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Biomedical subjects

J D Rodwell

Publications and source records attributed to J D Rodwell.

16 recordsLinked to original sources
PubMed1994

Inhibition of type 5 adenovirus infectivity by periodate oxidation.

Periodate oxidation of purified type 5 Adenovirus (Ad5) led to a mean loss of infectivity of 6.84 logs. There were no significant differences in adsorption and penetration between oxidized and mock-oxidized virus. However, after infection with oxidized virus, no synthesis of viral structural proteins could be detected and a 78.5% inhibition of viral DNA synthesis was observed. Labelling experiments performed by treating oxidized and mock-oxidized virus with tritiated sodium borohydride revealed that the fiber glycoprotein was one of the proteins labelled in oxidized virus whereas no labelled proteins were detected in non oxidized virus. In addition, it was found that one mol of formaldehyde generated during oxidation of sugar residues was bound per 500 base pairs in oxidized virus. One consequence of this in situ generation of formaldehyde is the formation of DNA-protein crosslinks. The DNA so crosslinked showed different patterns of restriction fragments with endonucleases such as Hpa I, Hind III and Kpn I but not with Xho I.

Adenoviruses, Human↗
PubMed1994

Thrombus imaging with technetium-99m synthetic peptides based upon the binding domain of a monoclonal antibody to activated platelets.

UNLABELLED: Monoclonal antibodies which recognize fibrin or platelets have enabled imaging of vascular thrombi, however, early imaging has been difficult because of the slow blood disappearance of even small antibody fragments. It was theorized that it might be possible to synthesize peptides which possess the same thrombus affinity as monoclonal antibodies, but which would leave the blood pool much more rapidly. METHODS: In this study, peptides were synthesized with amino acid sequences based on the primary binding region of the platelet glycoprotein IIb/IIIa-directed monoclonal antibody PAC1. Both termini of the peptides were blocked to prevent rapid proteolysis and a metallothionein-derived sequence was incorporated as a chelating agent for reduced technetium. RESULTS: Technetium-99m-labeled peptides produced images of fresh clots in the jugular veins of rabbits and day-old thrombi in the femoral veins of dogs within 2 hr after injection. In control experiments, a 99mTc-labeled nonspecific peptide failed to produce focal images of thrombus. Another control compound, 99mTc-glucoheptonate, did produce images of fresh clots in rabbits but failed to produce focal images of day-old thrombi. As was hoped, blood clearance of the 99mTc peptides was rapid, with excretion through the kidneys, however, none of the peptides studied had better thrombus-to-blood ratios than iodinated fibrinogen and all had significantly lower deposition in the thrombus. CONCLUSION: Using labeled synthetic peptides appears to be technically feasible but the absolute binding to thrombus is not yet sufficient for reliable imaging of pre-existing thrombi.

Amino Acid Sequence↗
PubMed1989

Radioimmunoscintigraphy and radioimmunotherapy in nude mouse models. Studies with site-specifically modified monoclonal antibodies.

Site-specific covalent modification of monoclonal antibodies at the oligosaccharide offers advantages over more conventional modification processes that involve direct attachment at tyrosine, lysine or glutamic/aspartic acid side chains. Using the site-specific modification process, attachment sites on the antibody are distal to the antigen-binding region. Thus, homogeneity of antigen-binding properties and affinity for the unmodified protein are preserved. Furthermore, higher derivatization ratios with no resultant loss of immunoreactivity can be achieved for monoclonal antibodies modified at the oligosaccharide. In vivo biodistribution and tumor localization studies in nude mouse models suggest that antibodies radiolabeled at their oligosaccharide might represent improved immunoscintigraphic reagents. In a variety of tumor xenograft models, site-specific modified 111In-labeled antibody conjugates localized to the tumor site with little non-specific localization in other tissues or organs. The degree of localization at the target site was substantially greater than that of 111In-labeled antibodies directly modified at the tyrosine side chain. Preliminary studies with 212Bi- and 90Y-labeled antibodies modified at the oligosaccharide indicate that both of these radioisotopes have immunotherapeutic potential. Because of its preferential uptake by the kidney, the use of 212Bi may be best suited for tumors localized within the peritoneal cavity, such as ovarian and colorectal carcinomas. The toxicity of 90Y at high specific activities suggests that a regimen of repeated smaller doses of this radioisotope is best suited for therapeutic use. Studies in tumor-bearing mouse models are currently underway to better define the optimal dosage and administration regimens for both of these radioisotopes when attached to site-specific modified antibodies.

Animals↗
PubMed1988

Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling.

Labeling an antibody site specifically through its carbohydrate residues preserves more of its antigen-binding activity than does labeling through protein moieties. To boost the amount of immunoglobulin G carbohydrate capable of being labeled, we treated hybridoma cells with a mannosidase inhibitor, deoxymannojirimycin (dMM). Polyacrylamide gel electrophoresis showed formation of a glycoprotein with high mannose content, in that endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) could digest the antibody from the dMM-treated cells, but not from control cultures. Carbohydrate analysis confirmed this conclusion, indicating that the antibody from the dMM-treated cells had twice as much mannose as did the control antibody. The glucosamine content of the treated-cells' antibodies was half that of the control, and no additional carbohydrate residues were detectable in the antibodies secreted by the dMM-treated cells. We conjugated both the dMM and control antibodies through their carbohydrate to a chelator. In labeling, the dMM antibody conjugate incorporated approximately threefold as much 111In isotope as the control conjugate. The two labeled antibodies were injected into mice and showed similar organ distributions.

1-Deoxynojirimycin↗
PubMed1986

Site-specifically modified 111In labelled antibodies give low liver backgrounds and improved radioimmunoscintigraphy.

Site-specific modification of monoclonal antibodies at their oligosaccharide had previously been demonstrated to produce excellent 111In imaging in a xenograft model using a Brown Norway (BN) rat lymphoma and a rat anti-BN MHC monoclonal antibody [Lee C. et al. Fed. Proc. Abstr. 43 3014 (1984)]. These results are due, in part, to lack of liver uptake, so we wanted to evaluate the extent of hepatic uptake observed with different monoclonal antibodies in normal mice. Biodistribution data were obtained for four monoclonal antibodies by first modifying each antibody at its carbohydrate with a diethylenetriaminepentaacetic acid (DTPA) derivative. The antibodies were then labelled with 111In and injected into normal mice. Images were obtained 24 h post-injection, and at 48 h the mice were dissected and the tissue-to-blood (T:B) ratios determined. T:B ratios were approximately 1 (or less) for every organ evaluated, indicating minimal non-specific uptake into these organs. Data is also presented for the BN-rat system which shows excellent localization into the tumor xenograft and low non-specific organ uptake. These data indicate that modification of antibodies site-specifically at their oligosaccharide results in minimal non-specific uptake into non-target tissues and enhanced localization into the tumor target, and that this may represent a preferred method for production of 111In labelled antibodies.

Animals↗
PubMed1986

Site-specific covalent modification of monoclonal antibodies: in vitro and in vivo evaluations.

A strategy for covalent modification of monoclonal antibodies utilizing the oxidized oligosaccharide moieties on the molecule was evaluated and compared to more conventional methods. As judged by quantitative in vitro measurements, a monoclonal antibody conjugate prepared via the oligosaccharides retained the homogeneous antigen binding property and affinity of the unmodified antibody. In contrast, conjugates of the same antibody, modified to the same degree on either lysines or aspartic and glutamic acid side chains, were heterogeneous in their antigen binding and had lowered affinity. In vivo biodistribution and nuclear-imaging experiments were also performed with a second monoclonal antibody and a tumor xenograft model. Antibodies modified on the oligosaccharides with either a peptide labeled with iodine-125 or a diethylenetriaminepentaacetic acid chelate with indium-111 localize into target tumors more efficiently than the same antibody radiolabeled on either tyrosines or lysines. These in vivo results, when compared to those reported in the literature for conventionally modified antibodies, suggest that oligosaccharide modification of monoclonal antibodies is a preferred method of preparing conjugates.

Animals↗
PubMed1980

Surface component of primate thymus-derived lymphocytes related to a heavy chain variable region.

In a study designed to determine whether T cells of man and higher primates express a surface component related to the variable region of immunoglobulin heavy chain (VH), chickens were immunized with the purified VH fragment of a monoclonal Waldenström macroglobulin. The antibody preparation reacted with a mu chain determinant contained in the Fd fragment and with individual determinants characteristic of the orginal Waldenström protein. As estimated by immunofluorescence analysis, a subpopulation of normal human peripheral T cells (approximately 30%) bound the anti-VH antibody. B-Cell lymphoma lines grown in vitro, as well as some T-cell leukemia lines of the cotton-topped marmoset (Sagiunus oedipus), also bound the anti-VH antibody. The VH-bearing component of the T-cell line 70-N-2 was labeled biosynthetically by incorporation of [3H]leucine and was precipitated specifically by anti-VH antibody. This component was characterized by an apparent mass of 70,000 daltons as assessed by polyacrylamide gel electrophoresis under reducing conditions in buffers containing sodium dodecyl sulfate. These data provide direct support for the hypothesis that some T cells express and synthesize a component related to immunoglobulin heavy chains.

Animals↗