Adoptive chemoimmunotherapy of a syngeneic murine lymphoma with long-term lymphoid cell lines expanded in T cell growth factor

Summary Recently techniques have been developed for the long-term growth of cytotoxic T-lymphoid cells in vitro with T cell growth factor (TCGF). We have investigated the use of these in vitro-expanded T cells for the immunotherapy of a disseminated syngeneic murine FBL-3 lymphoma. In this model, mice with disseminated tumor were treated on day 5 with 180 mg cytoxan/kg and then 5 h later were given lymphoid cells IP. In vivo-immunized lymphocytes resulted in significantly improved survival in three of three experiments, curing 52% of 38 animals, compared with treatment with cytoxan alone (0 of 31 cured) or cytoxan plus unimmunized cells (0 of 40 cured) (P<0.0005). In vivo-immunized lymphocytes were re-exposed to FBL-3 tumor in vitro for 5 days in complete medium (CM) or lectin-free TCGF (LF-TCGF). Both groups showed significantly improved survival in six of six experiments. Cytoxan cured 17% of 66 animals, while cytoxan plus normal lymphocytes after IVS cured 6% of 47 animals. In vivo-immunized cells resensitized in vitro to FBL-3 in CM or LF-TCGF cured 82% of 50 animals (P<0.001) and 72% of 61 animals (P<0.001), respectively. Cells from in vivo- and in vitro-sensitized lymphocytes exhibited no cytotoxicity in our in vitro ⁵¹Cr-release assay; expansion of these cells resulted in significant specific lysis of fresh FBL-3 targets. Adoptive transfer of immune lymphocytes resensitized to FBL-3 tumor in vitro and expanded in LF-TCGF conferred a significant survival benefit (P<0.001, curing 7 of 27 animals) compared with all controls. These expanded cells were then continuously grown in LF-TCGF for 2 1/2 months. Again, in vivo-immunized lymphocytes resensitized to FBL-3 tumor and expanded in LF-TCGF for 2 1/2 months cured 56% of the animals with disseminated tumor, significantly prolonging survival over that recorded in any control group (P<0.0002). Irradiation of these same cells totally abolished their efficacy. Clones were generated from IVS and continuously grown in LF-TCGF. Two of these clones were very cytotoxic for fresh FBL-3 (>4,000 lytic units/10⁶ cells). When adoptively transferred to mice in this chemoimmunotherapy model these cytotoxic clones significantly enhanced survival over that recorded following treatment with cytoxan alone (P<0.00001), though prolongation of survival was small. Implications of these results for application of these techniques to other less antigenic tumors and human cancers are discussed.     

Isolation and characterization of propionyl-CoA carboxylase from normal human liver. Evidence for a protomeric tetramer of nonidentical subunits

We have purified propionyl-CoA carboxylase from normal, postmortem human liver to homogeneity. The isolation procedure, which provided an approximately 3000-fold purification and an overall yield of 26%, employed initial centrifugation of a cetyltrimethylammonium bromide-treated homogenate, followed by sequential chromatographic separations using DEAE-cellulose, Blue Sepharose, and Bio-Gel A-1.5m. The native enzyme has a molecular weight of approximately 540,000 and is composed of nonidentical subunits (alpha and beta) of Mr = 72,000 and 56,000, respectively. When studied with analytical isoelectrofocusing techniques, it focuses as a single peak at pH 5.5. Each mole of native enzyme contains 4 mol of bound biotin, virtually all of which is found with the larger (alpha) subunit. The apparent Km values for ATP, propionyl-CoA, and bicarbonate are 0.08 mM, 0.29 mM, and 3.0 mM, respectively. The enzyme also catalyzes the carboxylation of acetyl-CoA and butyryl-CoA to a limited degree, but not that of crotonyl-CoA. Propionyl-CoA carboxylase is quite stable over a temperature range from -50--37 degrees C and over a pH range from 6.2 to 8.4. It has a broad pH optimum from pH 7.2 to 8.8. Limited proteolysis with trypsin results in slow, time-dependent deactivation of the enzyme with preferential cleavage of the smaller subunit. Antiserum prepared against the native enzyme is shown to be monospecific by immunodiffusion and immunoelectrophoresis.     

Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).     

Further studies of a new pathogenic mycobacterium (M. haemophilum sp. nov.).

Mycobacterium haemophilum is an acid-fast rod-shaped organism, originally isolated from deep subcutaneous granulomata of a patient with Hodgkin's disease. Like the other two mycobacterial skin-pathogens, M. ulcerans and M. marinum, M. haemophilum has a maximum temperature for growth below 37 degrees C. Mycobacterium haemophilum is distinguished from all other species examined by its requirement of haemin for growth and its complete lack of catalase activity. Extraneous catalase cannot replace haemin as a growth factor for this organism. Mycobacterium haemophilum can also be differentiated from other species by the patterns of electrophoresis of protein extracts and by gas-liquid chromatography of saponificated and methylated lipid extracts. A monospecific-agglutinating antiserum against M. haemophilum was obtained by adsorption of an immunoserum with M. intracellulare. A number of slow-growing mycobacterial species develop on monolayers of McCoy fibroblasts, and growth on these tissue cultures can be observed much earlier than on artificial media. Mycobacterium haemophilum is characterized by exclusively intracellular development.     

Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

The isolation and characterization of a specific antibody population directed against the prothrombin activation fragments F2 and F1 + 2

We have raised antisera against human prothrombin activation fragment F2 in rabbits and have chromatographed the respective immunoglobulin G fractions on prothrombin-Sepharose, Pr1-Sepharose, and F2-Sepharose immunoadsorbents. The specific antibody population obtained was utilized to construct a double antibody radioimmunoassay capable of measuring as little as 0.8 ng/ml of this component. Our studies suggest that the immunoreactive site defined by this antibody population is most probably located within the negatively charged COOH-terminal region of F2. The immunologic expression of this area is unaffected by denaturation or reduction-alkylation of F2 as well as by attachment of polypeptide to the NH2-terminal of this component. However, the presence of covalently bound polypeptide at the COOH-terminal of F2 reduces its immunologic reactivity by 300- to 400-fold. Prothrombin, Pr1, and Pr*1, which contain the F2 region as part of their covalent structure, are at least 4000 to 7000 times less immunoreactive than F2 on a molar basis. Conversion of these components to thrombin as well as activation fragments generates the theoretically predicted level of immunoreactivity. Masking of the immunoreactive site within these zymogens is due to two phenomena. Firstly, covalent attachment of polypeptides on the COOH-terminal of the F2 segment significantly depresses the reactivity of this region. Secondly, a critical S--S bridge aids in the sequenstration of the immunoreactive site. This cross-link may facilitate interactions between the COOH-terminal of the F2 segment and other regions of the zymogen.     

Extracellular endo-beta-1,4-glucanase in Cellvibrio vulgaris.

Endo-beta-1,4-glucanase of the cellulolytic bacterium Cellvibrio vulgaris is an actively secreted, truly extracellular enzyme, as supported by growth and secretion studies using filter paper as the sole carbon source.