GM1 Receptor Research Articles

Crucial role of carbon monoxide as a regulator of diarrhea caused by cholera toxin: Evidence of direct interaction with toxin

The present study evaluated the role of heme oxygenase 1 (HO-1)/carbon monoxide (CO) pathway in the cholera toxin-induced diarrhea and its possible action mechanism. The pharmacological modulation with CORM-2 (a CO donor) or Hemin (a HO-1 inducer) decreased the intestinal fluid secretion and Cl− efflux, altered by cholera toxin. In contrast, ZnPP (a HO-1 inhibitor) reversed the antisecretory effect of Hemin and potentiated cholera toxin-induced intestinal secretion. Moreover, CORM-2 also prevented the alteration of intestinal epithelial architecture and local vascular permeability promoted by cholera toxin. The intestinal absorption was not altered by any of the pharmacological modulators. Cholera toxin inoculation also increased HO-1 immunoreactivity and bilirubin levels, a possible protective physiological response. Finally, using fluorometric technique, ELISA assay and molecular docking simulations, we show evidence that CO directly interacts with cholera toxin, forming a complex that affects its binding to GM1 receptor, which help explain the antisecretory effect. Thus, CO is an essential molecule for protection against choleric diarrhea and suggests its use as a possible therapeutic tool.

Exploring the Role of Mentha in Gut Microbiota: A Modern Perspective of an Ancient Herb.

Gut microbiota includes the microbes present in the gut; these microbes are an essential component in maintaining a healthy gut. Gut microbiota has a wide range of functions, including effects on colonization, pathogen resistance, intestinal epithelial maintenance, metabolizing food and pharmaceutical chemicals, and influencing immunological functions. Every disease associated with the gut starts due to a disbalance in the composition of the gut microorganisms and can be managed by balancing the composition of gut bacteria using various herbal remedies. Mentha herbs are a variety of perennial herbs that are grown commercially in various parts of the world. Mentha is a potent herb that shows anticholinergic action and can block PGE2 and GM1 receptors and interact with cholera toxins; it is used traditionally in different systems of medicines to treat various gastrointestinal diseases associated with the gut. Mentha herbs have potent bactericidal, viricidal, and fungicidal properties. Mentha has been used to cure stomach and digestion issues as well as to treat a variety of disorders. This review article summarizes diseases associated with the gut, the composition, and function of gut microbiota, and mentha's effectiveness along with its extraction methods and traditional uses. Research findings have revealed mentha to be an essential source against a wide range of diseases, especially gastrointestinal disorders. From its ayurvedic claims to its present use, it has been confirmed to be clinically effective for human health. Mentha-based products are marketed in the form of oils, capsules, powder, etc. The present work describes the mechanism of action along with the bioactives present in this well-known herb from Ayurveda.

Affordable oral proinsulin bioencapsulated in plant cells regulates blood sugar levels similar to natural insulin

Diabetes Mellitus is a silent epidemic affecting >500 million, which claimed 6.7 million lives in 2021, a projected increase of >670% in <20 years old in the next two decades but insulin is unaffordable for the large majority of the globe. Therefore, we engineered proinsulin in plant cells to facilitate oral delivery. Stability of the proinsulin gene and expression in subsequent generations, after removal of the antibiotic-resistance gene, was confirmed using PCR, Southern and western blots. Proinsulin expression was high (up to 12 mg/g DW or 47.5% of total leaf protein), stable up to one year after storage of freeze-dried plant cells at ambient temperature and met FDA regulatory requirements of uniformity, moisture content and bioburden. GM1 receptor binding, required for uptake via gut epithelial cells was confirmed by pentameric assembly of CTB-Proinsulin. IP insulin injections (without C peptide) in STZ mice rapidly decreased blood glucose level leading to transient hypoglycemia, followed by hepatic glucose compensation. On the other hand, other than the 15-min lag period of oral proinsulin (transit time required to reach the gut), the kinetics of blood sugar regulation of oral CTB-Proinsulin in STZ mice was very similar to naturally secreted insulin in healthy mice (both contain C-peptide), without rapid decrease or hypoglycemia. Elimination of expensive fermentation, purification and cold storage/transportation should reduce cost and increase other health benefits of plant fibers. The recent approval of plant cell delivery of therapeutic proteins by FDA and approval of CTB-ACE2 for phase I/II human clinical studies augur well for advancing oral proinsulin to the clinic.

Anti-Cholera toxin activity of selected polyphenols from Careya arborea, Punica granatum, and Psidium guajava.

Careya arborea, Punica granatum, and Psidium guajava are traditionally used to treat diarrheal diseases in India and were reported to show anti-Cholera toxin activity from our earlier studies. As polyphenols are reported to neutralize Cholera toxin (CT), the present study investigated the inhibitory activity of selected polyphenols from these plants against CTB binding to GM1 receptor using in silico, in vitro, and in vivo approaches. Molecular modelling approach was used to investigate the intermolecular interactions of selected 20 polyphenolic compounds from three plants with CT using DOCK6. Based on intermolecular interactions, two phenolic acids, Ellagic acid (EA) and Chlorogenic acid (CHL); two flavonoids, Rutin (RTN) and Phloridzin (PHD) were selected along with their respective standards, Gallic acid (GA) and Quercetrin (QRTN). The stability of docked complexes was corroborated using molecular dynamics simulation. Furthermore, in vitro inhibitory activity of six compounds against CT was assessed using GM1 ELISA and cAMP assay. EA and CHL that showed prominent activity against CT in in vitro assays were investigated for their neutralizing activity against CT-induced fluid accumulation and histopathological changes in adult mouse. The molecular modelling study revealed significant structural stability of the CT-EA, CT-CHL, and CT-PHD complexes compared to their respective controls. All the selected six compounds significantly reduced CT-induced cAMP levels, whereas EA, CHL, and PHD exhibited > 50% binding inhibition of CT to GM1. The EA and CHL that showed prominent neutralization activity against CT from in vitro studies, also significantly decreased CT-induced fluid accumulation and histopathological changes in adult mouse. Our study identified bioactive compounds from these three plants against CT-induced diarrhea.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Both LTA and LTB Subunits Are Equally Important to Heat-Labile Enterotoxin (LT)-Enhanced Bacterial Adherence.

There is increasing evidence indicating that the production of heat-labile enterotoxin (LT) enhances bacterial adherence within in vitro and in vivo models. However, which subunit plays the main role, and the precise regulatory mechanisms remain unclear. To further elucidate the contribution of the A subunit of LT (LTA) and the B subunit of LT (LTB) in LT-enhanced bacterial adherence, we generated several LT mutants where their ADP-ribosylation activity or GM1 binding ability was impaired and evaluated their abilities to enhance the two LT-deficient E. coli strains (1836-2 and EcNc) adherence. Our results showed that the two LT-deficient strains, expressing either the native LT or LT derivatives, had a significantly greater number of adhesions to host cells than the parent strains. The adherence abilities of strains expressing the LT mutants were significantly reduced compared with the strains expressing the native LT. Moreover, E. coli 1836-2 and EcNc strains when exogenously supplied with cyclic AMP (cAMP) highly up-regulated the adhesion molecules expression and improved their adherence abilities. Ganglioside GM1, the receptor for LTB subunit, is enriched in lipid rafts. The results showed that deletion of cholesterol from cells also significantly decreased the ability of LT to enhance bacterial adherence. Overall, our data indicated that both subunits are equally responsible for LT-enhanced bacterial adherence, the LTA subunit contributes to this process mainly by increasing bacterial adhesion molecules expression, while LTB subunit mainly by mediating the initial interaction with the GM1 receptors of host cells.

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2

Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)—delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2.

In Vitro and In Vivo Inhibitory Activities of Selected Traditional Medicinal Plants against Toxin-Induced Cyto- and Entero- Toxicities in Cholera.

Careya arborea, Punica granatum, Psidium guajava, Holarrhena antidysenterica, Aegle marmelos, and Piper longum are commonly used traditional medicines against diarrhoeal diseases in India. This study investigated the inhibitory activity of these plants against cytotoxicity and enterotoxicity induced by toxins secreted by Vibrio cholerae. Cholera toxin (CT) and non-membrane damaging cytotoxin (NMDCY) in cell free culture filtrate (CFCF) of V. cholerae were quantified using GM1 ELISA and cell-based assays, respectively. Hydro-alcoholic extracts of these plants and lyophilized juice of P. granatum were tested against CT-induced elevation of cAMP levels in CHO cell line, binding of CT to ganglioside GM1 receptor and NMDCY-induced cytotoxicity. Significant reduction of cAMP levels in CFCF treated CHO cell line was observed for all extracts except P. longum. C. arborea, P. granatum, H. antidysenterica and A. marmelos showed >50% binding inhibition of CT to GM1 receptor. C. arborea, P. granatum, and P. guajava effectively decreased cytotoxicity and morphological alterations caused by NMDCY in CHO cell line. Further, the efficacy of these three plants against CFCF-induced enterotoxicity was seen in adult mice ligated-ileal loop model as evidenced by decrease in volume of fluid accumulation, cAMP levels in ligated-ileal tissues, and histopathological changes in intestinal mucosa. Therefore, these plants can be further validated for their clinical use against cholera.

Ingenious Action of Vibrio cholerae Neuraminidase Recruiting Additional GM1 Cholera Toxin Receptors for Primary Human Colon Epithelial Cells.

For five decades it has been known that the pentamer of B subunits (choleragenoid) of the cholera toxin (CT) of Vibrio cholerae binds with high preference to the ganglioside GM1 (II3Neu5Ac-Gg4Cer). However, the exact structures of CT-binding GM1 lipoforms of primary human colon epithelial cells (pHCoEpiCs) have not yet been described in detail. The same holds true for generating further GM1 receptor molecules from higher sialylated gangliosides with a GM1 core through the neuraminidase of V. cholerae. To avoid the artificial incorporation of exogenous gangliosides from animal serum harboring GM1 and higher sialylated ganglio-series gangliosides, pHCoEpiCs were cultured in serum-free medium. Thin-layer chromatography overlay binding assays using a choleragenoid combined with electrospray ionization mass spectrometry revealed GM1 lipoforms with sphingosine (d18:1) as the sole sphingoid base linked to C14:0, C16:0, C18:0 or C20:0 fatty acyl chains forming the ceramide (Cer) moieties of the main choleragenoid-binding GM1 species. Desialylation of GD1a (IV3Neu5Ac,II3Neu5Ac-Gg4Cer) and GT1b (IV3Neu5Ac,II3(Neu5Ac)2-Gg4Cer) of pHCoEpiCs by V. cholerae neuraminidase was observed. GD1a-derived GM1 species with stable sphingosine (d18:1) and saturated fatty acyl chains varying in chain length from C16:0 up to C22:0 could be identified, indicating the ingenious interplay between CT and the neuraminidase of V. cholerae recruiting additional GM1 receptors of pHCoEpiCs.

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

Binding of Cholera Toxin B-Subunit to a Ganglioside GM1-Functionalized PEG-Tethered Lipid Membrane.

We report neutron reflectometry (NR) studies of polyethylene glycol (PEG)-tethered model lipid membranes at the solid-liquid interface and of cholera toxin's B-subunit (CTxB) binding to tethered membranes containing ganglioside GM1 receptors. First, tethered polymer brushes were formed by grafting silane-functionalized PEG lipopolymers to quartz from solution. Subsequent deposition of lipids by Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) resulted in a tethered bilayer structure separated from the solid support by a hydrated PEG layer. NR revealed that the tethers formed a highly hydrated polymer brush, uniformly separating the bilayer from the underlying solid substrate. Further, the lipid bilayer did not significantly perturb the brush's conformation relative to a free brush. Biological functionality of the tethered bilayers was verified by interacting CTxB, with ganglioside GM1 receptors incorporated into the bilayer. The surface coverage of CTxB bound to the lipid membrane, θCTB= 0.58 ± 0.08, was consistent with the coverage predicted for random sequential absorption, and toxin binding did not impact the membrane conformation.

Evaluation of the Role of Probiotics As a New Strategy to Eliminate Microbial Toxins: a Review.

Probiotics are living microorganisms that have favorable effects on human and animal health. The most usual types of microorganisms recruited as probiotics are lactic acid bacteria (LAB) and bifidobacteria. To date, numerous utilizations of probiotics have been reported. In this paper, it is suggested that probiotic bacteria can be recruited to remove and degrade different types of toxins such as mycotoxins and algal toxins that damage host tissues and the immune system causing local and systemic infections. These microorganisms can remove toxins by disrupting, changing the permeability of the plasma membrane, producing metabolites, inhibiting the protein translation, hindering the binding to GTP binding proteins to GM1 receptors, or by preventing the interaction between toxins and adhesions. Here, we intend to review the mechanisms that probiotic bacteria use to eliminate and degrade microbial toxins.

Cholera Toxin as a Probe for Membrane Biology.

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.

Anti-diarrheal therapeutic potential of diminazene aceturate stimulation of the ACE II/Ang-(1–7)/Mas receptor axis in mice: A trial study

The angiotensin (Ang) II converting enzyme (ACE II) pathway has recently been shown to be associated with several beneficial effects on the body, especially on the cardiac system and gastrointestinal tract. ACE II is responsible for converting Ang II into the active peptide Ang-(1–7), which in turn binds to a metabotropic receptor, the Mas receptor (MasR). Recent studies have demonstrated that Diminazene Aceturate (DIZE), a trypanosomicide used in animals, activates the ACE II pathway. In this study, we aimed to evaluate the antidiarrheal effects promoted by the administration of DIZE to activate the ACE II/Ang-(1–7)/MasR axis in induced diarrhea mice models. The results show that activation of the ACE II pathway exerts antidiarrheal effects that reduce total diarrheal stools and enteropooling. In addition, it increases Na+/K+-ATPase activity and reduces gastrointestinal transit and thus inhibits contractions of intestinal smooth muscle; decreases transepithelial electrical resistance, epithelial permeability, PGE2-induced diarrhea, and proinflammatory cytokines; and increases anti-inflammatory cytokines. Enzyme-linked immunosorbent assay (ELISA) demonstrated that DIZE, when activating the ACE II/Ang-(1–7)/MasR axis, can still interact with GM1 receptors, which reduces cholera toxin-induced diarrhea. Therefore, activation of the ACE II/Ang-(1–7)/MasR axis can be an important pharmacological target for the treatment of diarrheal diseases.

Alternative Approaches to Oral Tolerance Induction to Factor FVIII

A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e &amp;gt;5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (&amp;lt;5 BU/ml). Interestingly, inhibitor formation in anti-CD3 treated mice showed a unique pattern, with two distinct outcomes. One subset of animals developed inhibitors of 0-15 BU/ml (on average 2.5-fold lower than control group), while the second subset showed increased titers of 41-89 BU/ml. There were no animals with intermediate titers. In general, FVIII-specific IgG titers followed the same patterns as inhibitor responses. When the 2 approaches (plant cells and anti-CD3) were combined, 50% of mice developed &amp;lt;5 BU/ml (as compared to 36% for anti-CD3 only). However, this did not represent an improvement over giving lettuce cells only. Finally, we tested anti-CD3 Fab fragment in place of FL anti-CD3. While a daily dose of 5 µg anti-CD3 Fab/mouse was unsuccessful in suppressing inhibitor formation, a dose of 0.5 µg marginally decrease titers. In summary, oral anti-CD3 delivery was not as effective in reducing inhibitor formation as plant cells expressing CTB-FVIII antigen fusions. While FL anti-CD3 showed promise in a subset of animals, it also posed a risk of an increased response in others, which complicates development of this approach. Distinct from all other treatment groups, lettuce-fed mice showed elevated frequencies in Foxp3+ Treg, a reduction in Teff cells, and the highest increase in LAP+ Treg. Further studies on induced CD4+LAP+FoxP3- Treg cells from lettuce-fed mice showed high expression of ICOS, CD69 and Ki-67, but not CTLA-4. Among the different subsets of Treg, LAP+ T cell contained the highest proportion of IL-10 expressing cells, and oral antigen delivery induced IL-10+LAP+ Treg, which are likely key to the suppression of the immune response against FVIII. Disclosures Daniell: Takeda Pharmaceuticals: Patents &amp; Royalties. Herzog:Takeda Pharmaceuticals: Patents &amp; Royalties.

SV40 Polyomavirus Activates the Ras-MAPK Signaling Pathway for Vacuolization, Cell Death, and Virus Release.

Polyomaviruses are a family of small, non-enveloped DNA viruses that can cause severe disease in immunosuppressed individuals. Studies with SV40, a well-studied model polyomavirus, have revealed the role of host proteins in polyomavirus entry and trafficking to the nucleus, in viral transcription and DNA replication, and in cell transformation. In contrast, little is known about host factors or cellular signaling pathways involved in the late steps of productive infection leading to release of progeny polyomaviruses. We previously showed that cytoplasmic vacuolization, a characteristic late cytopathic effect of SV40 infection, depends on the specific interaction between the major viral capsid protein VP1 and its cell surface ganglioside receptor GM1. Here, we show that, late during infection, SV40 activates a signaling cascade in permissive monkey CV-1 cells involving Ras, Rac1, MKK4, and JNK to stimulate SV40-specific cytoplasmic vacuolization and subsequent cell lysis and virus release. Inhibition of individual components of this signaling pathway inhibits vacuolization, lysis, and virus release, even though high-level intracellular virus replication occurs. Identification of this pathway for SV40-induced vacuolization and virus release provides new insights into the late steps of non-enveloped virus infection.

Autoantibody Biomarkers for Basal Ganglia Encephalitis in Sydenham Chorea and Pediatric Autoimmune Neuropsychiatric Disorder Associated With Streptococcal Infections.

Movement, behavioral, and neuropsychiatric disorders in children have been linked to infections and a group of anti-neuronal autoantibodies, implying dopamine receptor-mediated encephalitis within the basal ganglia. The purpose of this study was to determine if anti-neuronal biomarkers, when used as a group, confirmed the acute disease in Sydenham chorea (SC) and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). IgG autoantibodies against four neuronal autoantigens (tubulin, lysoganglioside GM1, and dopamine receptors D1 and D2) were detected in SC sera (N=8), sera and/or cerebrospinal fluid (CSF) from two groups of PANDAS cases (N=25 first group and N=35 second group), sera from Tourette's syndrome (N=18), obsessive-compulsive disorder (N=25), attention deficit hyperactivity disorder (N=18), and healthy controls (N=28) by direct enzyme-linked immunosorbent assay (ELISA). IgG specific for neuronal autoantigens was significantly elevated during the acute symptomatic phase, and the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) pathway was significantly elevated in human neuronal cells. Five assays confirmed the disease in SC and in two groups of children with PANDAS. In 35 acute onset PANDAS patients, 32 sera (91.4%) were positive for one or more of the anti-neuronal autoantibodies compared with 9 of 28 healthy controls (32.1%, p<0.0001). Importantly, CSF of 32 (91.4%) PANDAS patients had one or more detectable anti-neuronal autoantibody titers and CaMKII activation. Among healthy control subjects with elevated serum autoantibody titers for individual antigens, none (0%) were positively associated with elevated positive CaMKII activation, which was a striking contrast to the sera of PANDAS subjects, who had 76–89% positive association with elevated individual autoantibody titers and positive CaMKII activity. At 6 months follow-up, symptoms improved for more than 80% of PANDAS subjects, and serum autoantibody titers also significantly decreased. Results reported herein and previously published studies in our laboratory suggest the antibody biomarkers may be a useful adjunct to clinical diagnosis of SC, PANDAS, and related disorders and are the first known group of autoantibodies detecting dopamine receptor-mediated encephalitis in children.

Antidiarrheal activity of farnesol in rodents: Pharmacological actions and molecular docking

Diarrhea is a condition in which the individual has about three or more daily bowel movements, followed by changes in stool consistency. It is currently considered as one of the worst public health problems due to the number of cases and deaths involved and difficulty of treatment. Thus, the use of natural products is an alternative for new treatments. Among these possibilities is Farnesol (C15H26O), a sesquiterpene found in different herbal species that has known biological activities. The objective of this study was to evaluate the antidiarrheal activity of Farnesol (FOH). Initially, FOH activity was evaluated in models of diarrhea and enteropooling induced by castor oil and PGE2. To evaluate motility, the opioid and cholinergic pathways were studied. In addition, the effect of FOH was investigated in the secretion model in intestinal loops treated with cholera toxin. FOH was evaluated for the ability to absorb fluids in intestinal loops and interact with GM1 receptors using the ELISA method and molecular docking. The dose of 50 mg/kg of FOH showed the best results in all antidiarrheal activity tests with castor oil and PGE2, being considered as the standard dose, reducing motility by anticholinergic mechanisms. There was a reduction in fluid secretion when FOH interacted directly with GM1 receptors; cholera toxin and molecular docking showed strong interaction between farnesol and these targets. In view of the results presented, the antidiarrheal activity occurs through anticholinergic, anti-inflammatory and anti-secretory action, making farnesol a potential candidate for the development of a new drug to treat diarrheal diseases.

Biopolymer Extracted from Anadenanthera colubrina (Red Angico Gum) Exerts Therapeutic Potential in Mice: Antidiarrheal Activity and Safety Assessment.

Anadenanthera colubrina var. cebil (Griseb.) Altschul (Fabaceae family), commonly known as the red angico tree, is a medicinal plant found throughout Brazil’s semi-arid area. In this study, a chemical analysis was performed to investigate the antidiarrheal activity and safety profile of red angico gum (RAG), a biopolymer extracted from the trunk exudate of A. colubrina. Upon FT-IR spectroscopy, RAG showed bands in the regions of 1608 cm−1, 1368 cm−1, and 1029 cm−1, which relate to the vibration of O–H water molecules, deformation vibration of C-O bands, and vibration of the polysaccharide C-O band, respectively, all of which are relevant to glycosidic bonds. The peak molar mass of RAG was 1.89 × 105 g/mol, with the zeta potential indicating electronegativity. RAG demonstrated high yield and solubility with a low degree of impurity. Pre-treatment with RAG reduced the total diarrheal stool and enteropooling. RAG also enhanced Na+/K+-ATPase activity and reduced gastrointestinal transit, and thereby inhibited intestinal smooth muscle contractions. Enzyme-Linked Immunosorbent Assay (ELISA) demonstrated that RAG can interact with GM1 receptors and can also reduce E. coli-induced diarrhea in vivo. Moreover, RAG did not induce any signs of toxicity in mice. These results suggest that RAG is a possible candidate for the treatment of diarrheal diseases.

Quantitative Detection of Biological Nanoparticles in Solution via Their Mediation of Colocalization of Fluorescent Liposomes

Detection of biomolecules and biological nanoparticles by means of induced aggregation of larger nanoparticles using light scattering as readout was first accomplished in the middle of the last century. Since then, technical advances together with novel nanomaterials have enabled more sophisticated readout schemes, paving the way for methods exploiting dual-probe hybridization for biomolecular or nonoparticle recognition that today can compete with established bioanalytical methods. Herein, we present a quantitative assay, with single-nanoparticle readout, utilizing receptor-containing cell-membrane mimics in the shape of approximately 100-nm lipid liposomes rather than conventional antibody-modified nanoparticles to enable detection of virus particles in solution. Specifically, the method is based on virus-mediated aggregation of differently fluorescent-labeled liposomes that contain the ganglioside GM1 receptor for the Simian Virus 40 (SV40). The aggregation kinetics of the differently colored liposomes is studied by monitoring the spatial colocalization level of the liposomes and a theoretical model that successfully represents aggregation kinetics versus virus concentration is proposed. The limits of detection are identified experimentally for our current setup and theoretically in a more general context. The consistency between theory and experiments suggests that the approach will be generically applicable for similar biosensing applications or for the study of related systems where natural interactions with cell-membrane components can be used to induce liposome aggregation.