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Increased intestinal permeability has been implicated in various pathologies, has various causes, and can develop during vigorous athletic training. Colostrum bovinum is a natural supplement with a wide range of supposed positive health effects, including reduction of intestine permeability. We assessed influence of colostrum supplementation on intestinal permeability related parameters in a group of 16 athletes during peak training for competition. This double-blind placebo-controlled study compared supplementation for 20 days with 500 mg of colostrum bovinum or placebo (whey). Gut permeability status was assayed by differential absorption of lactulose and mannitol (L/M test) and stool zonulin concentration. Baseline L/M tests found that six of the participants (75%) in the colostrum group had increased intestinal permeability. After supplementation, the test values were within the normal range and were significantly lower than at baseline. The colostrum group Δ values produced by comparing the post-intervention and baseline results were also significantly lower than the placebo group Δ values. The differences in stool zonulin concentration were smaller than those in the L/M test, but were significant when the Δ values due to intervention were compared between the colostrum group and the placebo group. Colostrum bovinum supplementation was safe and effective in decreasing of intestinal permeability in this series of athletes at increased risk of its elevation.

Cholera is a severe diarrheal disease caused by ingestion of food or water contaminated with pathogenic Vibrio cholerae . Treatment for cholera includes rehydration therapy and antibiotics to avert death and reduce bacterial burden to prevent rapid transmission of the disease. In addition, in Indian subcontinent, there is historical evidence of using plants for treating cholera. This study was designed to investigate the cholera toxin-inhibitory properties of phytochemicals sourced from Indian medicinal plants. For this, three reported genotypes of cholera toxin subunit B (ctxB) associated with 7PET V. cholerae O1 El Tor strains were used as targets in molecular docking. Analysis results showed strong binding affinities (≤−7.5 kcal/mol) for 298 out of 7,607 phytochemicals, with minor variations for the ctxB genotype-specific targets. Multiple phytochemicals from the same plants were identified with high binding affinities, e.g. , 101 from Morus alba, 24 from Citrus aurantium, 17 from Emblica officinalis, and 16 from Capsicum annuum . However, further analyses, including drug-likeness, pharmacokinetics, and toxicity, identified five promising phytochemical candidates, namely, Abyssinone V ( Azadirachta indica ), Diosgenin ( Achyranthes bidentata ), Yamogenin ( Borassus flabellifer ), and two other unnamed phytochemicals (one from Azadirachta indica and one from Morus alba ) for cholera toxin inhibition. Molecular dynamics simulation using YASARA and GROMACS showed structural stability of the ctxB-phytochemical complexes, while exhibiting adaptive rearrangements of ligand within the active binding sites of the proteins. In the simulations, MM-PBSA binding free energies showed a favorable total binding energy for the complexes. Per-residue energy decomposition analysis identified different highly contributing sets of amino acids to the binding energy with variation for both ctxB genotypes and phytochemicals, suggesting bacterial evolutionary changes may affect binding patterns of the drug candidates. This study suggests five inhibitors of cholera toxin with varying genotypes, which may have potential as an alternative medication for cholera.

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes. Gangliosides such as GM1 present in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. Here the authors show that the acyl chain structure of GM1 determines the establishment of nanodomains when actively clustered by actin, which depended on membrane cholesterol and phosphatidylserine or superimposed by the GM1-binding bacterial cholera toxin.

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval. To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS). We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower. Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations. ISRCTN registry identifier 11806026.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

Mucosal vaccination for COVID‐19 to boost preexisting though insufficient systemic and local/mucosal immunity remains an attractive prospect but there are currently no licensed mucosal vaccines against this infection. Here, using a plant expression system, we developed a novel mucosal vaccine platform for respiratory viruses and demonstrated its application in the context of SARS‐CoV‐2 infection. In addition to the antigen itself, the PCF (Platform CTB‐Fc) vaccine candidate incorporates two molecular adjuvants, the IgG‐Fc antibody fragment and the nontoxic cholera toxin B subunit (CTB), with the first targeting the vaccine to IgG receptors on antigen‐presenting cells, and the second providing local adjuvanticity by targeting cellular gangliosides in the mucosae. We demonstrated that this vaccine candidate is highly immunogenic in mice, inducing virus‐neutralising systemic and mucosal antibodies as well as tissue resident memory T cells in the lungs. We also demonstrated that SRBD‐PCF is recognised by immune cells from exposed or vaccinated individuals, and that circulating antibodies also bind to the antigen within the vaccine, forming immune complexes (IC). Finally, with a view of respiratory delivery, we demonstrated that the vaccine can be aerosolised without loss of material or biological activity, and that it is noncytotoxic and nonhaemolytic to human cells. Furthermore, we demonstrate that the plant expression system represents a suitable platform to produce these complex, multifunctional macromolecules capable of simultaneously binding to multiple targets. Our data strongly support the case for a safe, self‐adjuvanting mucosal COVID‐19 vaccine development, as means to boosting both systemic and mucosal immunity.

Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases.

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout (KO) of B3GNT5 , which encodes an enzyme required for synthesis of lacto and neolacto series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. KO of B3GALT5 causes increased production of fucosylated O -linked and N -linked glycoproteins and leads to increased CT binding and intoxication. KO of B3GNT5 in B3GALT5 -KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into the molecular determinants regulating CT sensitivity of host cells. Fucosylation of host glycoproteins is revealed to sensitize cells to cholera intoxication while fucosylation of host glycolipids is protective. The glycosyltransferases encoded by B3GALT5 and B3GNT5 are identified as key regulators of this phenomenon.