Anti-Escherichia coli asparaginase antibody levels determine the activity of second-line treatment with pegylated E coli asparaginase: a retrospective analysis within the ALL-BFM trials.

Hypersensitivity reactions limit the use of the antileukemic enzyme asparaginase (ASE). We evaluated Ab levels against Escherichia coli ASE and ASE activity in 1221 serum samples from 329 patients with acute lymphoblastic leukemia who had received ASE treatment according to the ALL-BFM 2000 or the ALL-REZ BFM 2002 protocol for primary or relapsed disease. ASE activity during first-line treatment with native E coli ASE and second-line treatment with pegylated E coli ASE was inversely related to anti-E coli ASE Ab levels (P < .0001; Spearman rank order correlation). An effect on ASE activity during second-line treatment with pegylated E coli ASE was, however, only observed when anti-E coli ASE Ab levels were high (> 200 AU/mL). In the presence of moderate or intermediate Ab levels (6.25-200 AU/mL) the switch from native to pegylated E coli ASE resulted in a significant increase of ASE activity above the threshold of 100 U/L (P < .05). Erwinia chrysanthemi ASE activity was not correlated with anti-E coli ASE Ab levels. Erwinia ASE was found to be the best ASE alternative if Ab levels against E coli ASE exceed 200 AU/mL. This retrospective analysis is the first to describe the relationship between the level of anti-E coli ASE Abs and serum activity of pegylated E coli ASE used second-line after native E coli ASE.

Immunocytochemistry for drugs containing an aliphatic primary amino group in the molecule, anticancer antibiotic daunomycin as a model.

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH4, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues.

The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group.

Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.

Structure and function of P-glycoprotein in the normal liver and intestine.

Multidrug resistance (MDR) genes encode a family of membrane glycoproteins of approximately 170 kD (P-glycoproteins). In man and mouse, the MDR 1 (mdr 1) genes confer resistance to relatively hydrophobic cationic anti-cancer drugs (i.e., vinblastin, adriamycin). Anti-cancer drug sensitivity is restored by addition of other drugs (i.e., verapamil, reserpine) which are also P-glycoprotein substrates. Transfection of MDR 1 genes produces the resistance phenotype and overexpression of P-glycoprotein. Parenchymal cells in several normal tissues express P-glycoprotein in the secretory domain of the plasma membrane (i.e., bile canaliculus of hepatocytes, brush border of proximal tubular, and small intestinal cells). Studies using plasma membrane vesicles of different sidedness derived from the bile canaliculus and small intestinal brush border permit characterization of P-glycoprotein as a unidirectional, temperature dependent, saturable, ATP-dependent transporter which is competitively inhibited by various anti-cancer drugs and other compounds. Transport studies using single cell fluorescence microscopy with image analysis confirm observations in vesicles. No natural substrate has been identified. Structural studies indicate that the requirements for substrates are molecular weight of 350 to 100, hydrophobicity, two planar rings, and a weak cationic charge. Alternative mechanisms of transport function are considered. The identity of P-glycoproteins in normal rat and human tissues has not been established. Antibody reactions suggest that they may belong to the MDR 2 or 3 class. Studies using everted gut sacs suggest that inhibition of P-glycoprotein may facilitate accumulation of anti-cancer drugs in the tissue.

[Action of doxorubicin on immunogenesis]. / Deistvie doksorubitsina na immunogenez.

The effect of doxorubicin, carminomycin and rubomycin on some immunological reactions was studied comparatively on mice. It was shown that doxorubicin had an immunodepressant effect on the synthesis of antibodies and antibody-forming cells. By the character of its immunodepressant effect doxorubicin was similar to rubomycin. In a dose of 0.3 of the LD50 doxorubicin had no effect on the transplantation immunity in the system of "transplant versus host" and did not prolong the lifetime of the skin graft.

[Effect of rubomycin and carminomycin on suppressor cells participating in the regulation of the immune response]. / Vliianie rubomitsina i karminomitsina na kletki-supressory, uchastvuiushchie v reguliatsii immunogo otveta.

The effect of rubomycin and carminomycin on induction of suppressor cells in immunization of mice with high doses (2 . 10(9) cells) of sheep red blood cells (SRBC) or with the allogeneic spleen cells was studied. When the spleen cells of syngeneic animals hyperimmunized with SRBC were administered to intact cells, a marked specific suppression of both the antibody formation and the reaction of the delayed type hypersensitivity were observed. A decrease in the suppressor activity or its complete elimination as a result of exposure of the spleen cell donors to the antibiotics was indicative of a relatively selective effect of these drugs on the suppressor cells. This effect of the antibiotics increased after treatment of the spleen immune cells with the anti-T-serum and complement. However, the blocking effect of the antibiotics on the suppressor cells was not strictly specific, since they were detected in the syngeneic and allogenic systems with the cell transfer.

[Antitumor activity and the effect on immunogenesis of combinations of rubomycin and carminomycin with levamisole]. / Izuchenie protivoopukholevoi aktivnosti i vliianiia na immunogenez kombinatsii rubomitsina i karminomitsina s levamizolom.

The antitumor activity and effect on immunogenesis of rubomycin and carminomycin combinations with levamisole were studied comparatively. It was found that levamisole administered after carminomycin increased the intensity of delayed hypersensitivity in mice and only partially reduced immunoreactivity of the animals estimated by the number of antibody-forming cells in the spleen. No such effect was observed after injections of rubomycin. The antitumor effect of the combinations of the antibiotics wit levamisole was studied in experimental transplantable mouse lymphosarcoma L10-1. The use of rubomycin or carminomycin in combination with levamisole increased the survival of the animals and sometimes resulted in complete resolution of the tumors. No such effect was observed in treatment of the animals with the drugs alone. The data are indicative of a possible use of levamisole in combination with antitumor antibiotics in immunochemotherapy of tumors.

[Effect of rubomycin and carminomycin on the delayed hypersensitivity reaction in mice immunized with ram erythrocytes]. / Vliianie rubomitsina i karminomitsina na reaktsiiu gipershuvst-vitel'nosti zamedlennogo tipa u myshei, immunizirovannykh éritrotsitami barana.

The effect of rubomycin and carminomycin on the cellular immunity was studied on the model of delayed type hypersensitivity (DTH) in mice immunized with sheep red blood cells. It was shown that the antibiotics in the doses of 0.3-0.5 of the LD50 inhibiting the antibody formation induced intensive development of DTH. It was most pronounced on concurrent administration of the drugs or 24 hours after the immunization and did not depend on the antigen dose used for animal sensitization.

[Immunodepressive action of anthracycline antibiotics studied on 2 different transplantation immunity models]. / Issledovanie immunodepressivnogo deistviia antratsiklinovykh antibiotikov na dvukh razlichnykh modeliakh transplantatsionnogo immuniteta.

The effect of carminomycin and rubomycin in equitoxic doses on the reaction of rejection of the allogenic skin graft and the reaction of the transplant to the host in mice (RTAH) was studied. Carminomycin significantly hampered the rejection of the skin graft and under certain conditions prevented development of the RTAH which indicated suppression of the formation of the clone of the immune T-killers. Rubomycin almost did not hamper the rejection of the skin graft and had no effect on the RTAH which indicated the absence of the selective effect on the system of K-killers. Carminomycin eliminated the RTAH only when it was administered to the recipients of the allogenic lymphoid cells 24 hours after implantation of the cells to the mice treated with cyclophosphamide. When carminomycin was administered to the donors of lymphoid cells 24 hours before the beginning of the experiment, it did not suppress the ability of the cells from these donors to induce the RTAH. Therefore, its effect was directed to the precursors of T-killers activated in the host of the allogenic recipient.

[Immunodepressive activity of liposome-incorporated rubomycin]. / Immunodepressivnaia aktivnost' rubomitsina, zakliuchennogo v liposomy.

The effect of rubomycin incorporated into liposomes on the immune response of mice to sheep red blood cells was studied. It was found that an increase in the immuno depressant activity of the liposomal rubomycin was most pronounced, when the antibiotic was incorporated into large liposomes. The level of the immune response suppression by rubomycin incorporated into liposomes depended to a definite extent on the function of the macrophagal system.

[Functional activity of T- and B-lymphocyte populations after rubomycin and carminomycin exposure]. / Funktsional'naia aktivnost' populiatsii T- i B-limfotsitov posle vozdeistviia rubomitsina i karminomitsina.

The dynamics of antibody genesis at various periods after single administrations of the antibiotics to mice and the mice spleen capacity to cooperate with intact T- and B- lymphocytes and macrophages in an immune response to sheep red cells were studied. It was found that rubomycin in equal with carminomycin doses by the toxicity induced more significant and slower reducing shifts in the immune response. Analysis of the cell mechanism of the immunosuppressive effects of rubomycin and carminomycin at early periods after their administration showed that the antibiotics inhibited mainly the functional activity of B-lymphocytes and had not effect on the helper function of T-lymphocytes. The immunological activity of macrophages was inhibited insignificantly.

[Combined action of rubomycin and diuretics on the induction of antibody synthesis in mice]. / Sochetannoe deistvie rubomitsina i diuretikov na induktsiiu sinteza antitel u myshei.

The effect of 2 diuretics, diacarb and lasix on the intensity of immunodepressant action of low doses of rubomycin was studied. It was shown that diacarb increased the inhibitory effect of rubomycin on accumulation of the antibody-forming cells in the spleen. This effect was evident by the 3rd, 4th and 7th day after the antigen injection to the mice (the 4th, 5th and 8th day after the last injection of rubomycin). Combined use of rubomycin and diacarb also resulted in a low number of nuclear cells in the spleen as compared to control. Lasix did not increase the immunodepressant effect of rubomycin. Still, when used alone it had a capacity for changing the time course of the immune response development.

[Comparison of carminomycin and rubomycin action on the dynamics of the immune response to the T-independent Vi-antigen of S. typhi]. / Sravnenie deistviia karminomitsina i rubomitsina na dinamiku T-nezavisimomu Vi-antigenu S. typhi.

The effect of carminomycin and rubomycin on the dynamics of the primary immune response to T-independent Vi-antigen of S. typhi was studied. Differences in the character of the antibiotic effect indicative of the high selective effect of carminomycin on the multiplying cells or precursors of the antibody forming plasmocytes were noted. It was found that the carminomycin inhibitory effect on synthesis of hemagglutinins to Vi-antigen was higher than that to sheep red blood cells. Carminomycin was shown to impair the formation of the immunological memory, while rubomycin did not suppress the development of the "memory cell" clone to T-independent antigen.

[Rapid change in the dynamics of antibody-forming cells under the influence of a single injection of various anthracyclines]. / Bystroe izmenenie dinamiki antiteloobrazuiushchikh kletok pod vliianiem odnokratnoi in''ektsii razlichnykh antratsiklinov.

The dynamics of the titers and number of the antibody forming cells was studied within 26 hours after a single injection of anthracycline to immunized animals at various phases of the immune response. The specificity of the "rapid" effect of anthracyclines suggests that the low differentiated precursors of the antibody forming cells are the main target for the rubomycin effect and the immune competent cells in the state of multiplication and differentiation under the effect of the antigen are the main target for the carminomycin effect. The study was performed with noninbred mice immunized with sheep red cells.

Binding of anti-tumor immunoglobulins and their daunomycin conjugates to the tumor and its metastase. In vitro and in vivo studies with Lewis lung carcinoma.

The present study was undertaken in order to evaluate the possibility of using anti-tumor antibodies as specific carriers of chemotherapeutic drugs to tumor metastases. Xenogeneic and syngeneic antisera were prepared against a metastasizing C57BL tumor, the Lewis lung carcinoma (3LL). The binding of absorbed xenogeneic and syngeneic anti-3LL sera or their Ig fractions to 3LL tumor cells was assayed by direct and indirect methods. Antisera prepared against tumor cells from a local growth reacted to a similar extent with those obtained from the lung metastases. Radioiodinated immunoglobulins from syngeneic antisera injected into mice bearing metastases localized preferentially in metastasis-bearing lungs as compared to several other organs tested. No such preferential uptake was observed either in mice bearing metastases injected with iodinated normal Ig or in normal mice injected with iodinated anti-3LL Ig. This relative accumulation in the metastasis-bearing lungs was observed 3-4 days after the inoculation, when the whole Ig fraction was used, whereas a specific antibody-enriched preparation localized as early as 24 h after injection. Daunomycin--anti-3LL-Ig conjugates were effective inhibitors of tumor cells from both local and the metastatic growths. They were more active than either the free drug or drug conjugates of normal Ig, in in vitro assays.

[Immunodepressive action of carminomycin and rubomycin derivatives]. / Immunodepressivnoe deistvie proizvodnykh karminomitsina i rubomitsina.

The immunodepressive effects of carminomycin and its 3 semi-synthetic derivatives, as well as rubomycin and its derivative R-103 were compared. It was found that 14-hydroxycarminomycin was much superior to the other substances in the experiments with synthesis induction suppression of antibodies against sheep red cells in mice. Suppression of the rejection of the skin allogenic grafts in the mice by carminomycin was higher as compared to that by the other substances. Probably different populations of the immune competent cells have selective sensitivity to separate anthracyclines.

[Comparison of the action of carminomycin and rubomycin on the dynamics of the primary and secondary immune responses]. / Sravnenie deistviia karminomitsina i rubomitsina na dinamiku pervichnogo i vtorichnogo immunogo otveta.

The effect of rubomycin and carminomycin on the dynamics of the primary and secondary immune response and formation of the immunologic memory to sheep red cells in mice was studied. Differences in the character of the antibiotics effect indicative of the higher selective action of carminomycin on multiplying cells, precursors of the antibody-forming plasmids, were found. Theoretically interesting discrepancies in the effect of the antibiotics on the content of the antibodies in the serum and the antibody-producing cells in the spleen were shown. It was demonstrated that carminomycin had no effect on formation of the immunologic memory inspite of a noticeable decrease in the total number of the spleen nuclear cells and the number of the antibody-forming cells at the moment of immunization under the effect of the antibiotic.

[Effect of rubomycin and carminomycin on antibody formation during the immunization of mice with various antigens]. / Vliianie rubomitsina i karminomitsina na antiteloobrazovanie pri immunizatsii myshei raznymi antigenami.

The maximum immunodepressive effect of rubomycin and carminomycin was observed when the antibiotics were administered intravenously 24 hours after the immunization. The immune response induced by the sheep erythrocytes or the lipopolysaccharide was equally inhibited by rubomycin. Carminomycin in a dose of 0.5 mg/kg (0.1 of the LD50) to a larger extent inhibited the immune response stimulated by the lipopolysaccharide. Dependence of the immunodepressive effect of the antibiotics on their dose was found when the drugs were administered intravenously or orally.

[Effect of a UHF field on immunogenesis in mice with normal reactivity and with immunodepression induced by rubomycin]. / Vliianie UVCh-polia na immunogenez myshei s normal'noi reaktivnost'iu i s immunodepressiei, vyzvannoi rubomitsinom.

The experiments on male mice of SVA line showed that irradiation in the UHF-field 48 hours after the animal immunization with sheep erythrocytes increased the number of the antibody-producing cells in the spleen at least 3 times. On the background of immunodepression induced by rubomycin the UHF-field increased the primary immune response 2.5 times and the number of cariocytes in the spleen 1.8 times as compared to the control.