Antibody responses of remission leukemia patients receiving active specific and nonspecific immunotherapy.

A solid-phase radioimmunoassay was utilized to evaluate the antibody response of 21 acute myelogenous leukemia (AML) patients to active specific immunotherapy with either pooled allogeneic AML blast cells or leukemia-associated antigen (LAA), admixed with BCG cell-wall skeleton (CWS). Five of 13 patients treated with LAA had a significant antibody response to LAA after immunotherapy. Antibody response correlated with an increased remission duration (159+ vs. 75+ weeks) and an increased survival (164+ vs. 98+ weeks). Two of eight patients treated with cells responded to LAA, and three patients had initially high anti-LAA antibody levels. In the total study, eight of 11 patients surviving longer than 2 1/2 years and six of seven patients maintaining a complete remission longer than 2 years were antibody responders. Neither protocol induced significant antibody to a normal spleen extract, BCG-CWS, or a measles recall antigen. However, five of seven patients with initially high levels of antibody to BCG (following weekly BCG scarification) were long-term survivors. These data suggest that the humoral immune response to immunotherapeutic agents may be a useful parameter for monitoring immunotherapy of AML patients.

Quantitation of antibody binding to cell surface antigens by x-ray fluorescence spectrometry.

An X-ray spectrometric method has been developed to quantitate antibody bindng to whole cell surfaces in order to obtain a distribution of binding within a population of cells. The method involves incubation of target cells with ferritin-labeled antibody. Analysis of prepared samples in a modified transmission electron microscope with an X-ray detector and data analysis equipment, yields quantitative results on the binding of labeled antibody to individual cells. The binding of anti-2,4-dinitrophenol serum to Chinese hamster ovary cells with attached 2,4-dinitrophenol haptens was measured by X-ray spectrometry. Measurements of attached hapten by a radioisotopic marker correlated with the X-ray spectrometric determination of bound antibody. The use of synchronized cells in metaphase and G1 phases of the cell cycle permitted investigations into the binding per unit surface area. The distribution of antibody binding among a given population of cells was related to the surface area of the cells.

Callus formation versus differentiation of cultured barley embryos: hormonal and osmotic interactions.

The effect of various plant growth substances as single agents was evaluated in a complex tissue culture system: whole embryo culture of early differentiating barley embryos. Callus formation in unsupplemented medium derives from the mesocotyl and is uniquely characteristic of cultures initiated at this stage of embryonic development. This phenomenon could be prevented or reversed by incorporation of gibberellic acid in the medium resulting in plantlet formation. Indoleacetic acid enhanced callus growth, whereas kinetin did not promote either callus or meristematic development. Callus tissue markedly accumulated starch, effectively lowering the cellular osmolarity, while inducing a corresponding rise in the osmolarity of the culture medium. This osmotic pattern was reversed by gibberellic acid induction of shoot formation. These osmotic-hormonal interactions are interpreted relative to in vivo, in situ normal embryogeny or developmental lesions such as tumors.

Fetal antigens in human leukemia.

Immunization of BALB/c male mice with human peripheral leukemic blasts effectively reduced the later formation of syngeneic fetal liver, but not bone marrow hematopoietic colonies in the spleen when these mice were lethally irradiated and challenged i.v. Fetal antigen was detected in 6 of 6 lymphocytic leukemic patients and in 4 or 8 myelocytic leukemia patients and was correlated with low cellular levels of sialic acid. A rabbit antiserum ot BALB/c 15-day fetal liver cells labeled only 0 to 2% of normal donor peripheral leukocytes in indirect immunofluorescence but reacted with 10 to 21% of leukemic peripheral blasts. Active disease bone marrow on the same patients gave 7 to 40% fluorescent cells. Two remission bone marrow smaples were negative and 1 had 44% fluorescent cells. Using this antiserum coupled to sepharose, affinity column separation of KCl extracts from mouse and human fetal liver and from chronic lymphocytic leukemia has produced 4 common protein bands (identifiable on polyacrylamide gel electrophoresis).

Syngeneic and allogeneic mouse lymphoma antisera: specificity, reaction with fetal antigen and protective capacity.

Vaccination with modified lymphoma cells produced highly specific antisera in syngeneic murine hosts. These were C3H/HeJ anti-6C3HED and BALB/cJ anti-P1798. These antisera gave positive membrane immunofluorescence tests with the corresponding tumor cells as well as with drug-resistant cells derived from the parental line. Immune BALB/cJ, but not immune C3H/HeJ, antiserum was cytotoxic in the presence of complement. The antisera did not cross-react with the opposite tumor, Moloney virus-induced YAC lymphoma cells, or normal splenocytes from either mouse strain. Both immune and nonimmune sera gave a positive cytotoxic test with Gross virus-induced lymphoma cells, indicating exposure of all the animals to the virus. Mice periodically given booster injections served repeatedly as serum donors; BALB/cJ mice not boosted for 42 weeks after tumor rejection still had circulating anti-P1798 antibodies. Allogeneic C57BL/KsJ anti-P1798 gave immunofluorescence tests with P1798, 6C3HED and normal BALB/cJ lymphocytes; it was cytotoxic for P1798 but not for normal lymphocytes. After absorption with normal BALB/cJ tissues, the serum became highly specific for P1798 but lost it cytotoxicity. Syngeneic and absorbed allogeneic anti-P1798 inhibited fetal liver colony formation on the spleens of irradiated BALB/c mice, indicating reaction with fetal antigen, but 6C3H/HeJ anti-6C3HED did not give similar inhibition. The latter antiserum was used successfully for passive immunization as long as there was excess antibody; BALB/c anti-P1798 did not protect, while C57BL/KsJ anti-P1798 was only marginally protective.

Separation of ferritin labeled antibody from free antibody.

A procedure for the separation of ferritin-antibody conjugates from free antibody has been developed using isokinetic sucrose density gradient sedimentation. The gradient (5% w/w sucrose at the top to about 35% at the tube bottom) provides optimal separation of 7S free immunoglobulin from the ferritin (65S) and ferritin-antibody conjugate. Recovery of the conjugate band is simple and avoids problems of aggregation found by centrifugal pelleting and resuspension procedures. The method was tested by labeling human globulin and fractionating the gradient. Free antibody was detected at the top one-fifth of the gradient by immunodiffusion. Ferritin and the labeled antibody were found one-third to one-half of the way down the gradient. Immunoelectrophoresis was also used to demonstrate the separation of unconjugated ferritinand ferritin labeled antibody from free antibody.