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M Colombatti

Publications and source records attributed to M Colombatti.

At least 37 records · Page 2Linked to original sources
PubMed1992

Internalization and intracellular fate of anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human leukemic T cells.

We have investigated the entry and subsequent intracellular fate of T101 monoclonal antibody (MoAb) and T101-ricin A-chain (RTA) immunotoxin (IT) directed against the CD5 antigen (Ag) expressed on human leukemic CEM cells. We provide direct evidence for the internalization of T101 MoAb and the corresponding IT. Both the MoAb and IT were internalized at a relatively low rate. This slow internalization process could be related to the partial recycling of the MoAb/Ag or IT/Ag complexes. Analysis of the internalized molecules showed that their molecular weight was only partially altered after internalization and that no free A-chain could be found inside the cells, indicating that lysosomal degradation and cleavage of disulfide-linked conjugates is a quantitatively minor phenomenon compared with the localization of internalized anti-CD5 ITs in an endosomo-Golgi compartment, followed by their recycling to the cell surface. We believe that this is the major factor explaining the low efficacy of anti-CD5 IT when assayed in the absence of potentiating substances. Finally, we showed that the presence of ammonium chloride and monensin, which both dramatically enhance the kinetics of IT activity, did not affect the rate of internalization or the intracellular localization of the conjugate, suggesting that these activators could act at the postendocytotic level on a limited number of IT molecules.

Ammonium Chloride↗
PubMed1992

Blocking effect of human serum but not of cerebrospinal fluid on ricin A chain immunotoxin potentiation by monensin or carrier protein-monensin conjugates.

The potentiation of monoclonal antibody/ligand toxin (immunotoxin) cytotoxicity by the ionophore monensin (Mo) or by human serum albumin-monensin (HSA-Mo) conjugates was investigated. Since disulfide cross-linked HSA-Mo (HSA-SPDP-Mo) is rapidly inactivated by human serum (M. Colombatti et al., Cancer Res., 50: 1385-1391, 1990), we synthesized thioether cross-linked HSA-Mo conjugates (HSA-SIA-Mo). HSA-SIA-Mo is resistant to treatment with reducing agents (e.g., glutathione, dithiothreitol) and shows potentiating activity identical to that of Mo or of HSA-SPDP-Mo, enhancing immunotoxin (IT) cytotoxicity 45-35,000-fold. Human leukemic and tumor cell lines are highly sensitive to treatment with IT in combination with Mo, HSA-SPDP-Mo, or HSA-SIA-Mo (concentration required to inhibit protein synthesis by 50%, 10(-10)-2.5 x 10(-13) M). IT potentiation by both types of HSA-Mo conjugates, however, is inhibited by whole human serum. In contrast, human cerebrospinal fluid has no effect on the potentiation of IT by Mo or HSA-Mo conjugates. The serum blocking factors reside mostly in a Mr 40,000-90,000 protein fraction. Serum components of low molecular weight (less than 10,000) show no detectable effect upon the stability of HSA-Mo conjugates. The toxicity of HSA-SIA-Mo in vivo was investigated by intrathecal injections in rats. Concentrations of up to 60 micrograms/kg can be injected into the brain with only transient neurological sequelae. We therefore conclude that if the systemic delivery of HSA-Mo conjugates for the potentiation of ricin A chain-IT presents some limitations due to the blocking effect of serum, the application of HSA-Mo conjugates in combination with ricin A chain-IT for regional tumor therapy in the brain appears more promising.

Ammonium Chloride↗
PubMed1991

A comparative analysis of three different techniques for the detection of breast cancer cells in bone marrow.

Three different methods, morphologic, immunocytochemic, and fluorescence activated cell sorter (FC) analysis, were compared with respect to their efficiency in detecting breast cancer cells in bone marrow. In the first series of experiments, the three techniques were compared using bone marrow cells artificially mixed with a known amount of breast cancer cells, whereas in a second series bone marrow from breast cancer patients with bone metastases were used. The following results were obtained: When mixtures of the first series were analyzed, FC analysis detected from 1% to 10% of breast cancer cells in bone marrow (0.2% was a border line value), the morphologic method detected from 0.05% to 10%, and the immunocytochemic method, which was clearly superior, detected breast cancer cells in all mixtures (from 0.00025% to 10%). It was noted that, with both the morphologic and immunocytochemic methods, the percentage of breast cancer cells detected was 2 to 360 times higher than the percentage of added cells, and enrichment was inversely proportional to the percentage of added cells. This result could be a result of different separation of cells during centrifugation due to the different density of breast cancer cells. The superiority of the immunocytochemic method was confirmed in the second series of experiments.

Bone Marrow↗
PubMed1990

Distribution of endocytosed molecules to intracellular acidic environments correlates with immunotoxin activity.

We have investigated the internalization to low pH intracellular compartments of transferrin (Tfn), diphtheria toxin (DT) and of anti-cell surface antibodies (MAb) by a cytofluorometric assay based on low pH quenching of fluorescein (FITC) emission. FITC-labelled Tfn, anti-CD3, anti-CD5 and anti-Thy 1.2 MAb internalization resulted in a progressively lower FITC quenching effect. Following internalization, a distinction could be made between molecules that enter low pH compartments without undergoing intracellular degradation (e.g., Tfn, anti-CD3 MAb) and molecules that are internalized through low pH organelles and are then degraded within the cell (e.g., DT). A strict correlation was observed between quenching of internalized FITC-protein fluorescent emission and the cytotoxic activity of DT-based immunotoxins (IT).

Animals↗
PubMed1990

Morphological heterogeneity and phenotype modifications during long term in vitro cultures of six new human glioblastoma cell lines.

Long term in vitro cultures of six human malignant gliomas were established to obtain permanent lines and to assess, under conditions of prolonged culture, changes in morphology and phenotype of neoplastic cells and the extent of these modifications. We analyzed expression of the following markers by immunocytochemistry: glioma-specific antigens (GE2 and CG12), fibronectin, intermediate filaments (GFAP, vimentin, neurofilaments), class I and II histocompatibility antigens (HLA-ABC and HLA-DR), growth factor and receptor (alpha TGF and EGF-receptor), proliferation-associated antigen (Ki-67). Strong and stable staining with the two antiglioma monoclonal antibodies (GE2 and CG12) was seen, with coexpression of GFAP and fibronectin in five of six cell lines (after 20 passages) and presence of vimentin and neurofilaments. HLA-DR expression was heterogeneous, with a peculiar intracellular compartmentation in four of six cell lines. Cells showed clear cytoplasmic positivity for alpha TGF and strong membrane staining for EGF-receptor. In previous studies we showed that these cell lines have increased copies of chromosome 7; therefore we speculate that an autocrine pathway of stimulation may maintain the neoplastic growth. The percentage of Ki-67 positive proliferating cells ranged from 40 to greater than 60%, depending on cell line and passage. A slight decrease in the positivity of some markers (GFAP, vimentin and HLA-DR in 2/6 cell lines) was observed after prolonged in vitro culture (greater than 12 months), but morphophenotypic modifications, established within a few passages after explanation, were maintained with time. A clonogenic assay showed values of plating efficiency (PE) higher than corresponding values of other similar cell lines with a tendency to increase in the late passages. PE and Ki-67 positivity were not associated with tumorigenicity into nude mice (except the Hu 197 cell line). These results indicate that, in culture, all six cell lines acquired stable morphology, a well defined antigenic phenotype and high growth rate. Further studies will be performed on these permanent cell lines to clarify differentiation steps of malignant gliomas.

Antibodies, Monoclonal↗
PubMed1990

Carrier protein-monensin conjugates: enhancement of immunotoxin cytotoxicity and potential in tumor treatment.

We have investigated the potentiation of transferrin [Tfn]-toxin [Tfn-ricin toxin A chain (RTA) and Tfn-So6 saporin toxin] and monoclonal antibody-RTA conjugates by monensin (Mo) and by a human serum albumin (HSA)-monensin conjugate in vitro. The in vivo survival and in vitro and in vivo toxicity of HSA-Mo were also studied; monensin was chemically linked to HSA carrier protein via a disulfide bridge. HSA-Mo was 2-13-fold less toxic than Mo for cells in vitro. HSA-Mo was active in the same concentration range as Mo in potentiating mAb-RTA and Tfn-toxin conjugates reactive with Tfn receptors expressed by different cell lines in monolayer cell cultures. Multicell tumor spheroid cultures were used to investigate the target cell killing effect of cytotoxic conjugates and HSA-Mo in three-dimensional structures mimicking the properties of nonvascularized micrometastases. Spheroids 300-400 microns were as sensitive to Tfn-RTA and HSA-Mo in combination as monolayer cells. After 24 h incubation at 37 degrees C in human serum about 2% HSA-Mo molecules remained available for immunotoxin potentiation and about 10% after 24 h incubation in human cerebrospinal fluid. BALB/c mice tolerated injections of 2 mg/kg HSA-Mo i.v. and of 16 mg/kg i.p. The HSA-Mo half-life in the serum of BALB/c mice was 0.5 h. Following i.v. injection about 0.5% of the initial HSA-Mo persisted in the circulation at 24 h.

Animals↗
PubMed1989

HIV-1-specific B cell activation. A major constituent of spontaneous B cell activation during HIV-1 infection.

B cell activation is a well known consequence of HIV-1 infection, and seropositive subjects show high numbers of spontaneously activated Ig-secreting cells in circulation. To better define the importance of the HIV-1-specific response in this phenomenon, we first studied whether in vitro spontaneous anti-HIV-1 antibody production was accompanied by reactivation of memory B lymphocytes. Unstimulated PBL from HIV-1-infected individuals with prior history of hepatitis B and/or EBV infection did not consistently show spontaneous in vitro synthesis of anti-hepatitis B core Ag or anti-EBV antibodies; in addition, PWM-induced synthesis of anti-hepatitis B virus and anti-EBV antibodies was decreased compared to HIV-1-seronegative subjects. Moreover, in comparing the frequencies of activated HIV-1-specific B cell precursors and activated Ig-secreting precursors in limiting dilution experiments, a sizable fraction (20 to 40%) of circulating cells spontaneously secreting Ig produced antibody against HIV-1 determinants. The ratio between the two frequencies fitted in very well with the amount of Ig removed from unstimulated culture supernatants after HIV-1-specific antibody absorption with solid-phase HIV-1. These findings indicate that B cell activation during HIV-1 infection is mainly oriented toward a specific response to HIV-1 determinants; the possible relevance of this phenomenon to lymphomagenesis in AIDS patients is discussed.

Absorption↗
PubMed1989

Overexpression of N-ras oncogene and epidermal growth factor receptor gene in human glioblastomas.

Five human glioblastoma cell lines were analyzed for oncogene activation with a panel of probes. Abnormal expression of the epidermal growth factor receptor (EGFr) gene was detected in four of five lines; N-ras oncogene overexpression was found in all five cell lines. These results were subsequently confirmed with fresh brain tumor and nonneoplastic brain tissue biopsy samples; increased expression of the N-ras proto-oncogene was observed in five of five glioblastomas, all of which also showed EGFr gene overexpression, but not in well-differentiated gliomas or in nonneoplastic brain tissue specimens. No significant differences in Ha-ras and Ki-ras expression were observed. Preliminary histochemical observations showed that intracellular levels of transforming growth factor alpha, a putative biochemical link between these two oncogenes, were significantly higher in glioblastoma cells than in controls.

Blotting, Northern↗
PubMed1989

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1987

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication.

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.

Animals↗
PubMed1987

Identification and characterization of a monoclonal antibody recognizing a galactose-binding domain of the toxin ricin.

A monoclonal antibody raised against purified ricin B chain, 75/3B12, blocked ricin toxicity 30- to 100-fold in vitro. The 75/3B12 IgG and F(ab')2 blocked ricin binding to cell surface galactose-containing receptors. The 75/3B12 Fab bound ricin D with a Ka of 10(7) M-1, and this binding was blocked by asialofetuin, lactose, and N-acetylgalactosamine--molecules which interact with the ricin galactose-binding site--but not by fetuin, sucrose, or glucose. The 75/3B12 Fab contained no detectable carbohydrate and, according to several lines of evidence, did not bind ricin via Ig carbohydrate determinants. The monoclonal antibody appears to recognize a galactose-binding site on ricin D via the variable region of the antibody. The 75/3B12 Fab bound ricin E only 1/50 as well as ricin D and bound the Ricinus agglutinin only 1/80 as well as ricin D. The antibody specificity indicates that structural differences exist in the galactose-binding sites of the Ricinus communis lectins. Abrin and other lectins which bind galactose or N-acetylgalactosamine were not significantly bound by the monoclonal antibody. In vitro, the antibody blocked the nontarget toxicity of immunotoxins similarly to lactose. However, in vivo, unlike lactose, the 75/3B12 antibody protected mice from ricin toxicity.

Animals↗
PubMed1987

Hybridoma cells containing intracellular anti-ricin antibodies show ricin meets secretory antibody before entering the cytosol.

Hybridoma cells which synthesize monoclonal antibodies (mAb) that block ricin toxicity were 50-300-fold resistant to ricin compared with other hybridomas. Two of the mAb blocked two isozymes of ricin, D and E, to different and opposite extents, and the hybridoma cell resistance to the two forms of ricin closely corresponded with the mAb reactivity. The hybridoma cell resistance to ricin was therefore due to the binding activity of the mAb produced by the cells. Neither rabbit polyclonal antibodies, which neutralized extracellular anti-ricin mAb, nor quantitative removal of hybridoma cell surface IgG with papain affected the cellular resistance to ricin. Therefore, neither extracellular or cell surface antibodies contributed to the resistance of the hybridoma cells. In contrast, inhibition of protein synthesis by cycloheximide or puromycin, which selectively decreased levels of intracellular secretory IgG, decreased the hybridoma cell resistance to ricin. We conclude that intracellular mAb, synthesized de novo for subsequent secretion, block ricin toxicity. Ricin therefore must meet intracellular secretory antibodies before reaching the cytosol. The monoclonal antibodies can also be used to study toxin function within intracellular compartments. An antibody specific for the galactose-binding site of ricin blocks ricin intracellularly, showing that the ricin galactose-binding activity is required in an intracellular compartment for transport of ricin A chain to the cytosol.

Animals↗
PubMed1986

Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry.

The role of discrete domains of diphtheria toxin (DT) B chain in cytosol entry and cytotoxicity was investigated by linking a monoclonal antibody recognizing the human T cell-specific antigen T3 (UCHT1) to diphtheria toxin (UCHT1-DT), DT A subunit (UCHT1-DTA), or to a genetically engineered form of DT (UCHT1-MspSA) lacking the C-terminal 17-kDa portion of the B subunit. The N-terminal 21-kDa region of DT B chain increased toxicity of UCHT1-DTA 100-fold (UCHT1-MspSA) while addition of the C-terminal 17-kDa region (UCHT1-DT) increased toxicity 100-fold more. The cytotoxicity was dependent upon antibody binding as demonstrated by blocking toxicity with excess UCHT1. The differences in toxicity between these reagents were not due to differences in ADP-ribosylation activity of DT A chain, binding activity of the antibody moiety, extent of DT nicking, or the cross-linking method, so we conclude that the large differences in toxicity were due to the presence of different B chain domains. The large increase in toxicity by the C-terminal region of DT B did not appear to be caused by DT receptor binding. The lysosomotropic agent NH4Cl blocked the cytotoxic effect of DT, UCHT1-DT, and UCHT1-MspSA but not UCHT1-DTA.

Adenosine Diphosphate Ribose↗
PubMed1986

Monoclonal antibodies against ricin: effects on toxin function.

Monoclonal antibodies against ricin toxin were produced and some of their properties investigated. Antibodies 196 C12 and 197 C7 raised against A-chain reacted with a CnBr fragment probably comprised between amino acid 254 and 262. Antibodies 193 A9, 196 A3, and 191 B7 recognized a 6-7 kD CnBr peptide. A second set of antibodies was raised against whole inactivated ricin. Most of them bound in a solid phase radioimmunobinding assay only to ricin and few had a low activity against purified A-chain. Different effects were noted on toxin action in cultured leukemic cells. If cells were preincubated with ricin followed by antibodies, MAb 207 E5 and 216 B3 had a strong enhancing effect on toxin action. If antibodies and toxin were mixed and then added to sensitive cells, antibody 207 E5 gave a strong protection while 216 B3 maintained its enhancing activity. The effect of antibody 216 B3 was further investigated by quantitative cloning experiments which showed that toxin had a fivefold enhancement in its activity by a preincubation with this antibody. Binding of fluoresceinated ricin to leukemic target cells was inhibited by a preincubation with antibody 207 E5 while antibody 216 B3 had no effect.

Animals↗