Diazepam Impairs Innate and Adaptive Immune Responses and Ameliorates Experimental Autoimmune Encephalomyelitis.

Currently there is increasing attention on the modulatory effects of benzodiazepines on the immune system. Here, we evaluate how Diazepam (DZ) affects both innate and adaptive immunity. We observed that treatment with DZ and Lipopolysaccharide (LPS) on macrophages or dendritic cells (DCs) induced a defective secretion of IL-12, TNF-α, IL-6 and a lesser expression of classical activation markers as NO production and CD40 in comparison with LPS condition. More importantly, mice pre-treated with DZ and then challenged to LPS induced-septic shock showed reduced death. The DZ treatment shifted the LPS-induced pro-inflammatory cytokine production of peritoneal cells (PCs) to an anti-inflammatory profile commanded by IL-10. In agreement with this, DZ treatment prevented LPS-induced DC ability to initiate allogeneic Th1 and Th17 responses in vitro when compared with LPS-matured DC. Since these inflammatory responses are the key in the development of the experimental autoimmune encephalomyelitis (EAE), we treated EAE mice preventively with DZ. Mice that received DZ showed amelioration of clinical signs and immunological parameters of the disease. Additionally, DZ reduced the release of IFN-γ and IL-17 by splenocytes from untreated sick mice in vitro. For this reason, we decided to treat diseased mice therapeutically with DZ when they reached the clinical score of 1. Most importantly, this treatment ameliorated clinical signs, reduced the MOG-specific inflammatory cytokine production and prevented axonal damage. Altogether, these results indicate that DZ is a potent immunomodulator capable of controlling undesired innate and adaptive immune responses, both at the beginning of these responses and also once they have started.

MeCP2 Deficiency Disrupts Kainate-Induced Presynaptic Plasticity in the Mossy Fiber Projections in the Hippocampus.

Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events in vivo in the hippocampus of MeCP2 mutant mice. While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.

Diazepam treatment reduces inflammatory cells and mediators in the central nervous system of rats with experimental autoimmune encephalomyelitis.

Benzodiazepines are psychoactive drugs and some of them also affect immune cells. We here characterized the inflammatory and infiltrating immune cells in the central nervous system (CNS) during the acute phase of experimental autoimmune encephalomyelitis (EAE) in animals treated with Diazepam. Also, we evaluated the expression of Translocator Protein (18kDa) (TSPO), which is a biomarker of neuroinflammatory diseases. The results indicate that Diazepam exerts protective effects on EAE development, decreasing the incidence of the disease and reducing the number of inflammatory cells in CNS, with a concomitant decrease of TSPO levels in brain tissue and CNS inflammatory CD11b+ cells.

In vivo immunomodulatory effect of the lectin from edible mushroom Agaricus bisporus.

Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.

GABAergic Agonists Modulate the Glutamate Release from Frontal Cortex Synaptosomes of Rats with Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that mimics many of the clinical and pathological features of multiple sclerosis. We have previously described a significant diminution in the GABAergic regulation of glutamate release from synaptosomes of EAE rats isolated during the acute stage of the disease. In order to explore the possible metabolic pathways responsible for this alteration, in this work we evaluate the direct effect of different GABAergic agonists on the glutamate release and concomitant synapsin I phosphorylation in synaptosomes from the frontal cortex of control and EAE animals. The results show that GABA as well as the GABA receptor agonists Muscimol (GABAA agonist) and Baclofen (GABAB agonist) caused a decrease in glutamate release in control rats paralleled by a similar reduction in synapsin I phosphorylation. Meanwhile synaptosomes from EAE animals are responsive only to Baclofen with respect to nontreated EAE synaptosomes, since glutamate release from the synaptosomes treated with Muscimol was similar to that observed in EAE rat synaptosomes which was already reduced as consequence of the disease. In the case of the benzodiazepines Diazepam and Clonazepam (GABAA allosteric agonists), both of them induced a reduction in glutamate release in synaptosomes from the CFA rats, effect that was only observed in synaptosomes of EAE rats treated with Clonazepam. In all cases both benzodiazepines showed a higher effect on synapsin I phosphorylation than in glutamate release. These results indicate that the extent of GABAergic modulation of presynaptic terminals depends on the type of agonist employed and this regulation is altered in the frontal cortex during the acute phase of EAE with respect to control animals.

Diazepam Inhibits Proliferation of Lymph Node Cells Isolated from Rats with Experimental Autoimmune Encephalomyelitis.

OBJECTIVE: Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to human multiple sclerosis involving peripheral activation of autoreactive T cells which infiltrate the central nervous system and react to self antigens leading to damage. In previous studies, we have demonstrated that treatment with diazepam decreases the incidence and histological signs associated with the disease and diminishes immunological responses. The aim of the present work was to evaluate direct effects of diazepam on isolated T cells involved in immune responses during the development of EAE. METHODS: Animals were sensitized with whole myelin to induce EAE and sacrificed during the acute phase of the disease. In mononuclear cells isolated from popliteal lymph nodes, cell viability, apoptosis induction, proliferation and cytokine production were evaluated. RESULTS: Diazepam did not have a toxic or proapoptotic effect on the cells, at least up to the concentration of 25 µM, but proliferation, CD8+ T-cell activation and proinflammatory cytokine production were dose-dependently decreased. CONCLUSIONS: Diazepam has a direct inhibitory effect on the proliferation and activation of T lymphocytes isolated from the main lymphoid organ involved in disease onset and this could be one of the mechanisms that contribute to the beneficial effect previously observed with diazepam in vivo during EAE development.

Glutamate release machinery is altered in the frontal cortex of rats with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Novel antibodies reacting with two neighboring gangliosides are induced in rabbits immunized with bovine brain gangliosides.

Immunization of rabbits with bovine brain gangliosides induced an experimental neuropathy, with clinical signs resembling Guillain-Barré syndrome. All the immunized animals developed immunoglobulin G immunoreactivity to GM1 ganglioside. In a few (4 of 27) animals, an additional anti-ganglioside antibody population showing an unusual binding behavior was detected. Enzyme-linked immunosorbent assay and thin-layer chromatography immunostaining analyses showed that the binding of these unusual antibodies required the presence of two co-localized gangliosides. Maximal interaction was observed to a mixture of GM1 and GD1b, but the antibodies also showed "density-dependent" binding to GD1b. The antibodies were purified by affinity chromatography and displayed the ability to target antigens in biological membranes (rat synaptosomes).

Diphenyl diselenide-modulation of macrophage activation: down-regulation of classical and alternative activation markers.

Diphenyl diselenide (PhSe)(2), a simple organoselenium compound, possesses interesting pharmacological properties that are under extensive research. As macrophages respond to microenvironmental stimuli and can display activities engaged in the initiation and the resolution of inflammation, in the present report we describe the ability of (PhSe)(2) to modulate the macrophage activation. Our data indicate that (PhSe)(2) could inhibit the NO production in a dose-dependent fashion in peritoneal macrophages activated by LPS or treated with vehicle alone. We could demonstrate that this effect correlated with a reduction in the expression of the inducible NO synthase in (PhSe)(2)-treated cells. Furthermore, (PhSe)(2) suppressed the production of reactive oxygen species, diminished the activity of the arginase enzyme, and the accumulation of nitrotyrosine modified proteins in LPS-stimulated macrophages. This compound also diminished the antigen presentation capacity of classically activated macrophages, as it reduced MHCII and CD86 expression. In addition, (PhSe)(2) modulated the alternative activation phenotype of macrophages. Dexamethasone-activated macrophages presented higher production of IL-10 and CD206, which were both down-regulated by the addition of (PhSe)(2). These results suggest that (PhSe)(2) possesses antioxidant and anti-inflammatory activities in classically-activated macrophages. We could demonstrate that (PhSe)(2) can be also utilized to modulate the alternative activation phenotype of macrophages.

Synapsin peptide fused to E. coli heat-labile toxin B subunit induces regulatory T cells and modulates cytokine balance in experimental autoimmune encephalomyelitis.

We previously found that the preventive oral administration of a hybrid consisting of the C domain of synapsin and the B subunit of E. coli heat-labile enterotoxin (LTBSC) efficiently suppresses experimental autoimmune encephalomyelitis (EAE) development in rats. We investigated the effect of LTBSC on cytokine expression and on regulatory T (Treg) cells in rats with myelin induced EAE. LTBSC treatment increased the frequency of CD4(+)FoxP3(+) Treg cells in lymph nodes prior to challenge and in the EAE acute stage. LTBSC also up-regulated the expression of anti-inflammatory Th2/Th3 cytokines and diminished myelin basic protein-specific Th1 and Th17 cell responses in lymph nodes. CD4(+)CD25(+) Treg cells from LTBSC treated rats showed stronger suppressive properties than Treg cells from controls in vitro. Our observations indicate that LTBSC is a useful agent for modulating the autoimmune responses in EAE.

Effect of diphenyl diselenide on the development of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory and demyelinating disease of the central nervous system with clinical and pathological similarities with multiple sclerosis. The oxidative stress is one of the major mediators of demyelination and axonal damage in both, multiple sclerosis and EAE. Therefore, several studies are being performed to assess whether treatment with antioxidants prevents the progression of these diseases. Some organic forms of selenium that exhibit glutathione peroxidase-like activity have become good candidates for disease prevention and therapy since they catalytically remove oxidative stressors. Particularly, diphenyl diselenide ((PhSe)(2)) exerts antioxidant activity and has neuroprotective effects in several systems. The aim of the present study was to prove the therapeutic activity of (PhSe)(2) on the development of EAE. Intraperitoneally administered (PhSe)(2) (1-25 µmoles/kg body weight/day) reduced the incidence of the disease but was also deleterious for the animals. Conversely, (PhSe)(2) given orally (80 µmoles/kg body weight/day) produced a significant inhibition of EAE without any toxic effect. In addition, there was a reduction of the characteristic histological alterations and a diminished in vivo and in vitro T-cell response against the encephalitogenic myelin basic protein. These results show an effective suppression of the autoimmune response that could be the base for future developments of successful antioxidants therapies in EAE as well as in multiple sclerosis.

Synapsin-induced proliferation of T-cell lines against myelin basic protein obtained from rats with experimental autoimmune encephalomyelitis.

We have previously described that antibodies and T cells against myelin basic protein (MBP) rose under conditions to induce acute experimental autoimmune encephalomyelitis (EAE) bind other proteins present in the synaptosomal fraction, some of them identified as synapsin I. The aim of this study was to evaluate whether anti-MBP T-cell lines can be also activated by synapsin. The analysis of rat anti-MBP T-cell lines cultured with each antigen showed that these cells responded also to purified rat synapsin and to the amino terminal portion of this protein. This recognition originated a proliferative response with a concomitant pattern of cytokine secretion similar to that induced by MBP itself implicating that this recognition would be mediated by the T-cell receptor. On the other hand, anti-synapsin T-cell lines were not capable of responding to MBP stimulation. Therefore, the immunological cross-reactivity between both proteins occurs only in one direction and these cross-reactive cells would be elicited only in animals sensitized with MBP. A possible implication of immunological agents against MBP cross-reactive with extra-myelin proteins in the process of EAE is considered.

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting.

Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens for immunotargeting purposes.

Protective effect of a synapsin peptide genetically fused to the B subunit of Escherichia coli heat-labile enterotoxin in rat autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to multiple sclerosis that requires the activation of auto reactive T cells that infiltrate the central nervous system. In previous studies we have shown that intraperitoneal administration of synaptosomal antigens could suppress EAE. Herein we examined the effect in this animal model of a fusion protein comprising the C domain of synapsin Ia and the B subunit of Escherichia coli heat-labile enterotoxin (LTBSC). Oral administration to rats of low amounts of LTBSC induced immunological systemic tolerance to the encephalitogenic myelin basic protein. Treatment with LTBSC prior to EAE induction diminished disease incidence, DTH reaction to myelin basic protein, and central nervous system inflammation. LTBSC treatment also reduced the specific T-cell proliferative response to myelin basic protein, decreased nitric oxide production, and augmented arginase activity by peritoneal macrophages. All animals challenged for EAE developed antibody response specific for myelin basic protein, but rats treated with LTBSC showed a lower IgG2b/IgG1 ratio, indicating a shift to a Th2-type milieu. The data presented here suggest that well-conserved synapsin peptides conjugated to the B subunit of enterotoxins from the cholera toxin family have a protective role and provide a potential therapeutic tool for intervention in EAE as well as in multiple sclerosis.

Inhibition of Ca(2+)-dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis.

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca(2+)-dependent but not the Ca(2+)-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca(2+) using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca(2+) influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca(2+)-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca(2+).

Nitric oxide mediates the suppressive effect of testosterone on cell proliferative response to myelin basic protein.

This study assessed whether the in vitro effect of testosterone on the proliferative response of mononuclear cells to myelin basic protein (MBP) could be mediated by nitric oxide (NO). Testosterone but not cholesterol supplementation specifically suppressed the proliferative response of rat mononuclear cells to MBP and in parallel increased the NO level. NG-monomethyl 1-l-arginine, an inhibitor of NO synthesis, reverted the suppression of the testosterone-induced proliferative response to MBP. These results indicate that changes in the production of NO by testosterone are able to alter the specific T cell proliferation induced by the encephalitogenic MBP and in this way; it could be one of the molecular mechanisms that modulate the development of experimental autoimmune encephalomyelitis (EAE).

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide.

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.

Endogenous thyrocyte-produced nitric oxide inhibits iodide uptake and thyroid-specific gene expression in FRTL-5 thyroid cells.

Nitric oxide (NO) is a free radical that mediates a wide array of cell functions. It is generated from l-arginine by NO-synthase (NOS). Expression of NOS isoforms has been demonstrated in thyroid cells. Previous reports indicated that NO donors induce dedifferentiation in thyrocytes. However, the functional significance of endogenous thyrocyte-produced NO has not been explored. This work aimed to study the influence of endogenous NO on parameters of thyroid cell function and differentiation in FRTL-5 cells. We observed that treatment with the NOS inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), increased the TSH-stimulated iodide uptake. The TSH-induced sodium iodide symporter (NIS) and thyroglobulin (TG) mRNA expressions were increased after incubation with L-NAME. In transient transfection assays, TSH-stimulated transcriptional activities of NIS and TG promoters were increased by L-NAME. An increment of the TSH-stimulated cell proliferation was observed after NOS inhibition. Similar results were obtained when the action of another NOS inhibitor, N(g)-monomethyl-L-arginine, was analysed for most of these studies. The production of NO, which was not detectable in basal conditions, was increased by TSH. Our data provide strong evidence that endogenous NO could act as a negative signal for TSH-stimulated iodide uptake and thyroid-specific gene expression as well as proliferation in thyrocytes. These findings reveal a possible new inhibitory pathway in the regulation of thyroid cell function.

Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells.

Human colon adenocarcinoma cells (HT29-ATCC) and the clone HT29-5F7 were cultured under conditions that differentiate cells to a polarized intestinal phenotype. Differentiated cells showed the presence of junctional complexes and intercellular lumina bordered by microvilli. Intestinal brush border hydrolase activities (sucrase, aminopeptidase N, lactase and maltase) were detected mainly in differentiated HT29-ATCC cells compared with the differentiated clone, HT29-5F7. The presence of non-GM1 receptors of Escherichia coli heat-labile enterotoxin (LT-I) on both types of differentiated HT29 cells was indicated by the inability of cholera toxin B subunit to block LT-I binding to the cells. Binding of LT-I to cells, when GM1 was blocked by the cholera toxin B subunit, was characterized by an increased number of LT-I receptors with respect to undifferentiated control cells. Moreover, both types of differentiated cells accumulated higher amounts of cyclic AMP in response to LT-I than undifferentiated cells. Helix pomatia lectin inhibited the binding of LT-I to cells and the subsequent production of cyclic AMP. LT-I recognized blood group A-active glycosphingolipids as functional receptors in both HT29 cell lines and the active pro-sucrase form of the glycoprotein carrying A-blood group activity present in HT29-ATCC cells. These results strongly suggest that LT-I can elicit an enhanced functional response using blood group A-active glycoconjugates as additional receptors on polarized intestinal epithelial cells.