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Biomedical subjects

D M Neville

Publications and source records attributed to D M Neville.

At least 19 recordsLinked to original sources
PubMed1992

In vivo T-cell ablation by a holo-immunotoxin directed at human CD3.

We have evaluated the in vivo efficacy of anti-CD3-CRM9, a holo-immunotoxin constructed with a diphtheria toxin binding-site mutant. Eighty percent of established human T-cell subcutaneous tumors in nude mice completely regressed following intraperitoneal injection of immunotoxin at a dose set at half the minimum lethal dose assayed in toxin-sensitive animals. Similar regressions produced by a 137Cs source required a dose in excess of 500 cGy. The high degree of in vivo T-cell ablation produced by this immunotoxin is apparently due to maintenance of the toxin translocation function provided by CRM9 and a necessary intracellular routing function supplied by CD3. This immunotoxin may be useful in treating conditions caused by pathologic oligoclonal T-cell expansion such as graft-versus-host disease, autoimmune diseases, and possibly AIDS.

Animals↗
PubMed1992

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports.

We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.

Animals↗
PubMed1991

Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate.

The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.

Cell Compartmentation↗
PubMed1990

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.

Acyclovir↗
PubMed1989

Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants.

We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).

Animals↗
PubMed1989

New protein cross-linking reagents that are cleaved by mild acid.

New homo- and heterobifunctional cross-linking reagents have been synthesized. These reagents are based on ortho ester, acetal, and ketal functionalities that undergo acid-catalyzed dissociation but are base stable. The protein-reactive group in all the homobifunctional reagents is a maleimide group; the heterobifunctional acetal cross-linker has a maleimide group at one end and an N-hydroxysuccinimide ester at the other. These reagents have been used to cross-link diphtheria toxin (DT) to itself to give covalently cross-linked DT dimer or to conjugate DT monomer to the anti-CD5 antibody, T101. The hydrolysis of these cross-linked proteins was studied as a function of pH. Cleavage rates vary from minutes to hours at the pH of acidified cellular vesicles (approximately pH 5.4), ortho esters being the fastest, acetals the slowest, and ketals intermediate, but the cross-linked products are approximately 100 times more stable at the vascular pH of 7.4 and 1000 times more stable at a storage pH of 8.4 in all cases. The utility of these reagents in the reversible blockade of a toxic protein functional domain was demonstrated by using cross-linked DT dimer where the blocking and unblocking of toxin binding sites correlates with cellular toxicity. Of the different cross-linkers described, the acetone ketal, bis(maleimidoethoxy)propane (BMEP), appears to be the most promising in the construction of highly efficacious immunotoxins.

Antigen-Antibody Reactions↗
PubMed1988

A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins.

Immunotoxins, constructed by chemically cross-linking an antibody and protein toxin, do not possess the high efficacy of the native toxin. Decreases in toxicity are due in part to the steric constraints imposed on the two macromolecules, which result in both decreased antibody binding and toxin function. In examining the structural features that influence efficacy in holotoxin-antibody conjugates, it was found that the incorporation of a 29-residue polypeptide, derived from the insulin B chain between the antibody and ricin moiety, resulted in an increase in both potency and efficacy. In a murine model system, potency of the peptide spacer conjugate was increased nearly 10-fold; however, when examined by the procedure used to purge bone marrow, the peptide spacer conjugate was not demonstrably more toxic to nontarget cells than the nonspacer conjugate. Thus, in addition to increases of efficacy and potency, this novel immunotoxin demonstrated increased specificity by approximately 10-fold. To test the general utility of peptide spacer inclusion, a T101-ricin conjugate was constructed with the peptide spacer. It yielded a protein synthesis inhibition rate of -0.6 log/h on MOLT-3 cells, greater than 10-fold more efficacious than a previously constructed nonspacer T101-ricin conjugate examined under similar conditions.

Antibodies, Monoclonal↗
PubMed1988

Energy requirements for diphtheria toxin translocation are coupled to the maintenance of a plasma membrane potential and a proton gradient.

Translocation of diphtheria toxin (DT) or ricin to the cytosol is the rate-limiting step responsible for (pseudo) first-order decline in protein synthesis observed in intoxicated cell populations. The requirements for energy utilization in the translocation of both toxins are examined by perturbing the intoxication during this period of protein synthesis decline. Translocation of either toxin is blocked at 4 degrees C and requires energy. Ricin translocation is tightly coupled to ATP hydrolysis with no involvement of membrane potential. Cell depolarization slows the rate of DT translocation but does not block completely. Elimination of transmembrane pH gradients alone does not affect DT translocation; however, in combination with depolarization, translocation is blocked virtually completely. Energy requirements for DT intoxication are mediated by establishing a plasma membrane potential and a pH gradient across some cellular membrane. It is proposed that a postendocytotic vesicle containing processed DT fuses with the plasma membrane. Either component of the proton motive force across the plasma membrane then drives DT translocation. Ricin apparently utilizes a different energy coupling mechanism at a different intracellular site, thus demonstrating toxin specificity in the translocation mechanism.

Adenosine Triphosphate↗
PubMed1987

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release.

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.

Acetylcholine↗
PubMed1987

Temporal separation of protein toxin translocation from processing events.

Intoxication of Vero cells by ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A requires: 1) binding to cell surface receptors; 2) transport to the cytoplasm; and 3) enzymatic inactivation of a component of the protein synthetic machinery. The kinetic profiles of all four toxins consist of a lag followed by the apparent first-order decrease in protein synthesis. Autoradiographic analysis of DT-intoxicated cell populations has demonstrated that two subpopulations of cells exist during the period of decreasing protein synthesis: one population synthesizing at control levels and the other synthesizing little or no protein (Hudson, T. H., and Neville, D. M., Jr. (1985) J. Biol. Chem. 260, 2675-2680). The present study correlates the autoradiographic data with the rates of protein synthesis decline in cells intoxicated with modeccin, ricin, Pseudomonas exotoxin A, as well DT. In all cases, the first time point which exhibits a decrease in protein synthetic activity also exhibits two subpopulations of cells, one synthesizing protein at control rates and the other synthesizing little or no protein. As the intoxication progresses, cells leave the control population by the rapid cessation of all protein synthesis. These experiments demonstrate that transport of all four toxins to the cytosol is the rate-limiting step during the pseudo first-order decline in protein synthesis. Furthermore, the final step in the transport process (translocation) must result in the release to the cytoplasm of a quantity of toxin sufficient to rapidly inactivate all protein synthesis in that cell. The probability of a translocation event occurring in any cell of the population is established during the lag and remains constant throughout the first-order decrease in protein synthesis. The requirement for acidification during the intoxication by DT, Pseudomonas exotoxin A, or modeccin is restricted to the lag period. Acidification is therefore necessary to establish the probability of translocation, but it is not directly involved in the actual translocation of these toxins. The pseudo first-order passage of DT intoxications through antitoxin and NH4Cl- or monensin-sensitive stages are shown to have the same cellular basis as the pseudo first-order decrease in protein synthesis. A kinetic model is presented which defines the DT intoxication process from one of its earliest events (endocytosis) to its penultimate event (translocation of toxin to the cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)

ADP Ribose Transferases↗
PubMed1987

Graft-versus-host disease prevention in allogeneic bone marrow transplantation from histocompatible siblings. A pilot study using immunotoxins for T cell depletion of donor bone marrow.

Seventeen patients, ages 7-53 years were transplanted with histocompatible bone marrow that had been depleted of T lymphocytes by ex vivo immunotoxin (IT) treatment. Twelve patients had high-risk acute leukemias, and five had chronic myelogenous leukemia. No other graft-vs.-host disease (GVHD) prophylaxis was used. A mixture of three anti-T-cell monoclonal antibodies conjugated to ricin were used in this study: TA-1, UCHT-1 (anti-CD3), and T101 (anti-CD5). The mean number of bone marrow cells infused was 1.5 X 10(8) mononuclear cells/kg recipient weight. Thirteen of the 17 patients demonstrated complete and sustained engraftment. Four patients experienced autologous marrow recovery and/or graft rejection. Compared with an historical group of leukemic patients who received GVHD prophylaxis with methotrexate alone or combinations of methotrexate, and prednisone plus antithymocyte globulin, (ATG) or OKT3, the IT patients with stable engraftment demonstrated shorter time to recovery of leukocytes greater than or equal to 1000mm3 for three consecutive days (median, 20 days vs. 26 days, P = .03). The recovery of total lymphocytes, B and T cell subsets, and T cell function by day 28 was highly variable, but similar, for patients in both the IT-treated group and historical controls. Four patients (ages 13, 18, 21, and 38) developed grade II skin GVHD, but none had severe GVHD. Eight of the 13 patients with durable engraftment have had posttransplant leukemic relapse. Currently only four patients remain alive; two have not relapsed posttransplant, while the other two achieved remission following posttransplant relapse. We conclude that severe GVHD was not observed in this small series with ex vivo T cell depletion for GVHD prophylaxis, and that favorable recovery of hematologic and lymphocytic function was demonstrated for cases where primary engraftment was sustained. A larger randomized controlled study will be needed to establish whether T cell depletion of donor bone marrow with IT can significantly reduce GVHD, and/or improve disease-free survival.

Adolescent↗
PubMed1986

Kinetic comparison of ricin immunotoxins: biricin conjugate has potentiated cytotoxicity.

The plant toxin ricin was chemically coupled to an anti-Thy-1.1 antibody, and the resultant conjugates were fractionated by gel filtration. The cytotoxicity of the conjugate possessing two ricin molecules per immunoglobulin, yielding a first-order inactivation rate of protein synthesis of -0.4 log/h at 200 ng/mL, was well above that expected just from the increase in ricin per unit mass of conjugate, when compared to a conjugate possessing only one ricin per immunoglobulin. On a conjugate molar scale the biricin immunotoxin was determined to be 8 times more potent than the monoricin conjugate; thus, relative to the number of ricin molecules, the coupling of a second ricin to the immunoglobulin quadrupled the observed potency. The concentration of immunotoxin and the resultant inactivation rates of protein synthesis were found to be related through a power function. Additionally, the inactivation kinetics of these conjugates were found to be similar to those of native ricin.

Animals↗