Concentrations Of Cholera Toxin Research Articles

Evaluation of Cholera Toxin Adjuvanticity Effect on the Production of Specific Antibodies Induced by Avian Infectious Bronchitis Vaccine in Chickens.

Cholera toxin (CT) is one of the most well-known immunostimulants. Mammalian studies have shown that CT can generate immune responses against antigen. However, it has not exhibited a definite effect on poultry yet. In this study, focusing on a cost-effective method, the effect of different concentrations of CT obtained from Vibrio cholerae biotype El Tor and serotype Inaba was investigated on the immunogenicity of infectious bronchitis vaccine. After culturing and concentrating CT, different concentrations of CT (0.1, 1, 2, and 5 micrograms) were combined with avian infectious bronchitis vaccine strain H120 produced by Razi Vaccine and Serum Research Institute (RVSRI) and, at 7 days of age, inoculated via the eye drop administration in 42 specific-pathogen-free chickens (seven groups of six chicks that included four experimental groups, two negative control groups (PBS and toxin), and one positive control group). Blood samples were taken weekly from the wing veins of the chickens, and the immunoglobulin G (IgG) titer was checked by enzyme-linked immunosorbent assay. The results showed that 2 µg of CT in comparison with other concentrations caused a significant increase in the antibody titer against avian infectious bronchitis in the blood serums of the chickens. One-way ANOVA test showed that all the results of this study were significant at P<0.05 level. Our data show that CT has the potential to further stimulate the immune system of chickens and may increase the immunogenicity of the infectious bronchitis vaccine. However, more research is needed to examine all aspects of the use of this toxin in animal vaccines.

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.

Sorption Properties of Polymeric Enterosorbent and Its Specific Modified Analogue in Simulated Cholera Toxin Solutions in vitro

Carried out was comparative study of the sorption properties in polymeric enterosorbent - chitosan, and its specific modified analogue obtained through absorption of anti-toxin immunoglobulins onto the soluble chitosan template. It is determined that soluble chitosan and its specific counterpart express high sorption activity in relation to cholera toxin, thus making lower its initial concentrations. Equilibrium adsorption capacity of the chitosan and its modified variant increase with the raise of initial cholera toxin concentration in the solution. Cholera toxin extraction rate was identified under static conditions. Adsorbate substance recovery rate was registered in the progression. In the course of experiments proved was modified sorbent specific sorption efficiency, occasioned by the presence of the active component in the analogue composition - namely, anti-toxic anticholeraic immunoglobulins. The data obtained are of the importance for further development of specific anticholeraic enterosorbent.

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

Removal of Cholera Toxin from Aqueous Solution by Probiotic Bacteria

Cholera remains a serious health problem, especially in developing countries where basic hygiene standards are not met. The symptoms of cholera are caused by cholera toxin, an enterotoxin, which is produced by the bacterium Vibrio cholerae. We have recently shown that human probiotic bacteria are capable of removing cyanobacterial toxins from aqueous solutions. In the present study we investigate the ability of the human probiotic bacteria, Lactobacillus rhamnosus strain GG (ATCC 53103) and Bifidobacteriumlongum 46 (DSM 14583), to remove cholera toxin from solution in vitro. Lactobacillus rhamnosus strain GG and Bifidobacteriumlongum 46 were able to remove 68% and 59% of cholera toxin from aqueous solutions during 18 h of incubation at 37 °C, respectively. The effect was dependent on bacterial concentration and L. rhamnosus GG was more effective at lower bacterial concentrations. No significant effect on cholera toxin concentration was observed when nonviable bacteria or bacterial supernatant was used.

Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6nM) cause reduced hemocyte adhesion, while high levels (12–120nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120nM) and in vivo (12–120nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting a cAMP-independent mechanism stimulating the nodulation response.

One Step Highly Sensitive Piezoelectric Agglutination Method for Cholera Toxin Detection Using GM1 Incorporated Liposome

A novel piezoelelctric biosensor for the detection of Cholera Toxin (CT) has been developed, based on a lipid layer with GM1(Monosialoganglioside) and the subsequent binding of the specifically agglutinated network complex of GM1-incorporated liposomes in assay medium after the characteristic recognition of CT by the sensing interface. A lipid monolayer comprising a fixed DPPC: GM1 molar ratio was spontaneously formed on the gold surface by exposing it to an aqueous dispersion of GM1-incorporated phospholipid vesicles. The quartz crystal surfaces with GM1-containing supported lipid membranes can detect CT and probe the mass change on the sensing interface. The simple preparation of sensing interface with lipid monolayer makes the developed technique especially useful for rapid and renewable immunochemical determination. The frequency responses of the developed piezoelelctric biosensors are linearly correlated to CT concentration in the range of 0.25 – 1.0 colong mL-1 with a detection limit of 95ng mL-1. This could be practically used for the detection of CT.

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) is an AB(5) toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1.

A Simple and Sensitive Piezoelectric Immunosensor for Cholera Toxin Based on GM1‐Incorporated Liposome Agglutination

AbstractA sensitive piezoelelctric immunosensor has been developed for the direct detection of cholera toxin. The proposed technique is based on specific interface agglutination of GM1 incorporated liposome vesicles in the presence of the cholera toxin, which causes a frequency change and is monitored by a piezoelectric device. The interface agglutination reaction would take place as soon as the cholera toxin (CT) was captured by the antibodies modified on the probe surface and the GM1 incorporated liposome in the probe cell. Meanwhile, the agglutination reaction induced both the mass effect and viscoelastic effect acting on the probe surface. The results indicate that the probe signal can be observably multiplied. In addition, an optimization of liposome composition and assay medium were investigated. Moreover, no cross‐reactivates were observed with the common protein, such as goat anti‐mouse IgG (1 mg/mL), mouse IgG (1 mg/mL), goat anti‐human IgG (1 mg/mL), and thrombin (1 mg/mL). The frequency responses of the developed piezoelelctric immunosensor are linearly correlated to cholera toxin concentration in the range of 0.5 to 7.5 µg/mL with a detection limit of 0.27 µg/mL while showed no significant loss in activity over 3 d under storage at 4°C.

Sub-attomolar detection of cholera toxin using a label-free capacitive immunosensor

A label-free immunosensor for the direct detection of cholera toxin (CT) at sub-attomolar level has been developed based on potential-step capacitance measurements. Anti-CT antibody was adsorbed on gold nanoparticles (AuNPs) incorporated on a polytyramine-modified gold electrode. The concentration of CT was determined by detecting the change of capacitance caused by the formation of antibody–antigen complexes. By using AuNPs adsorbed to the sensing surface, the signal was dramatically increased leading to a significantly more sensitive assay. In fact, under optimum conditions the immunosensor could detect CT concentration with a limit of detection of 9 × 10 −20 M or 0.09 aM, with a dynamic range between 0.1 aM and 10 pM. Good analytical reproducibility could be obtained by injecting CT up to 36 times with an RSD of 2.5%. In addition, good performance of the developed immunosensor was achieved when applied to turbid water samples collected from a local stream that were spiked with CT.

Luminal Cholera Toxin Alters Motility in Isolated Guinea-Pig Jejunum via a Pathway Independent of 5-HT3 Receptors

Cholera toxin (CT) is well established to produce diarrhea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally. Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 or 12.5 μg/ml) in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by co-incubation with granisetron (5-HT3 antagonist, 1 μM), which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient-induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated.

A capacitive immunosensor for detection of cholera toxin

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0 × 10 −13 to 1.0 × 10 −10 M under optimized conditions. The limit of detection (LOD) was 1.0 × 10 −14 M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2 × 10 −12 M and a working range from 3.7 × 10 −11 to 2.9 × 10 −10 M whereas, the later had an LOD of 1.0 × 10 −11 M and a linearity ranging from 1.0 × 10 −9 to 1.0 × 10 −6 M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant ( K A) of 1.4 × 10 9 M −1 was estimated.

Detection of cholera toxin in seafood using a ganglioside-liposome immunoassay

Microbiological contamination of foods continues to be a major concern in public health. Biological toxins are one class of important contaminants that can cause various human diseases. Outbreaks related to contamination by biological toxins or toxin-producing microorganisms have made it extremely important to develop rapid (approximately 20min), sensitive and cost-effective analytical methods. This paper describes the development of a sensitive bioassay for the detection of cholera toxin (CT) in selected seafood samples, using ganglioside-incorporated liposomes. In this study, the assays were run with food samples spiked with various concentrations of CT. The limit of detection (LOD) increased by a factor of about 10-20 in most food samples, compared with the LOD in the buffer system previously reported. However, the LOD of toxins in food samples (8 × 10-3 × 10(3)fg/mL for CT) was still comparable to, or lower than, that previously reported for other assays. The results from this study demonstrate that the bioassays using ganglioside-liposomes can detect the toxin directly in the field screening of food samples rapidly, simply and reliably, without the need for complex instrumentation.

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi’s trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.

Manipulating FRET with Polymeric Vesicles: Development of a “Mix-and-Detect” Type Fluorescence Sensor for Bacterial Toxin

A simple "mix-and-detect" type of fluorescence sensor for cholera toxin (CT) is reported. The sensor consists of a BODIPY lipid dye and polydiacetylene (PDA) vesicles and utilizes the lipid insertion and FRET mechanism to offer a direct and fluorescence "turn-on" detection of the analyte. BODIPY conjugated GM1, dissolved in a Tris buffer through aggregate formation, demonstrated substantial fluorescence quenching with addition of PDA vesicle solution. The close proximity of the dye molecules to the conjugated chains as a result of lipid insertion enables energy transfer from dye to the polymer backbone, yielding the observed phenomenon. When CT is present, the binding of BO-GM1 to CT results in formation of a complex that prohibits it from membrane insertion, leading to the blocking of the quenching process. The fluorescence signal was found to be proportional to the CT concentration. The method is very simple and allows specific and sensitive detection of the protein toxin with just a few mixing steps. It can be further developed into a general sensing strategy for detection of other proteins with amplified FRET mechanism.

Biomimetic Particles for Isolation and Reconstitution of Receptor Function

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.

Differential Activities of Plant Polyphenols on the Binding and Internalization of Cholera Toxin in Vero Cells

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.

A kinetic study of analyte–receptor binding and dissociation for biosensor applications: a fractal analysis for cholera toxin and peptide–protein interactions

A fractal analysis is presented for (a) analyte–receptor binding and (b) binding and dissociation kinetics for biosensor applications. Examples analyzed include the binding of different concentrations of cholera toxin (CT) in solution to fluorophore-labeled ganglioside GM1 immobilized on a biomimetic membrane surface [16], and tyrosine kinase lck SH2 domain in solution to a phosphorylated peptide immobilized on a new cuvette-based surface plasmon resonance (SPR) instrument [14]. A single and a dual-fractal analysis is required to describe the binding kinetics for CT. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. A single and a dual-fractal analysis is required to describe the binding kinetics for tyrosine kinase lck SH2 domain in solution to the phosphorylated peptide. In this case, the dissociation kinetics may be described by a single-fractal analysis. Relationships are presented for the dissociation rate coefficient as a function of (a) the fractal dimension, D fd or the degree of heterogeneity that exists on the surface, and (b) as a function of the phosphotyrosine peptide, EPQY∗EEIPIYL concentration. When analyte–receptor dissociation is involved, an increase in the heterogeneity on the surface (increase in D fd) leads to an increase in the dissociation rate coefficient.

Cholera Toxin Suppresses Interleukin (IL)-12 Production and IL-12 Receptor β1 and β2 Chain Expression

Cholera toxin (CT) is a potent mucosal vaccine adjuvant, which has been shown to induce T helper cell type 2 (Th2) responses in systemic and mucosal tissues. We report that CT inhibits the production of interleukin (IL)-12, a major Th2 counterregulatory cytokine. IL-12 p70 production by stimulated human monocytes was inhibited by CT in a dose-dependent manner. This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect. CT also inhibited the production of IL-12 p70 by monocyte-derived dendritic cells, as well as the production of tumor necrosis factor alpha, but not IL-10, IL-6, or transforming growth factor (TGF)-beta1, by stimulated monocytes. The effects of CT were not due to autocrine production of IL-10, TGF-beta1, or prostaglandin E2. CT inhibited the production of IFN-gamma by anti-CD3-stimulated human peripheral blood mononuclear cell, due in part to suppression of IL-12 production, but also to the inhibition of expression of the beta1 and beta2 chains of the IL-12 receptor on T cells. In vivo, mice given CT before systemic challenge with lipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon gamma. These data demonstrate two novel mechanisms by which CT can inhibit Th1 immune responses, and help explain the ability of mucosally administered CT to enhance Th2-dependent immune responses.