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P Lake

Publications and source records attributed to P Lake.

At least 19 recordsLinked to original sources
PubMed2002

X-ray absorption spectroscopy measurements of thin foil heating by Z-pinch radiation.

Absorption spectroscopy measurements of the time-dependent heating of thin foils exposed to intense z-pinch radiation sources are presented. These measurements and their analysis provide valuable benchmarks for, and insights into, the radiative heating of matter by x-ray sources. Z-pinch radiation sources with peak powers of up to 160 TW radiatively heated thin plastic-tamped aluminum foils to temperatures approximately 60 eV. The foils were located in open slots at the boundary of z-pinch hohlraums surrounding the pinch. Time-resolved Kalpha satellite absorption spectroscopy was used to measure the evolution of the Al ionization distribution, using a geometry in which the pinch served as the backlighter. The time-dependent pinch radius and x-ray power were monitored using framing camera, x-ray diode array, and bolometer measurements. A three-dimensional view factor code, within which one-dimensional (1D) radiation-hydrodynamics calculations were performed for each surface element in the view factor grid, was used to compute the incident and reemitted radiation flux distribution throughout the hohlraum and across the foil surface. Simulated absorption spectra were then generated by postprocessing radiation-hydrodynamics results for the foil heating using a 1D collisional-radiative code. Our simulated results were found to be in good general agreement with experimental x-ray spectra, indicating that the spectral measurements are consistent with independent measurements of the pinch power. We also discuss the sensitivity of our results to the spectrum of the radiation field incident on the foil, and the role of nonlocal thermodynamic equilibrium atomic kinetics in affecting the spectra.

Journal Article↗
PubMed2001

A high-capacity alkaline phosphatase reporter system for the rapid analysis of specificity and relative affinity of peptides from phage-display libraries.

We describe a novel reporter enzyme cassette system which enables the analysis of large numbers of linear and cyclic peptides in terms of their binding to a specific target molecule. In this system, peptides selected for target binding from random peptide phage-display libraries are expressed as cloned fusion proteins with bacterial alkaline phosphatase. The binding specificity and relative affinity of each peptide-enzyme fusion protein is then evaluated in a target-specific ELISA. This strategy enables direct identification of the highest affinity peptides, specific for a given target, which can then be sequenced at the DNA level to derive their peptide sequences. This eliminates the need to sequence large numbers of clones to establish consensus sequences for binding peptides. This approach also eliminates the need for peptide synthesis or phage ELISA to determine relative binding affinities, which can be technically difficult. Identification of binding peptides based on specificity and relative affinity, rather than conforming to an amino acid consensus sequence, enables the rapid evaluation of hundreds of candidate peptides and identification of rare (non-consensus) binding peptides which may otherwise be missed.

Alkaline Phosphatase↗
PubMed2001

A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates.

We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.

Anti-Allergic Agents↗
PubMed2001

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.

ADP Ribose Transferases↗
PubMed2001

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein.

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin-based anti-(human CD3) immunotoxin, DT389-scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1-389 of diphtheria toxin, scFv is single-chain variable-region antibody fragment and UCHT1is an anti-(human CD3) monoclonal antibody]. Biochemical characterization of this molecule by MS and N-terminal sequencing by Edman degradation revealed that the protein was heterogeneous at the N-terminus, containing species both with (60%) and without (40%) the initiator methionine residue. In an attempt to generate an N-terminally homogeneous molecule, a panel of seven N-terminal variants was designed, based on the published specificity of bacterial methionine aminopeptidase. Following bacterial expression, partial purification and separation on SDS/PAGE, these proteins were subjected to N-terminal sequencing by Edman degradation. Three of the mutants yielded a 100% homogeneous amino acid sequence. By contrast, the original DT389-scFv(UCHT1) protein and four variant proteins yielded two sequences with varying ratios corresponding to species with and without methionine. The N-terminal sequences of the three homogeneous clones were MLADD and MLDD, where the methionine was completely retained, and SADD, where the methionine was completely removed. One of the homogeneous mutants (SADD) was expressed, refolded and purified and found to be equipotent with the parent immunotoxin. Thus, using a rational mutagenesis approach, three N-terminally homogeneous variants of DT389-scFv(UCHT1) have been identified, at least one of which is functionally indistinguishable from the parent immunotoxin. This approach is generally applicable to biopharmaceutical production and immunotoxin development in particular.

Antibodies, Monoclonal↗
PubMed2001

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Animals↗
PubMed2001

A high-capacity quantitative mouse model of drug-mediated immunosuppression based on rejection of an allogeneic subcutaneous tumor.

We describe a high-capacity in vivo assay to measure drug-mediated transplant immunosuppression using a mouse model of Sa1 tumor rejection. Sa1 grew poorly and was rejected by 14 days in immunocompetent allogeneic recipient mice. In nude (nu/nu) mice, Sa1 grew more rapidly and was not rejected, confirming the T cell dependence of this response. In immunocompetent animals, administration of immunosuppressive agents resulted in increased tumor growth relative to vehicle-treated animals. Treatment with immunosuppressive drugs such as cyclosporin A (CsA), 40-O-(2-hydroxyethyl)-rapamycin (SDZ RAD), or 2-amino-2-[2-(4-octylphenyl)ethyl]-1,2-propanediol hydrochloride (FTY720) produced dose-dependent inhibition of tumor rejection. By contrast, the drugs did not affect Sa1 tumor growth in nu/nu mice, which is consistent with their predicted indirect effect on tumor size by suppressing immunity, rather than by directly stimulating Sa1 growth. Drug potency, which is usually not described for immunosuppressive agents, was calculated from the linear relationship between drug dose and tumor volume. The potency of CsA was inversely related to the stringency of the histocompatibility barrier. Another advantage of this assay is that the endpoint is an objective size measurement over a short time period, compared with transplant models where the endpoint may not be reached for many weeks and may be more subjective. In addition, this model can measure the potency of combination drug treatments and compare new immunosuppressive drug regimens. For example, the administration of SDZ RAD or FTY720 with CsA resulted in a more than additive increase in potency, compared with the sum of the drugs as single agents.

Animals↗
PubMed1999

Effect of a conjugate of polymyxin B-dextran 70 in horses with experimentally induced endotoxemia.

OBJECTIVE: To determine the efficacy of polymyxin B-dextran 70 (PBD) for treatment of endotoxemic horses. ANIMALS: 15 horses during study 1 and 6 horses during study 2. PROCEDURES: 3 groups were used in study 1. Horses in groups 1 and 2 were given 30 ng of lipopolysaccharide (LPS)/kg of body weight, IV, over 60 minutes. Horses in group 3 were given saline (0.9% NaCl) solution. Beginning 15 minutes before LPS infusion and continuing for 75 minutes, horses in groups 1 and 3 were given PBD, IV. Horses in group 2 were given dextran 70. Blood samples were obtained for hemograms and determination of cytokine, lactate, and prostanoid concentrations. In study 2, horses were given ketoprofen (2.2 mg/kg) or saline solution 15 minutes before infusion of PBD. Fourteen days later, treatments were reversed, using a crossover design. Blood samples were obtained for measurement of thromboxane B2 (TXB2) concentration. RESULTS: For study 1, prior treatment with PBD completely blocked endotoxin-induced changes for heart and respiratory rates, rectal temperature, WBC count, and plasma tumor necrosis factor, interleukin 6, TXB2, and prostaglandin F1 concentrations. There was transient tachypnea, sweating, and increased plasma TXB2 concentration in horses given PBD (with or without LPS). Prior treatment with ketoprofen eliminated all PBD-induced signs and prevented the increase in plasma TXB2 concentration. CONCLUSIONS: Signs of endotoxemia were prevented in horses by treatment with PBD, although its use was associated with mild adverse effects. CLINICAL RELEVANCE: When used in combination with a cyclooxygenase-inhibiting drug, PBD has potential for treatment of horses with endotoxemia.

Animals↗
PubMed1995

Binding epitope of somatostatin defined by phage-displayed peptide libraries.

We have developed a versatile phagemid system to display peptides on the surface of M13 bacteriophage at a copy number which approaches monovalency. In this system, a phagemid encodes a peptide fused to the amino-terminus of the second domain (dII) of the minor coat protein pIII under control of the inducible lac promoter. The fusion protein is displayed in combination with several copies of wild-type pIII on the surface of phage. Two diverse random octapeptide libraries, one linear and one which contained flanking cysteines capable of forming disulfide bridges, were were generated using an in vitro mutagenesis approach and affinity selected on an anti-somatostatin mAb. Peptides with high affinity for the mAb were enriched only from the cyclic library and the tetrapeptide, FWKT, was identified by consensus as the binding epitope. The selected peptides exhibited not only the primary amino acid sequence but also shared structural features with somatostatin. One peptide, CRFWKTWC, also exhibited nanomolar affinities for the five known somatostatin receptor subtypes. This system can easily be adapted to display individual peptides or a wide range of custom peptide libraries.

Amino Acid Sequence↗
PubMed1995

Therapeutic efficacy of a polymyxin B-dextran 70 conjugate in experimental model of endotoxemia.

Numerous studies have suggested that lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, is responsible for the initiation of gram-negative septic shock. Previously, others have designed therapeutic regimens to target the biologically active lipid A region of LPS by either neutralization of the biological properties of LPS or enhancement of clearance of this molecule. One such compound capable of neutralizing lipid A is the antibiotic polymyxin B. However, the clinical utility of polymyxin B is limited by its toxicity. We therefore covalently conjugated this antibiotic to the high-molecular-weight polysaccharide dextran 70, resulting in reduced toxicity of polymyxin B but retention of its endotoxin-neutralizing ability. The studies described in this report were designed to test the in vivo efficacy of this compound in an experimental animal model of gram-negative septic shock. Mice were administered graded doses of Escherichia coli or Pseudomonas aeruginosa along with D-galactosamine and the antibiotic imipenem. We had previously determined that antibiotic chemotherapy provides significant protection against E. coli-mediated lethality with smaller doses of bacteria; however, the antibiotic does not provide protection against larger doses of bacteria, but it is effective at killing the bacterial inoculum in vivo. Administration of the polymyxin B-dextran 70 conjugate provided significant protection when given with an antibiotic but was not effective by itself. A requirement for a pretreatment period prior to E. coli challenge was shown to depend upon the bacterial challenge dose. In other studies using this D-galactosamine sensitization model, we demonstrated that the lipid A-specific conjugate had no effect on lethality caused by staphylococcus aureus or tumor necrosis factor alpha. The results of these studies indicate that this compound is effective in preventing lethal gram-negative septic shock in mice and may be useful as a potential therapeutic agent in humans as well.

Animals↗
PubMed1994

Primate antibodies to components of the human immune system.

The feasibility to raise nonhuman primate antibodies against selected components of the human immune system was tested. The immunogens were whole cells (human T lymphocytes) or purified, recombinant human proteins (cytokines: TNF alpha or GM-CSF; soluble forms of cell surface antigens: sCD4 or sCD25). Significant immunizations, yielding functionally relevant antibodies, were readily achieved in rhesus monkeys, but, not surprisingly, may be less frequent in chimpanzees. The results suggest a general strategy for production of therapeutically useful MAB.

Animals↗
PubMed1990

Isolation and characterization of a novel human monoclonal antibody that neutralizes tetanus toxin.

A human monoclonal antibody, designated 53-2-4, has been isolated and characterized in terms of its ability to interact with clostridial neurotoxins. In enzyme-linked immunosorbent assay assays the antibody reacted with native tetanus toxin, tetanus toxoid and the C fragment obtained from the carboxyterminus of the toxin (AA 864-1314). The antibody did not react with the B fragment of tetanus toxin (AA 1-863) or with six serotypes of botulinum neurotoxin (A to F). Both culture supernatant from the clonal line producing the antibody as well as homogeneous protein obtained by affinity purification of the antibody neutralized tetanus toxin. When tested in vivo, the antibody provided complete production against a supralethal injection of toxin; when tested in vitro, the antibody produced at least 99% inactivation of a 1 x 10(-9) M solution of toxin. The exceptional neutralizing activity of the antibody was attributed to its high affinity for the toxin (4.2 x 10(-10) mol/liter). Animal experiments revealed a novel phenomenon that has been labeled delayed intoxication. At the appropriate ratio of antibody to antigen, the toxin was retained in the host in a latent form. After several days the biological activity of the toxin became apparent and there was onset of nervous system poisoning. Isolated tissue experiments showed that each antibody molecule is capable of associating with two antigen molecules. The antibody has greater neutralizing activity when mixed with free toxin than when mixed with toxin already bound to plasma membrane receptors.

Animals↗