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MRL/lpr (lpr) mice spontaneously develop a lupus-like illness as well as massive lymphadenopathy. Attempts to transfer autoimmunity by adoptive transfer or radiation bone marrow chimeras have been unsuccessful. Since severe combined immunodeficiency (SCID) mice have been engrafted with human and rat xenografts without apparent graft-versus-host disease (GVHD), we subjceted SCID mice to low-dose irradiation and reconstituted the mice with spleen cells from young or old lpr mice or with lpr bone marrow. Fourteen out of twenty (70%) of SCID mice engrafted with spleen cells from old lpr mice produced autoantibodies (anti-DNA and anti-Sm) without evidence of the severe lymphoid atrophy previously described for lpr spleen→ +/+ chimeras. SCID mice engrafted with spleen cells from young lpr mice developed acute GVHD and 5/6 (83%) died within 4 weeks post transfer. Although 8/11 (73%) of lpr→SCID bone marrow allografts survived for at least 4 months, these mice developed a wasting disease characterized by lymphoid atrophy and fibrosis without the production of autoantibodies. None of the lpr→SCID grafts resulted in the transfer of double negative T cells or the lymphoproliferative syndrome characteristic of MRL/lpr mice. These findings indicate that SCID mice can be engrafted with splenocytes from old MRL/lpr mice and that B cells continue to secrete autoantibodies for several months in the SCID recipients. This study also demonstrates that, unlike i.p. transplant of xenogeneic cells, acute GVHD is a consistent feature of i.p. transplants of normal allogeneic mononuclear cells into SCID mice. [ABSTRACT FROM AUTHOR]

For comparison of cytotoxicity from alpha-particle irradiation with that from conventional x-irradiation, Bi, an alpha-emitting ra-dionuclide, was attached to a monoclonal antibody that recognizes a cell surface antigen on human pancreatic carcinoma cells. For a given level of survival, the Bi-antibody [ABSTRACT FROM PUBLISHER]

Objective: Our objective was to develop the alpha-emitting radionuclide bismuth 212 for possible intraperitoneal use against microscopic ovarian cancer., Study Design: The radiobiologic effectiveness of bismuth 212 was compared in vitro to x rays and chromic phosphate phosphorus 32). The distribution, toxicity, and maximum tolerated dose of bismuth 212 were determined after intraperitoneal administration in animal models. Dose estimates in animals and humans were made., Results: In in vitro studies bismuth 212 was three times more effective in eradicating tumor cells grown in monolayers and in 800 microm spheroids. In in vivo studies bismuth 212 was distributed uniformly after intraperitoneal administration. The maximum tolerated dose in rabbits was 60 mCi. There was reversible hematologic toxicity with minimal organ damage. Bismuth 212 prolonged survival and cured up to 40% of animals inoculated with Ehrlich carcinoma cells. Dose estimates made from these studies indicated that dosages administered were effective in eradicating tumor cells and were within the radiotolerance of normal human tissue., Conclusion: Bismuth 212 appears to be a suitable candidate for intraperitoneal use against microscopic ovarian cancer.

The response of cultured bovine aortic endothelial (BAE) cells after exposure to alpha-particle radiation from chelated 212Bi has been evaluated. The results suggest that even relatively high doses of alpha-particle radiation from 212Bi (20-72 Gy) cause only minor acute changes in the morphology of BAE cells (light and electron microscopy) under conditions of confluent monolayer growth. Significant morphological changes can be detected in cells that detach from the monolayer, though it is unclear whether these changes represent a genuine response to irradiation or reflect the causes or effects of monolayer detachment with the consequent loss of intercellular biochemical communication. After alpha-particle irradiation (20-40 Gy) angiotensin-converting-enzyme activity was not detectable in the monolayer culture medium but was significantly decreased within the cell monolayer. Neutral-elution-assay data demonstrated that DNA double-strand-break (DSB) damage occurred in these cells and that about 35% of the DSBs were repairable.

Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET alpha-radiation from 212Bi-DTPA or to gamma-radiation from a 137Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like gamma, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after alpha- and gamma-irradiation were comparable, though the dose-response relationships differed. The "RBE" value for G2M block for alpha- versus gamma-radiation was approx. 4. Electron microscopic studies of the cell samples where a large proportion of cells were arrested in G2M showed subcellular changes in nuclear membrane and the presence of morphologically apoptotic cells. Biochemical analysis of DNA from irradiated cells by agarose gel electrophoresis revealed more extensive DNA fragmentation for alpha- vs gamma-irradiation, even at relatively low total doses. We conclude that the high LET radiation is more efficient in inducing G2M block and apoptosis in EL4 lymphoma cells. The overall radiosensitivity of some high and low grade malignant lymphoma cells to radiation may correlate with these processes. The clinical implications of 212Bi-induced G2M delay may be particularly important for biologically targeted high LET radiopharmaceutical therapy.

MRL/lpr (lpr) mice spontaneously develop a lupus-like illness as well as massive lymphadenopathy. Attempts to transfer autoimmunity by adoptive transfer or radiation bone marrow chimeras have been unsuccessful. Since severe combined immunodeficiency (SCID) mice have been engrafted with human and rat xenografts without apparent graft-versus-host disease (GVHD), we subjected SCID mice to low-dose irradiation and reconstituted the mice with spleen cells from young or old lpr mice or with lpr bone marrow. Fourteen out of twenty (70%) of SCID mice engrafted with spleen cells from old lpr mice produced autoantibodies (anti-DNA and anti-Sm) without evidence of the severe lymphoid atrophy previously described for lpr spleen-->+/+ chimeras. SCID mice engrafted with spleen cells from young lpr mice developed acute GVHD and 5/6 (83%) died within 4 weeks post-transfer. Although 8/11 (73%) of lpr-->SCID bone marrow allografts survived for at least 4 months, these mice developed a wasting disease characterized by lymphoid atrophy and fibrosis without the production of autoantibodies. None of the lpr-->SCID grafts resulted in the transfer of double negative T cells or the lymphoproliferative syndrome characteristic of MRL/lpr mice. These findings indicate that SCID mice can be engrafted with splenocytes from old MRL/lpr mice and that B cells continue to secrete autoantibodies for several months in the SCID recipients. This study also demonstrates that, unlike i.p. transplant of xenogeneic cells, acute GVHD is a consistent feature of i.p. transplants of normal allogeneic mononuclear cells into SCID mice.

Though clinical results for radioimmunoconjugate therapy of most common epithelial tumors have been disappointing, dramatic responses have been observed repeatedly in the treatment of high- and low-grade malignant lymphomas. This high clinical responsiveness after radioimmunoconjugate therapy sometimes appears to be out of proportion to the calculated radiation dose absorbed by the lymphoma tissue. Here we describe some key aspects of the kinetics, dosimetry, and cellular radiobiology of murine lymphoma cells exposed to 212Bi-radiolabeled alpha-particle-emitting immunoconjugates specific for the differentiation antigen Thy 1.2. Approximately 25 cell-bound alpha-particle-emitting immunoconjugates per target cell were required to reduce clonogenic survival by 90% (the radiobiological D10). Serial kinetic analyses of the antibody and radioisotope components of the immunoconjugates revealed significant levels of dechelation and up to 7.5% cellular internalization of the isotope. Cellular radiation dosimetry performed by Monte Carlo computer simulation of alpha-particle energy deposition patterns based on the observed radiopharmacokinetics showed that the D10 resulted from approximately four alpha-particle traversals through the nucleus, corresponding to an absorbed radiation dose of approximately 0.95 Gy to the cell nucleus. Electron micrographs and DNA gel studies of murine lymphoma cells undergoing radioimmunoconjugate therapy in vivo and in vitro demonstrated bizarre blebbing patterns, condensation of chromosomal material, and internucleosomal DNA fragmentation patterns characteristic of programmed cell death (apoptosis). We conjecture that the efficacy of radioimmunoconjugates against responsive cell types may be the result of passive DNA damage by ionizing radiation and the initiation of apoptosis in response to radioimmunotherapy.

To assess the possibilities of using 114mIn as a therapeutic agent, the long-term biodistribution of 114mIn was studied, in tumor-bearing nude mice, after injection of labeled monoclonal antibody (MoAb) B72.3 IgG, either DTPA-coupled murine, DTPA-coupled chimeric, or macrocycle-coupled chimeric antibody. Although the biodistributions in all cases were similar, there were important differences. The use of DTPA-coupled chimeric antibody led to higher concentrations of radioactivity in tumor, and to lower concentrations in liver and bone, as compared to DTPA-coupled murine antibody. The use of macrocycle-coupled chimeric antibody led to higher concentrations of radioactivity in the liver and in bone as compared to the DTPA-coupled chimeric antibody. However, in this case there were no significant differences in tumor uptake or clearance. Radiation doses were calculated based on the organ retention and by neglecting source-to-target contributions. Radiation dose distribution was marginally favorable for therapy in the group injected with DTPA-coupled chimeric antibody.

Anti-Sm-positive sera from patients with systemic lupus erythematosus (SLE) recognize a major epitope located within the carboxyl-terminal 27 amino acids of a recombinant SmB fusion protein. To determine whether a synthetic peptide corresponding to this region could be used as an antigen to detect anti-Sm antibodies, sera were typed as anti-Sm positive or anti-Sm negative by counterimmunoelectrophoresis (CIE). Twenty-three SLE sera that were anti-Sm positive by CIE, 22 that were anti-Sm negative by CIE, and 42 sera from patients with other autoimmune diseases were tested for anti-Sm antibodies by enzyme-linked immunosorbent assay (ELISA), using either the synthetic peptide (C27) or a recombinant SmB (rSmB) fusion protein as the antigen. More than 90% of the sera that were anti-Sm positive by CIE were also positive by both the C27 and rSmB ELISAs, and an additional 2 SLE sera originally typed as anti-Sm negative were found to be positive (1 by the C27 ELISA, 1 by the rSmB ELISA), due to the greater sensitivity of the ELISAs. In the rSmB ELISA, anti-Sm antibodies were not detected in any of the sera from patients with other autoimmune diseases, whereas 3 patients with anti-U1 RNP antibodies (1 each with polymyositis, scleroderma, and mixed connective tissue disease) had a positive result in the C27 ELISA. These results indicate that both the C27 synthetic peptide and rSmB are excellent antigens for use in ELISAs to quantify anti-Sm antibodies.

Alpha emitter-labeled antibodies (Abs) are of considerable interest in cancer therapy. Alpha particles are densely ionizing and therefore have a high radiobiologic effectiveness, and the cell killing produced is influenced very little by dose rate or hypoxic conditions. LS174T human colon adenocarcinoma spheroids were used in this study to evaluate the efficacy of alpha emitter-labeled Abs in a three-dimensional model. NRLU-10, an IgG2b Ab to a pancarcinoma antigen, and its Fab fragment were used. Initial kinetic studies using 125I-NRLU-10 revealed that a large number of binding sites/cell and high Ab affinity led to slow Ab penetration. This effect could be overcome by increasing the Ab concentration ten-fold for Fab but not for intact Ab. Bismuth-212-NRLU-10 therapy was very effective in killing single cells (over 3 log reduction in surviving fraction) but was ineffective in spheroids (less than 1 log reduction). This was likely due to inadequate penetration into the spheroids before the 212Bi decayed. The use of higher Ab concentrations, tumors with fewer antigenic sites/cell for the Ab being used, lower affinity Abs, alpha emitters with longer half-lives, and pretargeting with bifunctional Ab are all potential ways of increasing the efficacy of alpha emitter-labeled Abs for cancer therapy.

For comparison of cytotoxicity from alpha-particle irradiation with that from conventional x-irradiation, 212Bi, an alpha-emitting radionuclide, was attached to a monoclonal antibody that recognizes a cell surface antigen on human pancreatic carcinoma cells. For a given level of survival, the 212Bi-antibody complex was found to be approximately 20 times more efficient in cell killing than x-irradiation and 5 times more cytotoxic when compared with the cytotoxicity of an antigen-negative cell line or an isotype-matched control antibody. High linear energy transfer radioimmunotherapy using alpha emitters linked to monoclonal antibodies may be useful in vivo and in vitro for selectively killing target cell populations, especially those resistant to other forms of treatment.

Short-lived isotopes that emit alpha particles have a number of physical characteristics which make them attractive candidates for radioimmunotherapy. Among these characteristics are high linear energy transfer and correspondingly high cytotoxicity; particle range limited to several cell diameters from the parent atom; low potential for repair of alpha-induced DNA damage; and low dependence on dose rate and oxygen enhancement effects. This report reviews the synthesis, testing and use in animal models of an alpha particle emitting radioimmunoconjugate constructed via the noncovalent chelation of Bismuth-212 to a monoclonal IgM antibody specific for the murine T cells/neuroectodermal surface antigen, Thy 1.2. These 212Bi-anti-Thy 1.2 immunoconjugates are capable of extraordinary cytotoxicity in vitro, requiring approximately three 212Bi-labeled conjugates per target cell to suppress 3H-thymidine incorporation to background levels. The antigen specificity afforded by the monoclonal antibody contributes a factor of approximately 40 to the radiotoxicity of the immunoconjugate. Animals inoculated with a Thy 1.2+ malignant ascites were cured of their tumor in an antigen-specific fashion by intraperitoneal doses of approximately 200 microCi per mouse. Alpha particle emitting radioimmunoconjugates show great potential for regional and intracavitary molecular radiotherapy.

Alpha particles are energetic short-range ions whose higher linear energy transfer produces extreme cytotoxicity. An alpha-particle-emitting radioimmunoconjugate consisting of a bismuth-212-labeled monoclonal immunoglobulin M specific for the murine T cell/neuroectodermal surface antigen Thy 1.2 was prepared. Analysis in vitro showed that the radioimmunoconjugate was selectively cytotoxic to a Thy 1.2+ EL-4 murine tumor cell line. Approximately three bismuth-212-labeled immunoconjugates per target cell reduced the uptake of [3H]thymidine by the EL-4 target cells to background levels. Mice inoculated intraperitoneally with EL-4 cells were cured of their ascites after intraperitoneal injection of 150 microcuries of the antigen-specific radioimmunoconjugate, suggesting a possible role for such conjugates in intracavitary cancer therapy.

We have developed an improved generator for the production of the alpha emitting radionuclide 212Bi and its parent, 212Pb. These radionuclides are well suited to use as radiotherapeutic agents due to their relatively short half lives and appropriate particle particle emissions. The parent, 224Ra, is available from a long-lived parent and can be isolated on a generator which produces the daughters in good yield and low breakthrough. The 212Bi can be eluted by itself or in equilibrium with its parent.

Greenfield, Leonard J. (University of Miami, Miami, Fla.), Janet J. Hines, and Linda L. Boral. Polymorphism in a marine bacterium in relation to population growth. J. Bacteriol. 84:357-363. 1962.-A marine pseudomonad, MB-1, from South Florida waters, exists in three forms: orange (y), lemon-yellow (xy), and white (x), each of which is polymorphic. Replicate growth curves of each form were obtained by culturing at 25 C in 1% peptone-sea water using a controlled inoculum. The controlled inoculum was obtained by growing an isolate on a 1% peptone-sea water agar (1.5%) slant for 24 hr at 25 C, transferring a wire loopful of culture to 50 ml of 1% peptone-sea water in a 125-ml flask, and incubating at 25 C for another 24 hr. The resulting bacterial suspension in 0.5-ml amounts was used to inoculate 50 ml of 1% peptone-sea water broths in which growth was measured. The logarithmic growth phase occurred in x and xy between 12 and 18 hr of incubation; in y, this period was between 18 and 24 hr. The stage of growth could also be characterized by the percentage of cell types present at a given period. In late lag phase, single cells outnumbered binary types by about two to one. During logarithmic growth, the single cells predominated by 2.5 or 2.4 to 1. In the stages following this ratio increased, but all cells showed considerable variation in size. Chains of cells were present in all stages but were reduced in number during the logarithmic phase.