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At least 19 recordsLinked to original sources

PubMed1991

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin.

An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [3H]leucine by CEM cells was inhibited by 50% at an M-T151--ricin-A-chain concentration (IC50) of 4.6 nM compared with an IC50 of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC50 values of 20-30 pM. The addition of ricin B chain to CEM cells treated with M-T151--ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly. Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151--ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [3H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.

Animals↗

PubMed1991

Comparative biochemical, cytotoxic and pharmacokinetic properties of immunotoxins made with native ricin A chain, ricin A1 chain and recombinant ricin A chain.

Immunotoxins were constructed by attaching native ricin A chain, ricin A1 chain and recombinant ricin A chain to the mouse monoclonal IgG2a antibody Fib75 by means of a disulphide linkage using the hetero-bifunctional cross-linker SPDP. The Fib75 immunotoxins were of similar composition and exerted identical cytotoxic effects against the EJ human bladder carcinoma cell line in tissue culture. All 3 immunotoxins broke down to the same extent upon incubation with glutathione in vitro. The clearance of the immunotoxins from the circulation of normal Wistar rats was determined following i.v. administration. The concentration of intact immunotoxin in serum samples taken at various intervals up to 48hr after injection was measured by a ricin A chain-specific ELISA. The Fib75 immunotoxin made with native ricin A chain was removed from the circulation most rapidly. Fib75-recombinant ricin A chain persisted in the circulation at a higher level than Fib75-ricin A1 chain. A higher proportion of the ricin A1 chain immunotoxin was lost from the bloodstream during the alpha-phase. The beta-phase half-lives of Fib75-recombinant ricin A chain and Fib75-ricin A1 chain were similar, consistent with the identical susceptibility of the immunotoxins to cleavage by glutathione. The presence of the complex-type oligosaccharide side-chain on the A1 chain may have accelerated the clearance of the A1 chain immunotoxin in relation to that of the immunotoxin made with the aglycosyl recombinant A chain.

Animals↗

PubMed1992

The specific cytotoxicity of immunoconjugates containing blocked ricin is dependent on the residual binding capacity of blocked ricin: evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated.

Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its ability to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibody-dependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of the ricin B-chain, is dependent on the minimal residual lectin activity of blocked ricin. Our findings support the notion that two functions of ricin, membrane binding and translocation, cannot be separated.

Antigen-Antibody Complex↗

PubMed1984

The role of binding ligand in toxic hybrid proteins: a comparison of EGF-ricin, EGF-ricin A-chain, and ricin.

To analyze the influence of ricin B-chain on the toxicity of hybrid-protein conjugates, the rate of cellular uptake of conjugates, and the rate at which ricin A-chain (RTA) is delivered to the cytoplasm, we have constructed toxic hybrid proteins consisting of epidermal growth factor (EGF) coupled in disulfide linkage either to ricin or to RTA. EGF-ricin is no more toxic on A431 cells than EGF-RTA. The two conjugates demonstrate similar kinetics of cellular uptake (defined as antibody irreversible toxicity). EGF-RTA and EGF-ricin, like ricin, required a 2-2 1/2 hour period at 37 degrees before the onset of protein synthesis inhibition occurred. Our results suggest that RTA determines the processes which carry it, either in conjugate or toxin, from the plasma membrane binding site to the cytoplasm following endocytosis, and the ricin B chain is not required for these processes.

Biological Transport↗

PubMed1987

The complete amino acid sequence of the B-chain of ricin E isolated from small-grain castor bean seeds. Ricin E is a gene recombination product of ricin D and Ricinus communis agglutinin.

The complete amino acid sequence of the B-chain of ricin E has been determined. The reduced and carboxymethylated B-chain was digested with trypsin, followed by separation and purification of the resulting peptides using reverse-phase HPLC. The amino acid sequence of each tryptic peptide was determined employing the DABITC/PITC double-coupling method. The B-chain of ricin E proved to consist of 262 amino acid residues. By comparing the amino acid sequence of the B-chain of ricin E with those of ricin D and of Ricinus communis agglutinin, it was found that the B-chain of ricin E was composed of the N-terminal half of ricin D and C-terminal half R. communis agglutinin. This result suggested that the gene recombination probably occurred at the center region of two B-chain genes of ricin D and R. communis agglutinin.

Amino Acid Sequence↗

PubMed1990

The influence of anti-(ricin toxin A chain) monoclonal antibodies on the pharmacokinetics of ricin toxin A chain and recombinant ricin A chain in mice.

Two monoclonal antibodies against ricin toxin A chain (RTA) have been examined for their effects on the blood survival and biodistribution of RTA and recombinant ricin A chain in mice. When admixed with the toxins at 1:1 molar ratios prior to intravenous injection, the antibodies prolonged blood survival and whole-body retention of both species of RTA, and this was due essentially to reduced renal clearance of the toxins. Immune complexes were identified by gel filtration chromatography and immune precipitation with anti-IgG antiserum in mixtures prior to injection and in the serum of mice injected with the mixtures. An irrelevant monoclonal antibody showed no complex formation, and no effect on biodistribution. These studies have shown that immune complexes formed between monoclonal antibodies and protein antigens of molecular mass up to at least 30 kDa survive in the circulation, rather than being cleared by the reticuloendothelial system. Such antibodies could be used to modulate the biodistribution of toxic molecules such as ribosome-inhibiting proteins like RTA. This might be exploited therapeutically, for example in the construction of bispecific antibodies against ribosomal inhibiting proteins and tumour-associated antigens.

Animals↗

PubMed1992

Biochemical studies on oral toxicity of ricin. V. The role of lectin activity in the intestinal absorption of ricin.

In order to investigate a possible role of lectin activity of ricin in its absorption from the small intestine, we prepared two ricin derivatives. BMH-ricin, prepared by crosslinking A and B chains of ricin with 1,6-bismaleimidohexane, was nearly non-toxic but the lectin activity was unaltered. And, NBS-ricin, prepared by the oxidation of tryptophanyl residues of ricin with N-bromosuccinimide, was not only non-toxic but also non-lectinic. After the oral administration of ricin derivatives to rats, their interaction with the digestive tract and absorption into the circulatory systems have been compared with those of ricin, immunochemically and histologically. It was shown by immunostaining that ricin and BMH-ricin could bind to the intestinal mucosa, whereas NBS-ricin could not. No appreciable damage in the small intestine from rats treated with either BMH-ricin or NBS-ricin has been observed, in contrast to ricin treatment where severe impairment of the small intestinal tissues resulted after 5 h. Immunoreactive ricin in the liver has been determined with the ricin enzyme immunoassay (EIA). When compared at 48 h after oral administration, NBS-ricin was not detected, whereas BMH-ricin was found to be 38 micrograms/liver and ricin 100 micrograms/liver. From these results, it was inferred that the lectin activity of ricin plays an important role in the absorption of ricin from the small intestine and that the absorption of ricin protein was enhanced by its high toxicity.

Administration, Oral↗

PubMed1991

Assessment of ligand effects in intracellular trafficking of ricin A chain using anti-ricin hybridomas.

Intracellular ricin and immunotoxin trafficking has been difficult to study as only one to two cytosolic ricin A chain (RTA) molecules are sufficient to cause cell death. Previous studies (R.J. Youle and M. Colombatti, J. Biol. Chem., 262: 4676-4882, 1987) using anti-ricin hybridomas identified the secretory pre-Golgi as a critical site for RTA intoxication. We used ricin and RTA immunotoxins constructed with transferrin (TF) or anti-murine TF receptor antibody (RI7/217) to compare patterns of cytotoxicity and intracellular trafficking in anti-ricin hybridomas. Anti-RTA and anti-ricin B chain (RTB) hybridomas bound similar amounts of ricin and secreted comparable amounts of anti-ricin immunoglobulin. Anti-RTA hybridomas were 50- to 500-fold more resistant to ricin than nonsecretory and anti-RTB hybridomas, defining a ricin-resistant phenotype. All hybridomas expressed similar levels of surface TF receptors. RTA immunotoxins were constructed using human TF or RI7/217 and a disulfide linker. In protein synthesis inhibition assays, ricin-resistant hybridomas were manyfold more resistant to RI7/217-RTA than were ricin-sensitive hybridomas. In contrast, all hybridomas were equally sensitive to TF-RTA. Monensin increased ricin cytotoxicity minimally against all hybridomas, but dramatically increased RI7/217-RTA cytotoxicity in ricin-resistant and ricin-sensitive hybridomas in a way that abrogated the ricin-resistant phenotype. In contrast, monensin increased TF-RTA cytotoxicity equally in all hybridomas. Ammonium chloride had little effect on ricin or RI7/217-RTA cytotoxicity, but increased TF-RTA cytotoxicity against all hybridomas. Taken together, these results suggest that RTA molecules mediating cytotoxicity pass through an anti-RTA antibody-containing pre-Golgi compartment when bound to RTB or RI7/217, but not when bound to TF. Monensin abrogates the ricin-resistant phenotype when RTA is linked to RI7/217, but not RTB. This suggests that monensin alters RI7/217-RTA processing proximal to the pre-Golgi and that passage through the pre-Golgi may not be necessary for translocation of RTA to the cytoplasm. Ammonium chloride alters toxin cytotoxicity only when RTA is linked to TF, suggesting that only TF trafficks RTA through an acid-sensitive compartment prior to cytoplasmic translocation. With the addition of potentiating agents, each toxin studied showed a unique cytotoxicity profile against the anti-ricin hybridomas, demonstrating a dominant role of the cell binding ligand in intracellular toxin trafficking.

Ammonium Chloride↗

PubMed1997

Cell lysis induced by ricin D and ricin E in various cell lines.

Ricin D, one of the two isolectins from small caster beans showed stronger cytotoxicity than another one, ricin E, based on the inhibition of colony formation and the inhibition of protein synthesis. Both ricin D and ricin E induced cell lysis to different extents in each cell line tested, albeit ricin E was slightly less effective than ricin D. DNA fragmentation, a characteristic feature of apoptosis, was also induced by ricin D and ricin E in Vero cells. Scatchard plot analysis showed that ricin D binds to cells with higher affinity than ricin E, while the number of binding sites per cell was not much different, suggesting that the differences in the cytotoxicity between ricin D and ricin E is mainly due to their differential binding affinity to cells. In Vero cells, the cytolytic activities of ricin D and ricin E were inhibited by brefeldin A (BFA), which is known to effect the Golgi apparatus, but not significant effect of BFA was observed in a BFA-resistant cell line, MDCK cells. These results suggest that the Golgi apparatus may be involved in ricin-induced cell lysis.

Animals↗

PubMed1981

Studies on the galactose-binding site of ricin and the hybrid toxin Man6P-ricin.

N-acetylimidazole (NAI) was used to O-acetylate the plant seed toxin ricin. O-acetylation of one to two tyrosine residues per molecule of ricin inhibited ricin binding to Sepharose 4B and decreased toxicity by 90% in a protein synthesis inhibition assay in HeLa cells. Lactose, known to block the binding site on the ricin B subunit, protected ricin from NAI modification of binding or toxicity. Thus NAI, under these conditions, can be a lactose site-specific inhibitor. The lactose site-specific modification of the hybrid toxin, Man6P-ricin, performed under the same conditions, exhibited the same 90% inhibition of Man6P receptor-mediated toxicity as the galactose-containing receptor-mediated toxicity of either Man6P-ricin or ricin. Thus the ricin B chain lactose-binding site appears to be essential for the high potency of Man6P-ricin via the new cell type-specific Man6P receptor. Treatment of fibroblasts with neuraminidase exposes galactose residues, thus increasing the sensitivity to ricin eight fold. The Man6P receptor-mediated toxicity of Man6P-ricin is not affected by this treatment, although the galactose-inhibited route is potentiated eight fold. The Man6P-ricin hybrid appears to require the ricin B chain galactose-binding site to enter the cytosol after initially binding to the Man6P receptor. These data provide some insights into the proper design of hybrid toxins. We discuss a number of possible models for hybrid toxin entry.

Acetylation↗

PubMed1987

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia.

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.

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↗

PubMed1995

Ricin toxicity and intracellular routing in tumoral HT-29 cells. I. Ricin routing and toxicity are related to the state of differentiation of HT-29 cells.

We previously showed that ricin, which is more cytotoxic to undifferentiated than to differentiated tumoral HT-29 cells, enters these cells by different routes. The final steps of ricin endocytosis were investigated in order to identify the translocation site from which ricin exerts its toxicity. Toxicity measurements and kinetic experiments followed by subcellular fractionation were run in parallel. In differentiated cells, from 20 min of internalization, radiolabeled ricin was found in a Golgi-enriched fraction. At 60 min, which corresponds to the lag time for ricin toxicity, the amount of radioactivity located in this fraction decreased without any concomitant increase in the other fractions. In undifferentiated cells, from 20 min of incubation, radiolabeled ricin was detected in the ER-enriched fractions. At 30 min, the lag time for ricin toxicity, the amount of radioactivity detected in these fractions decreased without any concomitant increase in the Golgi-enriched fraction. Monensin, which was used to confirm the passage of ricin through the Golgi, greatly increased ricin toxicity and diminished the lag time only in differentiated cells. Brefeldin A inhibited ricin toxicity when added before the end of the lag time in both cell populations and reduced the amount of ricin detected, respectively, in the Golgi- and ER-enriched fractions in differentiated and undifferentiated cells. We propose that ricin enters the cytosol from the Golgi apparatus and essentially from the ER in differentiated and undifferentiated HT-29 cells, respectively, and that these different intracellular routings might explain the differential toxicity of ricin.

Biological Transport↗

PubMed2004

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase.

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.

Auranofin↗

PubMed1992

Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats.

After oral administration of ricin in rats, its distribution in the gastrointestinal tract, body fluids and principal organs was determined by an enzyme immunoassay, and the immunoreactive ricin detected was identified by gel filtration followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, protein blotting and the immunobinding method. When ricin D (10 mg/kg rat) was given orally to a rat, which dose is equivalent to 1/3 LD50, about 75% of the ricin was found in the stomach and small intestine within 2 h, and most of it was transferred to the large intestine after 24 h. It was also demonstrated by an in vitro toxicity test of immunoreactive ricin in the blood and lymph obtained from the intoxicated rats that a part of the ricin was absorbed from the small intestine into the tissues and organs via the circulatory systems (lymphatic and blood vessels) as the active ricin. The participation of the blood vessels was greater in the absorption of ricin from the gastrointestinal tract than that of the lymphatic system. Ricin, after absorption, was detected in liver and spleen and ricin found in the liver was predominantly in the form of intact ricin, although an undetectable amount of ricin in other organs cannot be eliminated. These results infer that a small fraction of orally-given ricin was transferred to the circulating system and was responsible for rat's death as in the case of i.p. administration.

Administration, Oral↗

PubMed1982

Preparation and properties of chimeric toxins prepared from the constituent polypeptides of diphtheria toxin and ricin. Evidence for entry of ricin A-chain via the diphtheria toxin pathway.

A highly toxic conjugate of ricin A-chain and diphtheria toxin fragment B was prepared by disulfide exchange reaction. A similar conjugate between diphtheria toxin fragment A and ricin B-chain was nontoxic. Like native diphtheria toxin, the conjugate ricin A/diphtheria toxin B was much more toxic to Vero than to HeLa cells. Ricin was equally toxic to these cell lines. Lactose, which inhibits ricin binding, did not protect against the conjugate. Cells resistant to ricin, partly due to a reduced number of ricin-binding sites, were fully sensitive to the conjugate, indicating that the conjugate binds to diphtheria toxin receptors. The conjugate was fully toxic to two Vero cell mutants, resistant to diphtheria toxin because the elongation factor 2 could not be ADP-ribosylated by the diphtheria toxin A-fragment. Therefore, the inhibition of protein synthesis by the conjugate must be caused by the ricin A-chain. Ammonium chloride which prevents entry of diphtheria toxin, but not of ricin, also protected against the conjugate. Like diphtheria toxin, the conjugate was most toxic at low pH, whereas ricin is most active at pH above neutrality and inactive at low pH. The results indicate that the conjugate ricin A/diphtheria toxin B binds to diphtheria toxin receptors and inhibits cellular protein synthesis due to the action of ricin A-chain which appears to enter the cell by the diphtheria toxin pathway.

Ammonium Chloride↗

PubMed1995

Ricin toxicity and intracellular routing in tumoral HT-29 cells. II. Differential ricin toxicity from the apical and basolateral surfaces of differentiated HT-29 cells.

We previously showed that ricin, a toxin commonly used in the construction of immunotoxins, was more toxic to undifferentiated than to differentiated HT-29 tumoral cells. This results from differences in the intracellular routings of the toxin. As these studies concerned the entry through the apical pole of differentiated polarized HT-29 cells, we investigated and compared the intracellular routing of ricin from the apical and basolateral membranes of differentiated HT-29 cells and the toxicity of ricin depending on the pole of administration. For this purpose, we developed the culture of polarized HT-29 cells on porous membrane filters and demonstrated that differentiated HT-29 cells can establish a leakproof monolayer. Ricin is 2.5-fold less toxic when it is added at the basolateral than at the apical pole of the cells, which may result from different observations: (1) less ricin is bound at the basolateral membrane than at the apical one, leading to a lesser internalization of the toxin; (2) ricin sorting in the apical and basolateral endocytic compartments of HT-29 cells differs: apically internalized ricin is targeted intracellularly while basolaterally internalized ricin uses mainly the transcytotic pathway; using NH4Cl and monensin, we observed that ricin follows the same pathway from both sides of the cells, namely the endosomal system, to reach the Golgi apparatus from which toxin intoxication occurs; (3) kinetics studies showed that a delay exists before an efficient intoxication by the basolateral pole is observed. The use of monensin at low concentration in order to perturb only the Golgi functions indicated that this delay could account for a different presentation of the toxin toward the membrane of the apical and basolateral endocytic compartments. Together, our results showed that, in differentiated HT-29 cells, if the pathways carrying ricin from the apical and basolateral membranes to the Golgi apparatus appear identical, ricin exerts differentially its toxicity depending upon the surface of administration, i.e., the apical or the basolateral surface of the cells.

Cell Differentiation↗

PubMed1992

Blocked ricin-conjugated T cell immunotoxins: effect of anti-CD6-blocked ricin on normal T cell function.

The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked ("blocked ricin"), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC50) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC50 values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%-45% viable cells remained after 2 days yet only 3%-9% remained if phorbol ester and Ca2+ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 x 10(-3); when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10(-1). As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.

Antigens, CD↗