Quantitation of proteins and internal antigen pools by a monoclonal sandwich radioimmune assay.

We describe a monoclonal sandwich radioimmune assay (MSRA) that is capable of measuring 0.5 fmoles of protein antigens. The basis of the assay is the ability of most rabbit heteroantisera directed against protein antigens to precipitate labeled monoclonal Fab-antigen complexes. The assay offers the advantages of specificity, sensitivity, linearity and rapidity. The MSRA circumvents the need for preparation of either highly purified protein antigens for use in radioimmune inhibition assays or affinity purified antisera for sandwich immunoassays. Utilizing variations of MSRA protocols, we determined that 48% of FcR gamma 2b/gamma 1, 34% of an 90,000 Mr glycoprotein identified by monoclonal antibody 2D2C, and 24% of an 82,000 Mr glycoprotein identified by monoclonal antibody 2E2A were accessible to labeled Fab probes at the plasma membrane. The total amount of antigen per cell was determined by dividing the amount of antigen at the cell surface (determined from binding saturation assays) by the percentage of surface/total antigen. This value of total antigen agrees well with the total cellular antigen pool determined by MSRA after appropriate corrections for probe saturation and recovery of pre-formed Fab-antigen complexes were made.

Optimum field size and choice of isodose lines in electron beam treatment.

PURPOSE: A method is provided for the optimum field size and the choice of isodose line for the dose prescription in electron beam therapy. METHODS AND MATERIALS: Electron beam dose uniformity was defined in terms of target coverage factor (TCF) which is an index of dose coverage of a given treatment volume. The TCF was studied with respect to the field size, the beam energy, and the isodose level for prescription from the measured data for various accelerators. The effect of the TCF on air gap between electron applicator/cone and the surface was investigated. Electron beams from scattering foil and scanned beam units were analyzed for the target coverage. RESULTS: A mathematical method is provided to optimize a field size for target coverage by a given isodose line in terms of TCF which is strongly dependent on the type of accelerator and the design of the collimator. For a given type of collimating system, the TCF does not depend on the type of electron beam production (scattering foil or swept scanned beam). Selection of isodose line for dose prescription is very critical for the value of the TCF and the dose coverage. The TCF is inversely proportional to the isodose value selected for the treatment and nearly linear with field size and beam energy. Air gap between applicator and the surface reduces the dose uniformity. Tertiary collimator moderately improves the lateral coverage for high energy beams. CONCLUSIONS: To adequately cover the target volume in electron beam treatment, lateral and depth coverage should be considered. The coverage at depth is strongly dependent on the choice of isodose line or beam normalization. If the dose prescription is at dmax (i.e., the 100% isodose line is selected), the choice of beam energy is not critical for depth coverage since dmax is nearly independent of energy for smaller fields. The 100% isodose line should not be chosen for treatment because of the significant constriction of this isodose line and inadequate coverage at depth. For a higher TCF, a minimum air gap between the cone to the surface of the patient is desired. If such is not possible, then a tertiary collimator at the skin is required. Whenever, a tertiary collimator is used, it is advised to increase the collimator field size by a factor of 1.4.

Low dose rate versus high dose rate intraluminal brachytherapy for malignant endobronchial tumors.

Although the evolution from low dose rate to high dose rate brachytherapy for malignant endobronchial malignancies was primarily based on economy, patient convenience, and radiation protection, the difference in therapeutic index, if any, between these two modalities must be kept in mind. Our experience with both methods permits assessment of the feasibility of replacing low dose rate brachytherapy with high dose rate brachytherapy. Results with our first 110 patients (group 1) treated with low dose rate brachytherapy (133 procedures) were compared with results with our initial 59 consecutive patients (group 2) treated with high dose rate brachytherapy (161 procedures). In group 1, patients were treated with one or two sessions of 30-60 Gy each calculated at a 1 cm radius. In patients in group 2, we aimed at three weekly sessions of 7 Gy each calculated at a 1 cm radius. External beam irradiation therapy had previously been given to 88% of patients in group 1 and to 85% of patients in group 2. Laser bronchoscopy was performed in 36% of patients in group 1 and in 24% of patients in group 2 before brachytherapy. Clinical or bronchoscopic improvement was noted in 72% of patients in group 1 and in 85% of patients in group 2 (p > 0.05). Complication rates were low and comparable. Survival was similar in both groups (median < 6 months). Although both low dose rate and high dose rate brachytherapy appear equally effective in palliation for malignant endobronchial obstruction, we are now practicing the latter exclusively.

Treatment of high-grade low-stage prostate cancer by high-dose-rate brachytherapy.

BACKGROUND AND PURPOSE: The best management of patients with low-stage, high-grade prostate cancer remains unclear. In an attempt to improve the outcomes of this high-risk group, we have offered those with Gleason > or =7 cancers removable-source high-dose-rate (HDR) brachytherapy in combination with external-beam radiation. PATIENTS AND METHODS: We reviewed the clinical histories of 61 consecutive patients with high-grade clinical stage T1-T2 lesions who received the combination radiation therapy between March 1997 and November 1998. The average Gleason score was 7.5. The HDR brachytherapy was given in three sessions with removable-source afterloaded (192)Ir to a minimum peripheral dose of 6 Gy. Conformal external-beam radiation in 25 fractions to a dose of 50 Gy was given beginning 1 week later. Patients with prostate volumes >40 cc received a luteinizing hormone-releasing hormone analog before brachytherapy. RESULTS: Among the 52 patients available for follow-up (average duration 11.8 months), there has been one death from prostate cancer. After treatment, only one patient had an initial rise in serum prostate specific antigen (PSA) concentration. In addition to the patient who died, there have been three confirmed treatment failures. Toxicity was mild, with only two patients having RTOG grade 3 or 4 effects. Neither of them required surgery. CONCLUSION: Although long-term results are not available, available data suggest that HDR brachytherapy plus external-beam radiation is at least as effective as any single therapy for high-risk, low-stage prostate cancer. The toxicity is acceptable.

Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin.

Results from several systems indicate that correct protein folding and subunit assembly correlate with the transport of membrane and secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex. Because the site of oligomer assembly and its precise relationship to intracellular transport remain unclear, we have studied in detail the folding and trimerization of the influenza virus hemagglutinin (HA0) relative to its transport from ER to Golgi. Trimerization and transport were analyzed using several different methods, including transport inhibitors, temperature blocks, semi-intact cells, in vitro protein translocation, and immunocytochemistry. Taken together, the results clearly demonstrate that trimerization occurs at a point prior to exit from the ER. Before assembly, HA0 monomers were extensively folded and possessed intramolecular disulfide bonds, but monomers were not transported to the cis Golgi compartment. Thus, hemagglutinin progresses through at least two intermediate states before transport to the Golgi: highly folded monomers and trimers that have not yet left the ER.