FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2006736635> ?p ?o ?g. }

• W2006736635 endingPage "21957" @default.
• W2006736635 startingPage "21950" @default.
• W2006736635 abstract "T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxicin vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8–10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 μm brefeldin A, decreased 42% when microtubules were disrupted by 10 μm nocodazole, and decreased 74% at 18 °C. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17–22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not. T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxicin vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8–10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 μm brefeldin A, decreased 42% when microtubules were disrupted by 10 μm nocodazole, and decreased 74% at 18 °C. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17–22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not. Botulinum neurotoxin (BoNT), 1The abbreviations used are: BoNT/Abotulinum neurotoxin serotype ACaco-2human colon adenocarcinomaMDCKMadin-Darby canine kidneyT-84human colon carcinoma. which is the etiologic agent responsible for the disease botulism, can enter the body by several different routes, but the most common of these is the gastrointestinal system (1Sanders A.B. Seifert S. Kobernick M. J. Fam. Pract. 1983; 16: 987-1000PubMed Google Scholar, 3Hatheway C.L. Curr. Top. Microbiol. Immunol. 1995; 195: 55-75Crossref PubMed Scopus (160) Google Scholar, 4Montecucco C. Schiavo G. Rossetto O. Arch. Toxicol. 1996; 18: 342-354Crossref Scopus (52) Google Scholar). Thus, most cases of intoxication are due to ingestion of food contaminated with preformed toxin or ingestion of food contaminated with bacteria that can produce toxin in the gut. In either case, BoNT escapes the gastrointestinal system to reach the general circulation (lymph and blood). Toxin in the blood is then distributed to peripheral cholinergic nerve endings, which are the target cells for toxin action. botulinum neurotoxin serotype A human colon adenocarcinoma Madin-Darby canine kidney human colon carcinoma. There is now a substantial literature describing the cellular, subcellular, and even molecular aspects of toxin action on cholinergic cells (5Montecucco C. Schiavo G. Mol. Microbiol. 1994; 13: 1-8Crossref PubMed Scopus (496) Google Scholar, 6Jahn R. Hanson P.I. Otto H. Ahnert H.G. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 329-335Crossref PubMed Scopus (29) Google Scholar, 7Oguma K. Fujinaga Y. Inoue K. Microbiol. Immunol. 1995; 39: 161-168Crossref PubMed Scopus (130) Google Scholar, 8Rossetto O. Deloye F. Poulain B. Pellizzari R. Schiavo G. Montecucco C. J. Physiol. ( Paris ). 1995; 89: 43-50Crossref PubMed Scopus (44) Google Scholar, 9Schiavo G. Rossetto O. Tonello F. Montecucco C. Curr. Top. Microbiol. Immunol. 1995; 195: 257-274PubMed Google Scholar). This literature derives from a model that envisions the toxin proceeding through three major steps (2, 10Simpson L.L. J. Pharmacol. Exp. Ther. 1980; 212: 16-21PubMed Google Scholar, 11Simpson L.L. Pharmacol. Rev. 1981; 33: 155-188PubMed Google Scholar, 12Simpson L.L. Annu. Rev. Pharmacol. Toxicol. 1986; 26: 427-453Crossref PubMed Scopus (397) Google Scholar, 13Montecucco C. Papini E. Schiavo G. FEBS Lett. 1994; 346: 92-98Crossref PubMed Scopus (216) Google Scholar). During the first step, the toxin binds to receptors that are highly localized in the junctional region of cholinergic cells. The toxin molecule, which is composed of a heavy chain (Mr ∼100,000) and a light chain (Mr ∼50,000) linked by a disulfide bond, is thought to rely on the carboxyl terminus of the heavy chain to associate with cells. During the second step, the toxin is productively internalized by vulnerable cells. This process is actually a sequence of two events: 1) penetration of the plasma membrane by receptor-mediated endocytosis, and 2) penetration of the endosome membrane by pH-induced translocation. Although there is much that remains to be determined about productive internalization, there is suggestive evidence that the amino terminus of the heavy chain plays an important role in this process. During the third and final step in toxin action, the light chain that reaches the cytosol acts as a zinc-dependent endoprotease to cleave polypeptides that are essential for exocytosis (6Jahn R. Hanson P.I. Otto H. Ahnert H.G. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 329-335Crossref PubMed Scopus (29) Google Scholar, 8Rossetto O. Deloye F. Poulain B. Pellizzari R. Schiavo G. Montecucco C. J. Physiol. ( Paris ). 1995; 89: 43-50Crossref PubMed Scopus (44) Google Scholar, 9Schiavo G. Rossetto O. Tonello F. Montecucco C. Curr. Top. Microbiol. Immunol. 1995; 195: 257-274PubMed Google Scholar). Blockade of exocytosis leads to the failure of neuromuscular transmission that is characteristic of botulism. In contrast to our knowledge of events at the nerve ending, very little is known about the cellular and subcellular events that account for the ability of the toxin to cross membranes in the gastrointestinal system (14Lamanna C. Hillowalla R.A. Alling C.C. J. Infect. Dis. 1967; 117: 327-331Crossref PubMed Scopus (15) Google Scholar, 15Sugii S. Ohishi I. Sakaguchi G. Infect. Immun. 1977; 17: 491-496Crossref PubMed Google Scholar, 16Bonventre P.F. Rev. Infect. Dis. 1979; 1: 663-667Crossref PubMed Scopus (38) Google Scholar). Those data that are available suggest that the mouth is the least effective site of absorption, the stomach is intermediate, and the intestine is the most effective site of absorption. There are no studies that actually demonstrate a mechanism for botulinum toxin penetration of gut cells. However, there is one self-evident matter that can be deduced. The series of steps that underlie toxin movement at the level of individual gut cells cannot be identical to the steps that underlie toxin movement in individual nerve cells. If BoNT were to enter and remain inside cells in the gastrointestinal system, this would prevent it from reaching peripheral cholinergic nerve endings. Thus, there must be fundamental differences that distinguish toxin movement across gastrointestinal membranes and movement across neuronal membranes. There are two broad categories of toxin transport that could account for movement from the gastrointestinal system to the general circulation. As proposed by Bonventre (16Bonventre P.F. Rev. Infect. Dis. 1979; 1: 663-667Crossref PubMed Scopus (38) Google Scholar), transport could involve a specific, receptor-mediated mechanism or a nonspecific mechanism. In the present study, work has been done to test the hypothesis that BoNT crosses the gut wall by the process of specific binding and transcytosis. According to this hypothesis, there should be little or no pH-induced translocation of toxin molecules across intracellular membranes. Instead, this hypothesis requires that toxin bound on the lumenal side of cells must be delivered to the serosal side of cells, and furthermore the transcytosed toxin must be in a conformation that can act upon cholinergic nerves. This hypothesis has been tested by studying the binding, movement, and residual toxicity of BoNT type A (BoNT/A) added to a human epithelial cell line of gut origin (T-84). The resulting data provide the first insights into the cellular and subcellular events that account for toxin movement from the gastrointestinal system to the general circulation. T-84 human colon carcinoma cells, Caco-2 human colon carcinoma cells, and Madin-Darby canine kidney cells were obtained from The American Type Culture Collection (Rockville, MD). Tissue culture media and sera were purchased from Life Technologies, Inc. [3H]Inulin and 125I-Bolton-Hunter reagent were purchased from NEN Life Science Products. Reagents were purchased from Sigma, and tissue culture supplies were obtained from Fisher. Sephadex G-25 gel filtration columns were obtained from Amersham Pharmacia Biotech. Tetanus toxin was purchased from Calbiochem (Behring Diagnostics, La Jolla, CA). A sample of BoNT/A-associated proteins was kindly provided by Dr. Bibhuti DasGupta (University of Wisconsin-Madison, Madison, WI). Samples of BoNT/C were kindly provided by Dr. Y. Kamata (University of Osaka Prefecture, Osaka, Japan). BoNT/A and BoNT/B were purified according to procedures described in the literature (17Sakaguchi G. Pharmacol. Ther. 1982; 19: 165-194Crossref PubMed Scopus (297) Google Scholar, 18DasGupta B.R. Sathyamoorthy V. Toxicon. 1984; 22: 415-424Crossref PubMed Scopus (125) Google Scholar, 19Simpson L.L. Schmidt J.J. Middlebrook J.L. Methods Enzymol. 1988; 165: 76-85Crossref PubMed Scopus (22) Google Scholar). Swiss-Webster mice (female, 20–25 g), which were purchased from Ace Animals (Boyertown, PA), were housed in an American Association for Accreditation of Laboratory Animal Care-accredited animal colony and allowed unrestricted access to food and water. All procedures involving animals were reviewed and approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. T-84 cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium (1 g/liter d-glucose) and Ham's F-12 nutrient medium supplemented with 5% newborn calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 8 μg/ml ampicillin, and 15 mm Hepes. Cultures were maintained at 37 °C in 6% CO2. T-84 cells were fed every 3 days and passaged (1:2) when 95% confluent, approximately every 6 days. Passages 55 through 76 were used for experiments described in this report. Caco-2 cells were grown in Dulbecco's modified Eagle's medium (4.5 g/liter d-glucose) supplemented with 15% newborn calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 15 mm Hepes. Cultures were maintained at 37 °C in 6% CO2. Caco-2 cells were fed every 2 days and passaged (1:2 split ratio) when 95% confluent, approximately every 6 days. Passages 20 through 29 were used for experiments described in this report. MDCK cells were grown in Dulbecco's modified Eagle's medium (4.5 g/liter d-glucose) supplemented with 10% newborn calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 mm Hepes. Cultures were maintained at 37 °C in 6% CO2. MDCK cells were fed every 3 days and passaged (1:5) when 95% confluent, approximately every 3 days. Passages 56 through 75 were used for experiments described in this report. To control for membrane integrity and to estimate diffusion between cells, the rate of passage of [3H]inulin from the upper to the lower reservoir was determined essentially as described previously (20Madara J.L. Dharmsathaphorn K. J. Cell Biol. 1985; 101: 2124-2133Crossref PubMed Scopus (305) Google Scholar). According to this procedure, the medium in both reservoirs contained unlabeled 1 mm inulin. The assay was initiated by addition of [3H]inulin to the upper reservoir. Rates for inulin flux from the upper to the lower wells were determined for 2, 6, or 20 h. To control for nonspecific effects that might be caused by prolonged incubation of cells with toxin, inulin flux experiments were repeated on cultures that had been preincubated with 1 × 10−8m BoNT/A for 18 h. As an additional control for membrane integrity, each transwell was checked for leakage at the end of each experiment. For this purpose, medium was added to the upper chamber but not the lower chamber. The Transwell® apparatus was returned to the incubator at 37 °C, and evidence of fluid accumulation in the lower reservoir was monitored for 6 h. Leakage seldom occurred (<5% of wells); when there was leakage, the data from these wells were not included in the final analysis. BoNT/A, B, and C, and tetanus toxin, as well as toxin-associated proteins such as the hemagglutinins, were iodinated using 125I-Bolton-Hunter reagent (NEN Life Science Products) essentially according to manufacturer's instructions. The reaction time was reduced in order to diminish the loss of toxicity of the resulting product. The toxins were labeled to an average specific activity of 500 Ci/mmol with a residual toxicity of greater than 90% (21Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Scholar, 22Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Therap. 1994; 269: 256-262PubMed Google Scholar, 23Coffield J.A. Bakry N. Zhang R.D. Carlson J. Gomella L.G. Simpson L.L. J. Pharmacol. Exp. Ther. 1997; 280: 1489-1498PubMed Google Scholar). Pure neurotoxin (250 μg) in borate buffer (pH 7.8, 200 μl) was added to dried, iodinated ester and reacted on ice for 15 min. The reaction was terminated by addition of 50 μl of 1m glycine in borate buffer for 5 min. The total reaction mixture (250 μl) and rinse (250 μl) were loaded onto a Sepahadex G-25 column that was preequilibrated with filtration buffer (150 mm Na2HPO4, 150 mmNaCl, 0.1% gelatin, pH 7.4). The labeled toxin was eluted with filtration buffer, and 0.5-ml fractions were collected. An aliquot (5 μl) of each fraction was assayed for radioactivity. The labeled toxin peak, which eluted at void volume, was pooled and stored at 3 °C. Toxin concentration in the pooled fraction was determined spectrophotometrically at 278 nm using the following relationship, 1.63A278 = 1 mg/ml (18DasGupta B.R. Sathyamoorthy V. Toxicon. 1984; 22: 415-424Crossref PubMed Scopus (125) Google Scholar). A portion of this sample was counted in a γ-counter to ascertain the number of counts (disintegrations/min) present. Sample concentration and associated counts were used to calculate specific activity. Cells were grown, differentiated, and induced to polarize as described above. Cells were rinsed three times and then incubated in mammalian Ringers solution with 10 mmHEPES and 1 mg/ml bovine serum albumin, pH 7.4. The cells were slowly (∼2 h) cooled to 3 °C. The buffer in the upper well was replaced with cold buffer (3 °C) containing 125I-BoNT, with or without a 50-fold molar excess of unlabeled toxin. The cells were incubated at 3 °C for 3 h to allow sufficient time to reach equilibrium, after which cells and membranes were washed 3 times with Hanks' balanced salt soluiton plus 10 mm HEPES with 1 mg/ml bovine serum albumin. Membranes were then cut out of the Transwell® holders and placed in a γ-counter. At least three replicates per condition were included. For assay of botulinum neurotoxin transcytosis, cells were grown in Transwell® porous bottom dishes on polycarbonate membranes with a 0.4 μm pore size. The growth area within each insert was 1 cm2. Prior to being seeded with cells, the Transwells were coated with 10 μg/cm2 rat tail collagen type I. Briefly, rat tail collagen stock solution (6.7 mg/ml) was prepared in sterile 1% acetic acid and stored at 3 °C. The stock solution was diluted in acidified 60% ethyl alcohol, as needed, and 150 μl containing 10 μg of diluted collagen were added to each well. The coated wells were allowed to dry at room temperature overnight (18 h). After drying, the wells were sterilized under UV light for 1 h, followed by a preincubation with cell culture medium (30 min). The preincubation medium was removed immediately prior to addition of cells and fresh medium. Cells (T-84, Caco-2 or MDCK) were plated at confluent density (∼1.5 × 105 cells) into the Transwells with 0.5 ml of medium in the upper chamber and 1.0 ml in the lower chamber. In these experiments, medium in the upper chamber bathed the apical (or mucosal) surface of cells, and medium in the lower chamber bathed the basolateral (or serosal) surface of cells. Culture medium was changed every 2 days. The cultures were allowed to differentiate for a minimum of 10 days before assay of transcytosis. The formation of tight junctions was experimentally confirmed by measuring the rate of [3H]inulin movement from the upper chamber into the lower chamber (see above). Experiments were performed on cultures that were between 10 and 15 days old. The transcytosis assay was initiated by adding 1 × 10−8m125I-labeled botulinum neurotoxin either to the upper or lower chamber. Transport of radiolabeled toxin was monitored in two ways. Initially, the entire contents of the lower chamber were aspirated and placed on ice. Five μl of a 20 mg/ml stock of bovine serum albumin was added per ml of sample as a carrier (final concentration of 100 μg/ml) followed by trichloroacetic acid to a final concentration of 10%. Proteins in the samples were precipitated on ice overnight. The precipitates were washed three times with 10% trichloroacetic acid, and the radioactivity of pellets was measured in a γ-counter. Nonspecific counts were estimated by performing identical experiments at 10 °C. Subsequently, transport was monitored by collecting all of the medium from the appropriate chamber. An aliquot (0.5 ml) from each sample was filtered through a Sephadex G-25 column, and 0.5-ml fractions were collected. The amount of radioactivity in the fractions was determined in a γ-counter. Labeled toxin eluted at void volume, and the radioactivity contained in the void volume fractions was summed to determine the total amount of toxin present. Of the two methods, the latter was preferable due to better reproducibility among replicates. A minimum of two replicates per condition was included in each experiment. Cell fractions for assay of intracellular toxin were prepared by standard methods. Briefly, BoNT/A (1 × 10−8m) was added to the upper reservoir at 37 °C for 1 h. The wells were subsequently cooled to 18 °C over a 45-min time period, after which the cells were washed five times (upper and lower surface) with cold Dulbecco's phosphate-buffered saline without Ca2+ or Mg2+. Cells were trypsinized at 37 °C for 10 min, washed once with medium, then twice with homogenization buffer (255 mm sucrose, 1 mm EDTA, 20 mm Hepes, pH 7.4) containing protease inhibitors (5 μm antipain, 1 μmaprotinin, 10 μm leupeptin, 5 μm pepstatin A, 100 μm phenylmethylsulfonyl fluoride). Harvested cells were homogenized at 3 °C (on ice) in a glass-Teflon homogenizer (2 × 20 strokes). The homogenate was fractionated using an SS-34 rotor in a Sorvall RC5B centrifuge (Sorvall, Inc., Newtown, CT.). Homogenates were spun at 1000 × g for 5 min. The resulting supernatant (S-1) was recentrifuged at 10,000 ×g for 10 min. The second supernatant (S-2) was used for determination of cytosolic toxin. Samples for autoradiographic analysis were separated on 7.5% polyacrylamide gels according to Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), using reducing or non-reducing conditions. Gels were fixed for 30 min in destaining buffer, dried on blotter paper, and exposed to film (HyperfilmTM MP, Amersham Pharmacia Biotech). The toxicity of transcytosed BoNT/A that was collected from the lower reservoir was bioassayed either by intraperitoneal injection into mice or by addition to mouse phrenic nerve-hemidiaphragm preparations. For the in vivo bioassay, transcytosed toxin or native toxin was administered in a 100-μl aliquot of Dulbecco's phosphate-buffered saline with 1 mg/ml bovine serum albumin, per animal. Animals were checked for survival over the 12-h duration of the experiment. For the in vitro bioassay, tissues were excised and suspended in physiological buffer that was aerated with 95% O2, 5% CO2 and maintained at 35 °C. The physiological solution had the following composition (millimolar): NaCl, 137; KCl, 5; CaCl2, 1.8; MgSO4, 1.0; NaHCO3, 24; NaH2PO4, 1.0;d-glucose, 11; and gelatin, 0.01%. Phrenic nerves were stimulated continuously (1.0 Hz; 0.1–0.3-ms duration), and muscle twitch was recorded. Toxin-induced paralysis was measured as a 50% reduction in muscle twitch response to neurogenic stimulation. For both in vivo and in vitro bioassays, it was necessary to quantify the amount of toxin that was recovered and tested. This was done in two ways. First, the aliquot to be tested was submitted to gel filtration chromatography to isolate the toxin peak. The specific activity of the starting material added to the upper chamber of the Transwell® apparatus was calculated immediately after iodination (see “Iodination”). The number of counts in the purified fraction from the aliquot taken from the lower chamber and the specific activity were used to calculate the amount of toxin. Second, the actual rates of transcytosis as determined in this study (e.g.Table I) were used to calculate the amount of the toxin in the aliquots that were tested.Table ICharacterization of 125 I-BoNT/A transcytosis in polarized epithelial cell culturesCell lineConditionsTranscytosisPercent of controlpfmol/h/cm2%T-8437 °C(A→B)11.29 ± 0.3010037 °C(B→A)8.98 ± 0.2080<0.00118 °C(A→B)2.26 ± 0.4620<0.001Caco-237 °C(A→B)8.42 ± 0.4975<0.001MDCK37 °C(A→B)0.32 ± 0.072.8<0.001Transcytosis was assayed in T-84, Caco-2, or MDCK cell cultures. Assay was initiated by addition of 1 × 10−8m125I-BoNT/A to the upper or lower chamber. Cultures were subsequently incubated for 6–24 h at the temperature indicated. At the end of each experiment aliquots were collected and gel-filtered. Void volume fractions were assayed for radioactivity, and the toxin peak was summed to determine total counts. The amount of transcytosis was calculated based on the specific activity of labeled toxin. The rates reported are mean ± S.E. for T-84: 37 °C(A→B) n = 14; 37 °C(B→A) n = 4; 18 °C n = 7; Caco-2 n = 4; MDCKn = 9. All other details are provided under “Experimental Procedures.” Open table in a new tab Transcytosis was assayed in T-84, Caco-2, or MDCK cell cultures. Assay was initiated by addition of 1 × 10−8m125I-BoNT/A to the upper or lower chamber. Cultures were subsequently incubated for 6–24 h at the temperature indicated. At the end of each experiment aliquots were collected and gel-filtered. Void volume fractions were assayed for radioactivity, and the toxin peak was summed to determine total counts. The amount of transcytosis was calculated based on the specific activity of labeled toxin. The rates reported are mean ± S.E. for T-84: 37 °C(A→B) n = 14; 37 °C(B→A) n = 4; 18 °C n = 7; Caco-2 n = 4; MDCKn = 9. All other details are provided under “Experimental Procedures.” All data derived from three or more separate experiments are presented as means ± S.E. The equality of variances was tested using the F test. Subsequentlyp values were calculated using either a two-samplet test for independent samples with equal variances or a two-sample t test for independent samples with unequal variances (Cochran's method). Experiments were done both to confirm the formation of tight junctions in cell cultures and to assess the rate of movement of molecules across these tight junctions. For this purpose, [3H]inulin flux was measured over periods of 2–20 h. The average values obtained in three experiments indicated that the rate of [3H]inulin flux from the apical to the serosal side of T-84 cells typically fell within the range of 0.01- 0.07 nmol/h/cm2. The rates were slightly lower for Caco-2 and MDCK cultures. These rates compare favorably with those reported by others (20Madara J.L. Dharmsathaphorn K. J. Cell Biol. 1985; 101: 2124-2133Crossref PubMed Scopus (305) Google Scholar), and they are a strong indication that tight junctions had been formed and molecular diffusion was hindered. Identical experiments were done on cultures that had been pretreated with 1 × 10−8m BoNT/A for 18 h. Exposure to toxin did not alter inulin flux in either T-84 or MDCK cultures. Several serotypes of botulinum toxin and tetanus toxin were iodinated, and the ability of these ligands to bind to intact cells was measured. As shown in Fig.1, there was substantial and nearly equivalent binding of serotypes A (19.44 ± 2.35 fmol/cm2) and B (18.33 fmol/cm2) to T-84 cells. In contrast, the binding of serotype C (0.22 fmol/cm2) and tetanus toxin (0.47 fmol/cm2) was negligible. A more limited series of experiments was done with MDCK cells, in which binding of only serotype A was studied. Interestingly, there was no evidence of significant high affinity toxin binding (0.02 fmol/cm2) to these cells (Fig. 1, inset). Experiments were done to test the ability of differentiated, polarized T-84 epithelial cells to transcytose botulinum neurotoxin types A, B, and C, as well as tetanus toxin. Similar experiments were done on MDCK cells, which have the ability to differentiate, polarize, and transcytose, but which have not been implicated in clostridial toxin action. Fig.2, which illustrates data obtained for iodinated, homogeneous serotype A, clearly shows that T-84 cells are capable of uptake, transcytosis, and release of toxin. In contrast, MDCK cells did not transcytose serotype A at a significant rate. T-84 cells were also assessed for their ability to transcytose other serotypes of botulinum toxin and tetanus toxin. Interestingly, the cells showed a substantial ability to discriminate among the toxins. Botulinum toxin type B was efficiently transcytosed (83% of A; n = 5), whereas neither serotype C nor tetanus toxin (8–10% of A) was efficiently transcytosed. Data presented thus far indicate that T-84 cells transcytose BoNT/A. The experiments illustrated in Fig.3 show that increases in toxin concentration were associated with increases in transcytosis in T-84 cells. The rates of apical → basolateral transcytosis increased 600-fold from 0.262 fmol/cm2/h at 10−10m to 168.50 fmol/cm2/h at 10−7m toxin. Conversely, the fractional amount of toxin added to the top well (Fig. 3, numbers in parentheses) that was transported across cells decreased 2-fold from 12% at 10−10m to 6% at 10−7m in T-84 cultures. When equivalent experiments were done on MDCK cells, there was much less indication of a dose-dependent capacity for transcytosis. Data presented thus far demonstrate that polarized human epithelial cells transcytose certain clostridial toxins. Therefore, serotype A was selected as a prototype, and further work was done to establish rate constants. In addition, an effort was made to compare apical → basolateral transcytosis with basolateral → apical transcytosis. The data on rate constants are presented in TableI. T-84 cells were shown to transcytose iodinated BoNT/A at a rate of 11.29 fmol/h/cm2 when toxin was added to the upper reservoir. Transcytosis from the basolateral surface to the apical surface was less efficient (8.98 fmol/h/cm2). Cooling cultures to 18 °C reduced the rate of transcytosis to 2.26 fmol/h/cm2 (apical → basolateral). As shown in Table I, transcytosis of BoNT/A in Caco-2 cells (apical → basolateral) was less than that observed in T-84 cells, but still substantial. In contrast, transcytosis of the toxin by MDCK cells was negligible. Several drugs and procedures were used to further characterize the process of toxin movement across cells. Each drug and procedure was selected on the basis that previous studies have shown them to increase or decrease transcytosis (25Hunziker W. Male P. Mellman I. EMBO J. 1990; 9: 3515-3525Crossref PubMed Scopus (128) Google Scholar, 26Matter K. Bucher K. Hauri H.P. EMBO J. 1990; 9: 3163-3170Crossref PubMed Scopus (78) Google Scholar, 27Lencer W.I. de A.J. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed" @default.
• W2006736635 created "2016-06-24" @default.
• W2006736635 creator A5023439959 @default.
• W2006736635 creator A5065330318 @default.
• W2006736635 date "1998-08-01" @default.
• W2006736635 modified "2023-10-12" @default.
• W2006736635 title "Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells" @default.
• W2006736635 cites W1494548428 @default.
• W2006736635 cites W1538065055 @default.
• W2006736635 cites W1563623332 @default.
• W2006736635 cites W1586678823 @default.
• W2006736635 cites W1866032923 @default.
• W2006736635 cites W1949707762 @default.
• W2006736635 cites W1969755027 @default.
• W2006736635 cites W1972894937 @default.
• W2006736635 cites W2007142841 @default.
• W2006736635 cites W2010455127 @default.
• W2006736635 cites W2025931131 @default.
• W2006736635 cites W2032724670 @default.
• W2006736635 cites W2039418825 @default.
• W2006736635 cites W2041069875 @default.
• W2006736635 cites W2041408554 @default.
• W2006736635 cites W2041528452 @default.
• W2006736635 cites W2047426750 @default.
• W2006736635 cites W2075449190 @default.
• W2006736635 cites W2099635745 @default.
• W2006736635 cites W2100837269 @default.
• W2006736635 cites W2124347780 @default.
• W2006736635 cites W2125095112 @default.
• W2006736635 cites W21261928 @default.
• W2006736635 cites W2134625663 @default.
• W2006736635 cites W2138352514 @default.
• W2006736635 cites W2145879362 @default.
• W2006736635 cites W2147421583 @default.
• W2006736635 cites W2173682852 @default.
• W2006736635 cites W2284013506 @default.
• W2006736635 cites W2329186577 @default.
• W2006736635 cites W2331738825 @default.
• W2006736635 cites W4321059765 @default.
• W2006736635 cites W995999460 @default.
• W2006736635 doi "https://doi.org/10.1074/jbc.273.34.21950" @default.
• W2006736635 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9705335" @default.
• W2006736635 hasPublicationYear "1998" @default.
• W2006736635 type Work @default.
• W2006736635 sameAs 2006736635 @default.
• W2006736635 citedByCount "119" @default.
• W2006736635 countsByYear W20067366352012 @default.
• W2006736635 countsByYear W20067366352013 @default.
• W2006736635 countsByYear W20067366352014 @default.
• W2006736635 countsByYear W20067366352015 @default.
• W2006736635 countsByYear W20067366352016 @default.
• W2006736635 countsByYear W20067366352017 @default.
• W2006736635 countsByYear W20067366352018 @default.
• W2006736635 countsByYear W20067366352020 @default.
• W2006736635 countsByYear W20067366352021 @default.
• W2006736635 countsByYear W20067366352022 @default.
• W2006736635 crossrefType "journal-article" @default.
• W2006736635 hasAuthorship W2006736635A5023439959 @default.
• W2006736635 hasAuthorship W2006736635A5065330318 @default.
• W2006736635 hasBestOaLocation W20067366351 @default.
• W2006736635 hasConcept C1491633281 @default.
• W2006736635 hasConcept C185350488 @default.
• W2006736635 hasConcept C185592680 @default.
• W2006736635 hasConcept C2777010668 @default.
• W2006736635 hasConcept C2777367657 @default.
• W2006736635 hasConcept C2780358027 @default.
• W2006736635 hasConcept C28005876 @default.
• W2006736635 hasConcept C2994342964 @default.
• W2006736635 hasConcept C55493867 @default.
• W2006736635 hasConceptScore W2006736635C1491633281 @default.
• W2006736635 hasConceptScore W2006736635C185350488 @default.
• W2006736635 hasConceptScore W2006736635C185592680 @default.
• W2006736635 hasConceptScore W2006736635C2777010668 @default.
• W2006736635 hasConceptScore W2006736635C2777367657 @default.
• W2006736635 hasConceptScore W2006736635C2780358027 @default.
• W2006736635 hasConceptScore W2006736635C28005876 @default.
• W2006736635 hasConceptScore W2006736635C2994342964 @default.
• W2006736635 hasConceptScore W2006736635C55493867 @default.
• W2006736635 hasIssue "34" @default.
• W2006736635 hasLocation W20067366351 @default.
• W2006736635 hasOpenAccess W2006736635 @default.
• W2006736635 hasPrimaryLocation W20067366351 @default.
• W2006736635 hasRelatedWork W113230002 @default.
• W2006736635 hasRelatedWork W2051855939 @default.
• W2006736635 hasRelatedWork W2083002540 @default.
• W2006736635 hasRelatedWork W2161466677 @default.
• W2006736635 hasRelatedWork W2322719911 @default.
• W2006736635 hasRelatedWork W2353486218 @default.
• W2006736635 hasRelatedWork W2370265995 @default.
• W2006736635 hasRelatedWork W2417106366 @default.
• W2006736635 hasRelatedWork W2795943227 @default.
• W2006736635 hasRelatedWork W4317677645 @default.
• W2006736635 hasVolume "273" @default.
• W2006736635 isParatext "false" @default.
• W2006736635 isRetracted "false" @default.
• W2006736635 magId "2006736635" @default.
• W2006736635 workType "article" @default.