One-step fluorescent probe product-enhanced reverse transcriptase assay.

Recently developed PCR-based reverse transcriptase (RT) assays are useful in the detection of retroviruses since they are approximately a millionfold more sensitive than conventional RT assays. However, these assays are both labor- and time-intensive. The previously described product-enhanced reverse transcriptase (PERT) assay involves a two-step RT-PCR followed by detection and quantitation of PCR products by either Southern blot or enzyme-linked immunosorbent assay (ELISA). We have modified the PERT assay to be a one-step, fluorescent probe, PCR-based RT assay that can be completed from sample dilution to final quantitative assay results in approximately 5 h without loss of assay sensitivity or specificity. The assay has a dynamic range of 6 logs, and therefore, extensive sample dilution is not necessary for quantitation. This newly enhanced fluorescent PERT assay can play an important role in the high-throughput detection of retroviral infection and characterization of RT activity.

Anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries.

A phage display library screening approach was used to identify peptide sequences that could bind to anti-HIV-1 MAbs whose binding specificities are complex. Most of the antibodies used recognize discontinuous epitopes in gp120 and one recognizes gp41. Both a 15-mer and a 21-mer display library (each with a complexity of greater than 60 x 10[6]) and two constrained, V3 region-biased libraries, all expressed as recombinant pIII protein of filamentous phage, were used. The unmapped anti-gp120 human MAb A32 recognized a set of related linear sequences and repeatedly identified a single phage sequence that could form a cyclic disulfide structure. Selection methods were also developed so that phage could be obtained by competition selection in the presence of antibody bound to native, monomeric gp120 antigen (used with MAb IgG1b12 and the anti-gp120 V3 region MAb 447-52D) or gp120 variable region 3 synthetic peptides (used with anti-gp120 V3 region MAb 19b). The potent, virus-neutralizing MAb IgG1b12 recognized numerous sequences and, when used in competition with gp120, recognized only one sequence. These studies extend the range of antibody determinant studies that can be performed with display phage libraries, demonstrate a workable experimental strategy for use of competition ligands to discriminate among phage mimotopes, and provide a large number of mimotopes that bind potent virus-neutralizing MAbs for HIV-1 vaccine studies.

Higher ploidy in Saccharomyces cerevisiae supports enhanced hepatitis B virus S cloned gene expression at the pilot scale.

The effect of host strain ploidy on the production of hepatitis B surface antigen (HBsAg) in Saccharomyces cerevisiae was evaluated at the pilot scale (75 L). We found that the accumulation of HBsAg normalized to cell protein was 2-fold higher for the diploid strain compared to its isogenic haploid. No detectable differences in many fermentation parameters were observed (e.g., rate of fermentation, growth rate, final cell yield). However, the enhancement of productivity in the diploid strain appeared to be associated with a slower rate of plasmid shedding (2 microns element) and, thus, a higher average copy number (2-fold at stationary phase) compared to those of the haploid strain.

Neutralization of divergent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody.

The antiviral characteristics of monoclonal antibody IAM-41-2F5 (2F5) were determined in cell culture. The antibody had been previously shown to bind a specific sequence, ELDKWA, within the external domain of the gp41 envelope glycoprotein human immunodeficiency virus type 1 (HIV-1). Selection by 2F5 of recombinant phage from an epitope library confirmed the identification of the antibody's binding determinant. The antibody was found to be capable of neutralizing a broad range of lymphoid cell culture-adapted HIV-1 variants as well as HIV-1 primary isolates. Sequence analysis of the latter showed that neutralization was related to the presence of the antibody binding site. From kinetic measurements using an epitope-containing peptide or gp41, the half-time of dissociation for 2F5 was determined to be 122 min for the peptide and 156 min for gp41. The region of gp41 expressing this sequence exhibits greater conservation among HIV-1 isolates than do the variable domains of gp120.

Identification of HIV vaccine candidate peptides by screening random phage epitope libraries.

Most synthetic HIV-1 gp120 V3 loop peptides that are used as immunogens in experimental HIV-1 vaccine studies are modeled from the naturally occurring viral gp120 V3 loops. In experimental animals these immunogens generally elicit type (or variant)-specific neutralizing antibodies that are not broadly reactive among HIV-1 variants. In an attempt to find a more general structure for the V3 loop, we have obtained candidates that mimic V3 loop sequences by screening random epitopes displayed in a fusion phage 15-residue epitope library. Human monoclonal antibody 447-52D, a highly potent and broadly reactive virus-neutralizing antibody that recognizes the conserved V3 loop tip motif GPXR, was the probe. By using a screening method that was designed specifically for this work, we identified hundreds of reactive phage clones, 70 of which were sequenced. Over 98% of the epitopes contain the motif GPXR, yet none of the 70 are an identical match to any V3 variant loop described to date. One of these sequences was synthesized as the beta-maleimidopropionyl 15-mer peptide, covalently conjugated to a carrier and used to immunize rabbits. High anti-peptide titers were obtained in all animals with three of four individual responses also binding to a peptide that is representative of the "North American consensus" V3 loop. The sera from these three positive rabbits neutralized HIV-1 variant SF-2 in vitro. In addition, one of them was capable of neutralizing variant AL-1. Both of these variants are considered to have V3 loops of the North American consensus type. Thus, neutralizing responses were obtained by use of an immunogen that was selected for its ability to bind a broadly reactive human monoclonal antibody rather than modeled from an HIV-1 gp120 V3 loop sequence.

Cloning of the cDNA and expression of moubatin, an inhibitor of platelet aggregation.

Moubatin, a new type of specific inhibitor of collagen-induced platelet aggregation, has been isolated from the soft tick Ornithodoros moubata (Waxman, L., and Connolly, T. M. (1993) J. Biol. Chem. 268, 5445-5449). A polymerase chain reaction-generated hybridization probe, produced using primers based on moubatin protein sequence, identified phage containing the entire cDNA sequence of moubatin. Analysis of the predicted amino acid sequence yielded a mature protein of 156 amino acids with a putative prepeptide of 15 amino acids. Comparison of the sequence of moubatin to that of other proteins in the Swiss PROT data base revealed no significant homology. The cDNA sequence was cloned into the yeast expression vector pKH4 alpha 2, producing a biologically active protein which inhibited collagen-stimulated aggregation of washed human platelets with an IC50 of about 100 nM, which is similar to the potency of native tick moubatin. A concentration of recombinant moubatin that fully inhibited collagen-stimulated aggregation did not inhibit aggregation induced by a variety of other platelet agonists, again demonstrating comparable properties of the recombinant and native proteins. Moubatin did not inhibit platelet adhesion to collagen even at a concentration up to 16 times its IC50 for the inhibition of aggregation. This specificity for inhibiting collagen-stimulated aggregation and not adhesion to collagen indicates that moubatin is unique among the natural product inhibitors of collagen stimulation of platelets. Further examination of the mechanism of moubatin-mediated inhibition of collagen-stimulated aggregation revealed that 1-6 microM moubatin diminished the second phase of aggregation induced by ADP, inhibited aggregation in response to submaximal concentrations of the thromboxane A2 mimetic U46619, and competed for the binding of a thromboxane A2 receptor antagonist to platelet membranes. Therefore, at higher concentrations, moubatin may affect more than one aspect of platelet signal transduction including the thromboxane A2 receptor. The availability of recombinant moubatin will allow further investigation of its unique activities in vitro and in vivo.

Use of GM-CSF in children after high-dose chemotherapy.

The toxicity and efficacy of recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) is established in adults, but limited information is available on its use in children. The profound myelotoxicity induced by cisplatin (40 mg/m2 daily x 5) and etoposide (150 mg/m2 daily x 3) provides a model to test the clinical value of recombinant human granulocyte-macrophage colony-stimulating factor in pediatric cancer patients; myelosuppression occurred (absolute neutrophil count [ANC] < 500/microL) during 99 of 118 (84%) courses given to 44 children with refractory solid tumors. Fifty-nine courses (50%) resulted in hospitalizations for fever. Subsequently, rhuGM-CSF was added to this treatment regimen to: (i) determine the dose-limiting toxicity of this agent in children; and (ii) to determine whether it can decrease the duration and severity of neurtropenia and attendant complications. Here we summarize and update our experience with this glycoprotein in children with relapsed solid tumors.

Granulocyte colony-stimulating factor corrects the neutropenia associated with glycogen storage disease type Ib.

A young woman with glycogen storage disease, type Ib, and chronic neutropenia had severe recurrent infections. In a life-threatening situation, treatment with granulocyte colony-stimulating factor (G-CSF) resulted in the prompt correction of neutropenia. Subsequently, daily G-CSF therapy has allowed the maintenance of a normal neutrophil count and marked clinical improvement over a period of 18 months. The spectrum of neutropenic conditions which are responsive to G-CSF should include this inherited metabolic disorder.

Characterization of the point spread function and modulation transfer function of scattered radiation using a digital imaging system.

A digital radiographic system was used to measure the distribution of scattered x radiation from uniform slabs of Lucite at various thicknesses. Using collimation and air gap techniques, [primary + scatter] images and primary images were digitally acquired, and subtracted to obtain scatter images. The scatter distributions measured using small circular apertures were computer fit to an analytical function, representing the circular aperture function convolved with a modified Gaussian point spread function (PSF). On the basis of goodness of fit criterion, the proposed Gaussian function is a very good model for the scatter PSF. The measured scatter PSF's are reported for various Lucite thicknesses. Using the PSF's, the modulation transfer functions are calculated, and this spatial frequency information may have value in analytical scatter removal techniques, grid design, and air gap optimization.

Noise analysis of a digital radiography system.

The sources of noise in a digital video subtraction angiography system were identified and analyzed. Signal-to-noise ratios of digital radiography systems were measured using the digital image data recorded in the computer. The major sources of noise include quantum noise, TV camera electronic noise, quantization noise from the analog-to-digital converter, time jitter, structure noise in the image intensifier, and video recorder electronic noise. A new noise source was identified, which results from the interplay of fixed pattern noise and the lack of image registration. This type of noise may result from image-intensifier structure noise in combination with TV camera time jitter or recorder time jitter. A similar noise source is generated from the interplay of patient absorption inhomogeneities and patient motion or image re-registration. Signal-to-noise ratios were measured for a variety of experimental conditions using subtracted digital images. The measured signal-to-noise ratios were found to fluctuate on repeat trials with about a 10% standard deviation. Averaging of video frames was found to reduce the noise level by the expected square root N relation, where N is the number of frames averaged. Image-intensifier structure noise was shown to be a dominant noise source in unsubtracted images at medium to high radiation exposure levels. A total-system signal-to-noise ratio (SNR) of 750:1 was measured for an input exposure of 1 mR/frame at the image intensifier input. The effect of scattered radiation on subtracted image SNR was found to be greater than previously reported. The detail SNR was found to vary approximately as one plus the scatter degradation factor. Quantization error noise with 8-bit image processors (signal-to-noise ratio of 890:1) was shown to be of increased importance after recent improvements in TV cameras. The results of the analysis are useful both in the design of future digital radiography systems and the selection of optimum clinical techniques.

The effect of phosphor K x-rays on the MFT of rare-earth screens.

The LSF's and MTF's of two high resolution rare-earth screen-film combinations were measured at two beam qualities. The two beam qualities were chosen to provide x-ray spectral distributions either above or below the K-edge of the screen phosphor. The LSF's were found to be photon energy dependent. This energy dependence is attributed to the generation and reabsorption of phosphor K x-rays resulting in a broadening of the LSF.

Magnification mammography: a low-dose technique.

Image quality and radiation exposures of a mammographic technique using direct radiographic magnification at 2 X with a microfocal spot x-ray tube and a fast, double screen-film system were compared to those of conventional contact mammography with a rare-earth screen and molybdenum target tube. The results indicate that the magnification technique yields improved detection of microcalcifications and comparable visualization of soft-tissue details, with a large reduction in radiation exposure. This technique has demonstrated the feasibility of carrying out high-quality mammography with an entrance dose of 1.35 X 10(-3) Gy (135 mrad) for the average breast.

Postero-anterior radiography: a method for reduction of eye dose.

The radiation dose to the eyes during cerebral angiography can be reduced by a factor of 50--100 by radiographing the patient in the postero-anterior projection. Test pattern and phantom tests demonstrated no loss of image quality.

Evaluation of mammographic screen-film systems.

Four screen-film systems were evaluated for their imaging properties in mammography, Modulation-transfer functions were measured at 40 kVp. Absolute screen-film sensitivities in mR and entrance exposures were measured with tungsten and molybdenum target tubes. Five radiologists viewed radiographs of a phantom containing microgranules of SiC ranging in diameter from 590 to 120 micrometer. The Rarex-B screen--composed of yttrium oxysulfide--performed best, allowing phantom radiographs at 185 mR with image quality sufficient to demonstrate microgranules greater than 330 micrometer in dimension.

Measurements of reciprocity law failure in green-sensitive X-ray films.

Reciprocity law failure was measured for four brands of medical x-ray films exposed with intensifying screens. Three of the films are green light-sensitized for use in combination with green light-emitting rare-earth screens. These films showed larger reciprocity failure effects than one conventional blue-sensitive film, Dupont Cronex-2. Development conditions had a small effect on reciprocity failure. As part of the investigation, a detector was constructed with a response that accurately monitors the light emission from the double screen-cassette combination over a wide range of x-ray photon energies.

The LSF and MTF of rare-earth oxysulfide intensifying screens.

The line spread function (LSF) and modulation transfer function (MTF) of 9 rare-earth screen/film systems were measured and compared with those of two fast calcium tungstate systems, using double-emulsion films sandwiched between two screens and mounted in regular cassettes. The LSFs were found to fit exponential functions. These results indicate that the increased sensitivity of rare-earth phosphors over calcium tungstate can be used to construct screens with a higher MTF or increased speed. The fast rare-earth systems allow the use of smaller focal spots for increased resolution while reducing the radiation dose to the patient.