Establishment of a human B-cell tumor in athymic mice.

Human B-cell tumors have been established in athymic, BALB/c mice using the EBV-positive Burkitt lymphoma cell line Namalwa. One-hundred-one of 104 animals (97%) developed tumors 10-14 days following s.c. injection of a mixture of 20 x 10(6) Namalwa and 5 X 10(6) irradiated human fibrosarcoma (HT-1080) cells. Tumors developed at the site of injection and reached approximately 300 mm2 (product of cross-sectional diameters) after 21 days; no metastases were found. Histological analysis showed that tumors consisted solely of lymphoid cells. Immunofluorescence assays demonstrated that while 85% of the tumor cells retained reactivity with the monoclonal B-cell antibody BA-1, 96% retained reactivity with antibody BA-2 and 43% with BA-3. A similar reactivity profile was observed with cultured Namalwa cells. Tumors were passaged serially 10 times without significant change in BA-1, BA-2, or BA-3 reactivity. Indirect immunofluorescence demonstrated that antibody BA-2 reached tumor cells within 2 h following i.p. injection; antigen modulation was not observed. These results demonstrate the suitability of this B-cell model for testing the in vivo efficacy and stability of anti-B-cell immunoconjugates.

Monoclonal antibodies to purified ricin A-chain: production and properties.

Ricin A-chain was purified from native ricin using lactosyl-Sepharose. It was non-toxic to whole cells at a concentration of 1 microM yet nearly as effective as an equimolar concentration of ricin in blocking in vitro protein synthesis. Hybridomas secreting monoclonal antibodies to ricin A-chain were produced using the murine myeloma cell line NS-1. These anti-ricin A-chain antibodies cross-reacted with whole ricin but exhibited little cross-reactivity with purified ricin B-chain. Antibodies 2F2 and 2F5 both immunoprecipitated ricin A-chain. Both antibodies also precipitated ricin B-chain, as did the irrelevant control antibodies MOPC-21 and MPC-11. Pre-incubation of B-chain with 0.1 M galactose eliminated greater than 90% of precipitation by 2F2, MOPC-21 and MPC-11 but effected minimally precipitation by 2F5. Enzyme-linked immunosorbent assays using antibodies 2F2 and 2F5 to detect ricin A-chain in murine or human serum were linear between 40 and 800 ng ricin A-chain per ml. Anti-ricin A-chain antibodies 2F2 and 2F5 produced some inhibition of in vitro A-chain catalytic activity. Specific monoclonal antibodies to A-chain hemitoxin will be useful for characterization of functional hemitoxin domains, in in vitro assays for the stability of A-chain immunotoxins, and in characterizing the cellular internalization and processing of conjugates containing ricin or ricin A-chain.

Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli.

D-Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/permease, we have tested a number of sugar alcohols and their derivatives as substrates and/or inhibitors of this protein. Our results show that the mannitol permease is highly, but not absolutely, specific for D-mannitol. Compounds accepted by the enzyme include those with substitutions in the C-2(= C-5) position of the carbon backbone of the natural substrate as well as D-mannonic acid, one heptitol and one pentitol. All of these compounds were both inhibitors and substrates for the mannitol permease except for D-mannoheptitol, which was an inhibitor but was not phosphorylated by the enzyme. No compound examined, however, exhibited an affinity for the enzyme as high as that for its natural substrate. We have also investigated the phospholipid requirements of the mannitol permease using phospholipids purified from E coli. The purified protein was significantly activated by phosphatidylethanolamine, but little activation was observed with phosphatidylglycerol or cardiolipin. These observations partially delineate requirements for interaction of sugar alcohols and phospholipids with the mannitol permease. They suggest approaches for the design of specific active site probes for the protein, and strategies for stabilizing the enzyme's activity in vitro.