Selective immunotoxins prepared with mutant diphtheria toxins coupled to monoclonal antibodies.
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Animals↗
Biomedical subjects
G Tridente
Publications and source records attributed to G Tridente.
Heterogeneity and modulation of tumor-associated antigens in human glioblastoma cell lines.
Seven human glioblastoma cell lines established in vitro from primary tumor explants were studied. A marked heterogeneity of glial fibrillary acidic protein was observed whereas vimentin was uniformly expressed by all cell lines. Indirect immunofluorescence and flow cytofluorometry revealed a heterogeneous distribution of surface GE 2 and CG 12 tumor-associated antigens (TAA's): three cell lines were positive (greater than 69% TAA-positive cells) and three cell lines were negative (less than 9% TAA-positive cells). One cell line (Hu 228) was moderately positive at early culture passages and subsequently acquired a TAA-negative phenotype. The difference in the relative amounts of surface TAA's of the three positive cell lines was less than twofold. In spite of the heterogeneous distribution of surface TAA's, all cell lines exhibited considerable amounts of intracellular TAA. Treatment with phorbol esters and density-dependent growth arrest decreased the percentage of the TAA-positive cells and the amount of cell-surface TAA's in one cell line (Hu 195). Interferon-gamma treatment in vitro increased the percentage of CG 12-positive cells by 12% and the amount of cell-surface CG 12 antigens by 38% as compared to untreated cells. The percentage of TAA-positive cells among phorbol ester-treated cells of the Hu 195 cell line was lowest 48 hours after treatment, but returned to normal values within the next 48 hours. Reduction of 3H-thymidine incorporation preceded the decrease in number of TAA-positive cells by about 18 hours. Two-color fluorescence analysis performed in positive cell lines for simultaneous determination of surface TAA's and deoxyribonucleic acid content or reactivity with the proliferation-associated Ki67 intracellular marker indicated that GE 2 and CG 12 antigens are expressed preferentially by actively proliferating glioma cells. The results of this study indicate the existence of two different phenotypes in cultured human glioblastoma cells: surface TAA-positive/cytosol TAA-positive and surface TAA-negative/cytosol TAA-positive cell populations. In addition, modulation of TAA expression was dependent on the cell-cycle differentiation stage, culture conditions, and proliferative state of the cells.
Animals↗
Human recombinant interleukin-4 inhibits lymphokine-activated killer activity of sheep erythrocyte rosette-forming (E+) and -non-forming (E-) human lymphocytes.
The effect of human recombinant interleukin-4 (rIL-4) on lymphokine-activated killer activity (LAK) of peripheral blood lymphocytes (PBL) as well as sheep erythrocyte rosette-forming (E+) and -non-forming (E-) lymphocytes stimulated by human recombinant interleukin-2 (rIL-2) has been investigated. rIL-4 drastically inhibited LAK activity of PBL cultured for 18 hr and for 5 days in the presence of rIL-2. Distribution of T, B and IgG Fc-receptor-bearing lymphocytes, as assessed by immunofluorescence and flow cytometry, was no different at the end of the culture in the presence of rIL-2 plus rIL-4 than with rIL-2 alone. LAK activity of E+ and E- lymphocytes was similarly inhibited. Finally, rIL-4 did not affect the natural killer (NK) activity of rIL-2-activated PBL as assessed by the capacity of these cells to kill the appropriate NK target.
Animals↗
Sensitivity of human glioma cells to cytotoxic heteroconjugates.
Several anti-human glioma cytotoxic conjugates were studied in vitro. Monoclonal antibodies (MAbs) to the GE2 glioma-associated antigen (anti-GE 2) and MAbs to HLA-DR antigens (D1/12) or human diferric transferrin (Tfn) were linked to the potent cytotoxin ricin (anti-GE 2-ricin) or to its A subunit (anti-GE 2-RTA, D1/12-RTA, Tfn-RTA). Anti-GE 2-RTA had low cytotoxic activity in both the absence and the presence of lysosomotropic substances inhibiting intracellular degradation. Anti-GE 2-ricin was about 1,000 times more toxic than RTA alone, but showed only 14-fold target specificity. D1/12-RTA was about 20 times more toxic than RTA and its cytotoxic effect increased about 6- to 7-fold when cell-surface HLA-DR antigen expression was enhanced by IFN-gamma treatment. Human diferric Tfn linked to RTA demonstrated the highest cytotoxic activity, being about 5,000 times more toxic than RTA alone for glioma cells and about 6,000 times more toxic for Jurkat cells in the presence of the carboxylic ionofore monensin. Ricin toxin was only about 5 times more toxic for Jurkat and glioma cells than Tfn-RTA-monensin. Tfn-RTA was over 100,000 times more potent than the chemotherapeutic agent BCNU in reducing glioma cell survival in vitro. Addition of 80% human pooled cerebrospinal fluid (CSF) reduced Tfn-RTA toxicity about 10-fold. Kinetics of Tfn-RTA cytotoxicity at non-saturating concentrations indicated that over 80% of target cells could be killed within 8-10 hr in the absence and within 10-12 hr in the presence of human pooled CSF.
Animals↗
Establishment and characterization of a new human melanoma cell line (HU 214) with a high growth potential and stable properties.
A human melanoma cell line (HU 214) with high growth potential was established from a lymph node metastasis of a patient with advanced cutaneous melanoma. The cells of this line were able to grow in monolayer (according to the Rosenblum technique) and in agar (according to the Courtenay-Mills method), and formed tumors when injected in nude mice. The line has been maintained in culture for more than 47 passages. The cell cultures were periodically characterized (every 6-8 passages) by immunohistochemistry using a panel of monoclonal antibodies (MoAbs) including MoAbs against tumor-associated antigens (antimelanoma, antiglioma and anti-LLA), against vimentin, and against major histocompatibility antigens, and including also Ki 67, a MoAb which reacts with a nuclear antigen associated with cell proliferation. The results of this characterization indicate that we have established a human melanoma cell line with a stable antigenic phenotype during subculturing, poorly differentiated cells, and a high growth potential.
Animals↗
A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity.
The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.
Animals↗
DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry.
A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.
Cell Cycle↗
Human glioma cell lines: tumour associated antigens distribution and sensitivity to antibody-toxin or ligand-toxin conjugates. A preliminary report.
We have investigated the phenotype of seven human glioma cell lines established in vitro from primary tumour explants. Indirect immunofluorescence and flow cytofluorimetry revealed a heterogeneous distribution of surface GE 2 and CG 12 Tumour Associated Antigens (TAA). In one group of cell lines TAA were detected both at the cell surface and in the cytosol, whereas in a second group of glioma cell lines TAA were found only in the cytosol. We have also investigated the sensitivity of glioma-derived cell lines to antibody-toxin and ligand-toxin conjugates (Immunotoxins). Monoclonal antibodies anti GE 2 antigen linked to ricin toxin A subunit (RTA) showed poor cytotoxicity, which increased about 50 fold when the whole toxin was linked to anti GE 2 monoclonals. Treatment with human recombinant interferon gamma (IFN-gamma) greatly augmented the percentage of HLA-DR+ cells and the amount of HLA-DR antigens per cell. IFN-gamma treatment resulted in a net increase of sensitivity to anti HLA-DR Immunotoxins (IT). Human diferric transferrin linked to RTA exhibited a potent cytotoxic effect against human glioma-derived cells when used in the presence of the lysosomotropic carboxylic ionophore monensin.
Antibodies, Monoclonal↗
Heterogeneity of lymphokine-activated killer (LAK) populations at the clonal level: both NK and CD3+, CD4-, CD8- clones efficiently mediate tumor cell killing.
To investigate at the clonal level the phenotypic and functional properties of interleukin 2 (IL-2) activated killer cells (LAK), recombinant IL-2 activated peripheral blood lymphocytes were cultured under limiting conditions. Among 56 clones that lysed P815 in the presence of phytohemagglutinin (PHA) (22% of total proliferating microcultures) 36 clones lysed also the natural killer (NK)-sensitive K562 and the NK-resistant Hu126 glioma cell lines and one clone lysed only the K562 cell line. Several LAK clones were further assayed for both phenotype and functional activity. Of 22 clones, 10 were CD3-, CD4-, CD8-, and expressed the CD16 marker of NK cells; only one clone had the conventional phenotype of cytolytic T cells (CD3+, CD4-, CD8+), while 11 clones were CD3+, CD4-, CD8- and did not express alpha/beta heterodimer of T-cell antigen receptor as identified by WT31 monoclonal antibody. Only one of the latter clones was CD16+. Endogenous production of IL-2 after stimulation with PHA and phorbol myristate acetate was positive in 3/9 CD3- and in 8/8 CD3+, CD4-, CD8- clones. CD3- mediated strong antibody-dependent cellular cytotoxicity, a function exerted also by some CD3+, CD4-, CD8- T-cell clones to a lower extent. CD3+, CD4-, CD8- T-cell clones lysed different major histocompatibility complex unrelated tumor targets; moreover, this lytic activity seems to be CD3 dependent.
Antibody-Dependent Cell Cytotoxicity↗
Biochemical characterization of a T-lymphoma-specific 90,000 molecular weight disulfide linked dimeric glycoprotein.
The biochemical features of a membrane antigen detected by a mouse monoclonal antibody (A1) raised against the murine thymoma cell line EL4 are described. This reagent detected a novel disulfide-linked 90,000 mol. wt dimeric membrane glycoprotein composed of two chains of approx 45,000 mol. wt. Endo-beta-N-acetylglucosaminidase F digestion generated a single 28,000 polypeptide, thus suggesting that the A1 molecule is a homodimer. No structural homology between the A1 molecule and the human T 90/44 protein (9.3 antigen) could be revealed by peptide mapping analysis. In view of the fact that three polypeptides of mol. wts 28,000-30,000, 21,000 and 15,000 respectively co-precipitated with the A1 antigen, the possible relationship of the A1 molecular complex to other known T-cell surface antigens including the antigen receptor is discussed.
Animals↗
Immunocytochemical characterization of long-term medulloblastoma cultures: preliminary report.
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Antibodies, Monoclonal↗
Myasthenia gravis: immunohistological heterogeneity in microenvironmental organization of hyperplastic and neoplastic thymuses suggesting different mechanisms of tolerance breakdown.
Four samples of thymoma obtained from patients affected by myasthenia gravis have been immunohistologically analysed on cryostat sections using a panel of antisera and monoclonal antibodies specific for antigens which define different stages of intrathymic lymphocyte differentiation and antigens specific for different types of thymic epithelial cells (cortical, medullary). When the thymoma samples were compared to age-matched normal thymuses and hyperplastic thymuses obtained from patients with myasthenia gravis some evident microenvironmental differences could be demonstrated using these reagents. In all the thymoma samples in fact the neoplastic lobules appeared as grossly enlarged cortical-type areas, formed by accumulations of T lymphocytes exhibiting the cortical immature phenotype (TdT+, T6+, etc.) within a network of putatively neoplastic epithelial cells characterized by cortical phenotype as defined by reactivity with various monoclonal antibodies (RFD4-, MR3+). These 'cortical' epithelia showed some abnormal features such as lack or irregular distribution of HLA-DR and enhanced keratin expression. Small areas of 'medullary' differentiation could be observed in 3/4 thymoma samples. In thymic hyperplasia, on the other hand, the cortical areas appeared somewhat compressed (but comparable to those observed in normal age-matched samples) by enlarged medullary areas. The expansion of medullary areas was due to the infiltration of 'peripheral' lymphoid tissue intruding through the extraparenchymal zone and forming organized B and T areas. These observations are discussed in the light of the clinical heterogeneity observed in myasthenia gravis.
Adolescent↗
In vitro analysis of BCNU-sensitivity in human malignant gliomas. II. Cross-resistance studies with cisplatinum and nitrosoureas.
With the use of the Human Brain Tumor Stem Cell Assay (HBTSCA) in a cross-resistance study, four early (3-4) culture passages of human malignant gliomas (glioblastoma multiforme) were tested for in vitro chemosensitivity with three of the most effective single agents for brain tumor chemotherapy: BCNU, CCNU and cisplatinum (DDP). The shapes of the dose-response curves indicated complete cross-resistance between BCNU and CCNU, i.e. two chloroethyl-nitrosoureas sharing a common alkylating-carbamoylating activity, with no evident cross-resistance between the two nitrosoureas and the DDP, a DNA binder with a putatively different antitumor action. Probably because of differences in drug delivery kinetics or in the cytotoxic mechanism, DDP might play a role in the treatment of nitrosourea-resistant gliomas.
Brain Neoplasms↗
In vitro analysis of BCNU-sensitivity in human malignant gliomas. I. A model study with alkylating, cross-linking and carbamoylating agents in anaplastic astrocytomas of pediatric age.
Like all chloroethyl-nitrosoureas of major clinical use, 1,3 bis-(2-chloroethyl)-1-nitrosourea (BCNU) - which is one of the most effective chemotherapeutic agents for CNS malignancies - biologically degrades into active alkylating and carbamoylating moieties. Using a human brain tumor stem cell assay, we analyzed a series of anaplastic astrocytomas of pediatric age, characterized by different degrees of BCNU-resistance. Early (2-4) passage cultures from these tumors were treated in vitro with model drugs for alkylation (BCNU, CHLZ (2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose), ENU (N-ethyl-N-nitrosourea), cross-linking (BCNU, CHLZ) and carbamoylation BHCNU (1,3 bis (trans-4-hydrocyclohexyl)-1-nitrosourea): dose-schedules were compatible with clinically achievable levels. Results of chemosensitivity tests confirmed that - as previously reported in malignant gliomas of the adult - cellular resistance to BCNU was closely related to the cross-linking activity of alkylating species. However, in pediatric gliomas the levels of cell kill after treatment with the purely carbamoylating agent BHCNU, even at the highest doses tested, were lower than expected.
Age Factors↗
Immunopathology of the human thymus.
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Antigens, Surface↗
Specific IgE antibodies in twenty-eight workers with diisocyanate-induced bronchial asthma.
A specific IgE-mediated response was evaluated in twenty-eight workers exposed to TDI or MDI, with diagnosis of occupational asthma and positive to bronchial provocative challenge. The presence of anti-diisocyanate IgE was observed in 27% of subjects exposed to TDI and 83% of those exposed to MDI, particularly in individuals who experienced an acute massive exposure. An immediate-type response to bronchial provocative test was found in 66% of individuals with specific antibodies. Specific IgE are prevalent (91%) in subjects who developed symptoms before 6 years of exposure to isocyanates. The results suggest an association between the presence of specific IgE, early asthmatic symptoms and heavy episodic exposure.
Asthma↗
Immunohistochemical and enzyme histochemical contributions to the problem concerning the role of the thymus in the pathogenesis of myasthenia gravis.
Normal fetal and postnatal thymuses, as well as thymuses from patients with myasthenia gravis (MG), were investigated by immunohistochemical and enzyme histochemical techniques and their morphology reviewed. Intrathymic B-cells, detected by ATPase activity, were found to be markedly increased in number in MG thymuses. They were scattered in the medulla and accumulated around the junctional and medullary vessels and Hassall's corpuscles (HCs). Large epithelial cells, singly or within HCs, were found to be unevenly distributed in the medulla of all the thymuses examined. The striated muscle-like nature of some of these cells was revealed by the presence of myoglobin in their cytoplasm. In myasthenics these cells and small developing HCs characteristically surrounded lymph follicles and were in direct contact with the expanded cap of the follicle mantle, without interposition of reticulin fibres. The close association of immune reactive foci (lymphoid follicles, junctional and medullary vessels, and HCs) with structures involved in autoimmune responses in the thymus (muscle-like and true myoid cells, HCs) strongly suggests that the autoimmune reactions against AChR (acetylcholine receptor) and other muscular components, which constitute the basic defects in myasthenia gravis, may begin in the thymus.
Adenosine Triphosphatases↗
Thymic involvement in myasthenia gravis. Study by immunofluorescent and immunoperoxidase staining.
Changes in the thymus are often encountered in myasthenia gravis (MG), together with neuromuscular pathology. In the present study, the cell population of 28 thymuses from patients with MG were analyzed by immunofluorescent and 49 thymuses by immunoperoxidase techniques. We were able to show the presence of sIg receptor positive, SpA reactive medium and large B lymphocytes of light specific density in the majority of thymuses, and to demonstrate a characteristic pattern of distribution in the gland. PAP analysis showed that the infiltrating B cells appeared in the interlobular septa, then reached the medulla, occasionally the cortex which mostly revealed signs of atrophy. These findings are consistent with an autoimmune reaction occurring against a thymic antigen; such an antigen could be acetylcholine receptor (AchR) of the thymic structures, since anti-AchR antibody or serum from patients with MG will react with 60% thymocytes and some epithelial cells and Hassall's corpuscles.
Acetylcholine↗