model had a greater predictive ability. The results of a blind trial with the lactate assay in two laboratories indicated that the techniques required were transferable between laboratories. The lactate results were reproducible between laboratories, even when cultures derived from different passage human corneal cells were tested, provided that the passage number was below 20.

Comparison of the cleaning efficacy of passive sonic activation and passive ultrasonic activation after hand instrumentation in molar root canals.

The purpose of this study was to compare the cleaning efficacy of passive ultrasonic activation with that of passive sonic activation after hand instrumentation. Sixty curved molar canals were hand-instrumented to size 35 and divided into three groups. Group 1 received no further treatment. Group 2 received 3 min of passive sonic activation. Group 3 received 3 min of passive ultrasonic activation. The roots were split and photomicrographs (x20) were made of the apical 6 mm of canal. A transparent grid was placed over projected images, and the total number of squares covering the apical 6 mm of canal space and the number of squares containing debris were counted. A debris score was calculated for each specimen by dividing the number of squares with debris by the total number of squares. The mean debris scores were 31.6% for hand instrumentation only, 15.1% for the sonic group, and 16.7% for the ultrasonic group. The debris scores for the sonic and ultrasonic activation groups were significantly lower than that for the hand instrumentation only group (p < 0.01); however, there was no significant difference between the sonic and ultrasonic activation groups. Passive sonics after hand instrumentation produces a cleaner canal than hand instrumentation alone and is comparable with that of passive ultrasonics.

Juvenile-onset distal myopathy in Rottweiler dogs.

Two juvenile Rottweiler siblings were presented with the complaint of decreased activity and various postural abnormalities, including plantigrade and palmigrade stance and splayed forepaw digits. The neurologic examinations were otherwise normal. Electromyography revealed rare fibrillation potentials and positive sharp waves. Motor nerve conduction velocities were normal, whereas compound muscle action potentials from the interosseous muscles were decreased. These findings were consistent with a primary myopathy. A 3rd pup from a different litter and a 4th pup from a litter with 3 of 8 affected dogs had similar clinical presentations. Histopathologic changes in fresh-frozen muscle biopsy samples were similar in all pups and consisted of myofiber atrophy with mild myonecrosis, endomysial fibrosis and replacement of muscle with fatty tissue. These changes were more severe in distal muscles than in proximal muscles. Plasma carnitine concentrations (total and free) were decreased in all pups. Muscle carnitine concentrations (total and free) were decreased in 3 of 4 pups and the least affected pup had a borderline low free muscle carnitine concentration. Abnormalities involving major metabolic pathways were not found on quantification of organic and amino acids. Dystrophin immunocytochemistry was normal in 2 dogs tested. Distal myopathies in humans are classified under the dystrophic group of muscle disorders. These 4 cases represent a form of muscular dystrophy apparently not previously reported in dogs.

Enhanced anticancer therapy mediated by specialized liposomes.

It has been a central aim of experimental and clinical therapeutics to deliver therapeutic agents as close as possible to, or if possible within, a diseased cell. Such targeting achieves two major aims of drug delivery, the maximum dose of therapeutic agent to the diseased cell and avoidance of uptake by and, usually, accompanying side-effects to normal, healthy cells. Conventional liposomes, originally used for studies in membrane biophysics and biochemistry, have been used in therapy for the past two decades. However, when applied to deliver drugs into cells, conventional liposomes proved inefficient and so novel unconventional or specialized liposomes are constantly being prepared to enhance cell-specific delivery in-vivo. One possible way of achieving better targeting is combination of the positive attributes of more than one specialized type of liposome into one vesicle. Although a limited number of studies has examined the combined effect of such dual-specialty liposomes, more studies are warranted using appropriate models. Liposomes are composed of one, a few, or many concentric bilayer membranes which alternate with aqueous spaces. The drugs are encapsulated within the aqueous internal volume if they are hydrophilic or in the lipid bilayers if they are hydrophobic (Kim 1993). Liposomes range in size from 25 nm to more than 20 microns (Sugarman & Perez-Soler 1992). Depending on their solubility and method of formulation antimicrobial, cytotoxic and other conventional drugs, hormones, antigens, enzymes, genetic material, viruses and bacteria can be incorporated in either the aqueous or hydrophobic phase. This review discusses the types and characteristics of non-conventional liposomes used in various modes of cancer therapy, mainly chemotherapy and gene therapy. It concludes with suggestions on improving these novel liposomal to effect better targeting to cancer cells.

Bronchoalveolar lavage: comparison of nucleopore filters and direct smear preparations.

To determine whether cytopreparation affects the diagnostic yield of bronchoalveolar lavage specimens, we compared 50 lavage samples by using two methods. One nucleopore filter preparation and four direct slides were prepared on each sample submitted. All slides were stained by the Papanicolaou method. Assessment of cellularity, cellular preservation, and an independent diagnosis were rendered on each sample for both preparatory methods. Statistical analysis showed no difference in cellularity between the two methods (P = .06). The degree of nuclear and cytoplasmic preservation was higher using the direct method, although this did not appear to affect diagnosis in this study. One major discrepancy in diagnosis was observed between the two methods. By prospectively comparing nucleopore filter and direct preparation of bronchoalveolar lavages, we found that there was minimal affect on either cellularity or diagnosis. We conclude that either method delivers reliable cytologic results.

The efficacy of doxorubicin microspheres for hepatic micrometastases in a rat tumour model.

The use of sustained-release microspheres is of potential benefit as an adjuvant treatment for patients with occult hepatic micrometastases. This study investigates the response of a model of implantable adenocarcinoma micrometastases in the livers of DA rats following the intraportal injection of doxorubicin-incorporated ion-exchange microspheres compared to free drug bolus administration. A point-counting technique was used to determine the percentage of liver consisting of tumour 13 days after treatment. This was used as an indicator of tumour response, as was the derived tumour mass. There was a significantly higher tumour response in animals treated with the microspheres compared to animals treated with free drug delivered at the same concentration. This effect, however, was shown to decrease with a delay in the time of treatment. The tumour response of the sustained-release microspheres was achieved in the absence of any detectable local or systemic toxicity. This study demonstrates the potential of sustained-release microspheres in the treatment of patients with hepatic micrometastases.

Evaluation of a human corneal epithelial cell line as an in vitro model for assessing ocular irritation.

A human corneal epithelial cell line, 10.014 pRSV-T (HCR-T cells), has been used to develop a three-dimensional in vitro model of the human corneal epithelium (HCE-T model). HCE-T cells form a stratified culture when grown at the air-liquid interface on a collagen membrane in serum-free medium. This model served as the basis for assays which supported the ocular irritancy assessment of water-soluble test substances. Cellular alterations in the HCE-T model were measured following 5-min topical exposures to 20 chemicals [listed in the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) Reference Chemicals Data Bank] and 25 surfactant-based product formulations [utilized in the Cosmetic, Toiletry, and Fragrance Association (CTFA) Alternatives Program Phase III]. In vitro assays used were transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). These measured alterations in the barrier function of this corneal epithelial equivalent. Barrier function is a well-developed property in the HCE-T model that supports the mechanistic relevance of these assays. In vitro data, averaged from replicate assays, were compared to respective Draize rabbit eye irritation data from the publicly available ECETOC and CTFA databases using linear regression with Pearson's correlation analysis. For chemicals, Pearson's correlation coefficients, r, from comparisons of Draize maximum average scores (MAS) to TEP and TER data were 0.71 and 0.55, respectively. For product formulations, Pearson's correlation coefficients from comparisons of Draize MAS to TEP and TER data were 0.86 and 0.80, respectively. Data indicated that barrier function alterations in the HCE-T model correlated with ocular irritancy and corneal toxicity. While the irritancy of the chemicals tested was effectively assessed only by the TEP assay, that for the surfactant-based product formulations was effectively assessed by both the TEP and TER assays. Results also suggested that the HCE-T TEP and TER assays vary in their effectiveness for evaluating specific classes of test materials.

Evaluation of chemically induced toxicity using an in vitro model of human corneal epithelium.

Stratified cultures of human corneal epithelial cells were used as an in vitro model for the evaluation of chemical damage to the ocular surface. Plasmid-transfected human corneal epithelial cells (HCE-T cells; 10.014 pRSV-T), cultured on a collagen membrane at the air-liquid interface, form a stratified epithelium (the HCE-T model). Results showed the HCE-T cell line to be comparable to primary human corneal epithelial (HCE) cells in morphology, keratin expression, and calcium-mediated modulation of morphology. Intercellular junctions and other ultrastructural features common to human corneal epithelium were identified in stratified HCE-T cultures. Chemical effects on morphology and cell viability indicated that the HCE-T model was more resistant to chemical toxicity than HCE-T monolayer cultures. Barrier function established by the HCE-T model was determined by measuring transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). Previous results demonstrated similar baseline TEP and TER values for HCE and HCE-T cultures. Stratified HCE-T cultures retained 96.4 +/- 2.2% of the fluorescein applied to the apical surface for 30 min, and attained a TER of 468 +/- 89 ohms x cm(2); these baseline values were maintained over a 20-day culture period. Chemically induced alterations were determined by measuring TEP and TER after 5-min exposures to sodium dodecyl sulfate, benzalkonium chloride, ethanol or isopropanol. These exposures resulted in dose-dependent increases in TEP, and reductions in TER and cell viability (MTT assay). Transmission electron microscopy revealed dose-dependent mechanisms of toxicity. Two days after toxicant treatments, some cultures recovered barrier properties related to TEP, but most had not repaired tight junctions (TER). Cell viability either did not recover, or continued to decline. The results indicate that TEP, TER and the MTT assay measure different properties of the cultures, and are useful endpoints for the evaluation of chemically-induced damage in the HCE-T model. (c) 1997 Elsevier Science Ltd.

Tumor Gene Targeting Using Microspheres: Cell Culture and in vivo Studies.

In a previous study, Dass et al. (Pharm. Sci. 2:401-405, 1996), it was shown that 1 mg of two types of ion-exchange microspheres was capable of binding and releasing plasmids in a continuous-flow system to the order of 10(11) copies in phosphate-buffered saline at 37°C. However, the functionality of the plasmids was not evaluated. In this study, one of the plasmids, pCMV-CAT, was bound to both microspheres and the functionality of the biomol-ecule was examined in cell culture and in vivo transfection studies. Rat tumor cells incubated with the hydroxyapatite (HA) plasmids were transfected 5.4-fold better than when incubated with free plasmids and 56.0-fold better than polystyrene divinylbenzene (PDB) microspheres. Cells incubated with PDB microspheres were transfected 10.4-fold less than cells incubated with free plasmids. However, HA microspheres were highly cytotoxic to the cells whereas PDB spheres had no effect on cell numbers compared with control cells. Based on expression levels of delivered plasmids in the in vivo study, delivery on microspheres to kidneys was 2.7-fold better than plasmids delivered free. Microspherical delivery of plasmids to tumors was 1.6-fold better than free delivery. However, these results were not significantly different (p >. 05). The tumor/normal tissue ratio of gene expression was 4.5:1 for free delivery and 2.6-fold when delivered on microspheres. Although the difference between deliveries in the two tissues was significant (p > 0.005) for free delivery, it was not so for micro-spherical delivery. Expression of the CAT gene was not noted in either liver or spleen of any of the animals. This present study has proved that ion-exchange microspheres have no detrimental effect on released plasmid DNA expression. In an in vivo setting, resistance to enzymatic degradation of plasmids that are bound to microspheres and subsequent release of plasmids once embolization has occurred in the tumor vascular bed effected better transfection than delivery of free plasmids.

Tumour cells surviving in vivo cisplatin chemotherapy display elevated c-myc expression.

The c-myc oncogene has been extensively implicated in cell proliferation, cell differentiation and programmed cell death. Aberrant expression of the c-myc gene product has been observed in a range of tumours and has also been implicated in cisplatin (cis-dichlorodiammineplatinum)-mediated chemoresistance. A solid transplantable tumour model in syngeneic DA rats was subjected to treatment with cisplatin to determine the impact of such therapy on endogenous c-myc gene expression. Serially transplanted tumours were intravenously treated with a single cisplatin dose (1 mg/kg) and c-myc expression analysed 2 and 7 days after treatment. The surviving tumour cells display a significant 2-fold elevation in c-myc expression at 48 h and 7 days after treatment. Primary cell cultures have been derived from untreated in vivo tumours of the same model and subjected to treatment with a c-myc phosphorothioate antisense oligomer. Administration of 5 microM c-myc antisense oligomer directed at the initiation codon and first four codons of c-myc mRNA results in total inhibition of c-myc expression and coincident suspension of cell growth for a period of 4 days in culture. Antisense therapies directed at the c-myc gene may well prove an effective tool for treating tumours in conjunction with cisplatin as these findings show that tumour cells surviving cisplatin chemotherapy display elevated c-myc expression.