Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.     

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house aminopropyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37 degrees C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P < 0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 microm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P < 0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications.     

LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication

A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.     

Acute surgical unit safely reduces unnecessary after-hours cholecystectomy

Introduction

The acute surgical model has been trialled in several institutions with mixed results. The aim of this study was to determine whether the acute surgical model provides better outcomes for patients with acute biliary presentation, compared with the traditional emergency surgery model of care.

Methods

A retrospective review was carried out of patients who were admitted for management of acute biliary presentation, before and after the establishment of an acute surgical unit (ASU). Outcomes measured were time to operation, operating time, after-hours operation (6pm – 8am), length of stay and surgical complications.

Results

A total of 342 patients presented with acute biliary symptoms and were managed operatively. The median time to operation was significantly reduced in the ASU group (32.4 vs 25.4 hours, p=0.047), as were the proportion of operations performed after hours (19.5% vs 2.5%, p<0.001) and the median length of stay (4 vs 3 days, p<0.001). The median operating time, rate of conversion to open cholecystectomy and wound infection rates remained similar.

Conclusions

Implementation of an ASU can lead to objective differences in outcomes for patients who present with acute cholecystitis. In our study, the ASU significantly reduced time to operation, the number of operations performed after hours and length of stay.     

Transseptal pressure gradient with leftward septal displacement during the Mueller manoeuvre in man.

Septal displacement is postulated as an important mediator of ventricular interdependence. During acute right ventricular loading with the Mueller manoeuvre the septum flattens and shifts leftward. To investigate the mechanism of this septal deformation, we measured transseptal pressures in nine patients during Mueller manoeuvres with simultaneous right and left ventricular micromanometers, and left ventricular configuration with two-dimensional echocardiograms. Data were analysed throughout diastole and at end-systole during control and maximum Mueller manoeuvre (-40 to -80 mmHg airway pressure). Leftward septal displacement during the Mueller manoeuvre was evidenced by an increase in septal radius of curvature at end-diastole persisting through end-systole. The left ventricular free wall radius of curvature was unchanged. During the Mueller manoeuvre, the left ventricular cavity area decreased significantly in the cross-sectional view. All Mueller manoeuvres were associated with a decrease in left-to-right ventricular transseptal pressure gradient throughout diastole. There was no significant change in the gradient at end-systole; septal flattening persisted, however, despite a pronounced left to right pressure gradient. Thus, diastolic septal flattening during right ventricular loading is associated with a decreased transseptal pressure gradient but does not require right ventricular diastolic pressure to exceed left ventricular diastolic pressure. The persistence of flattening in systole suggests that once septal shift occurs during diastole, other forces during systole maintain the deformity despite a large intracavitary transseptal gradient.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.