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Simple Summary: Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies. Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies. [ABSTRACT FROM AUTHOR]

The use of available treatments for Fabry disease (FD) (including enzyme replacement therapy [ERT]) may be restricted by their limited symptom improvement and mode of administration. Lucerastat is currently being investigated in the MODIFY study as oral substrate reduction therapy for the treatment of FD. By reducing the net globotriaosylceramide (Gb3) load in tissues, lucerastat has disease-modifying potential to improve symptoms and delay disease progression. MODIFY is a multicenter, double-blind, randomized, placebo-controlled, parallel-group Phase 3 study (ClinicalTrial.gov: NCT03425539); here we present the rationale and design of this study. Eligible adults with a genetically confirmed diagnosis of FD and FD-specific neuropathic pain entered screening. Patients were randomized (2:1) to receive either oral lucerastat twice daily or placebo for 6 months; treatment allocation was stratified according to sex and ERT treatment status. The main objectives of MODIFY are to assess the effects of lucerastat on neuropathic pain, gastrointestinal (GI) symptoms, FD biomarkers, and determine its safety and tolerability. Neuropathic pain and GI symptoms are key features of FD that have a significant impact on quality of life. Despite various tools available to assess pain and GI symptoms, there are currently limited tools available to assess neuropathic and GI symptoms in FD, validated according to health authority guidelines. Based on FDA recommendations, we undertook a patient-reported outcome (PRO) validation study, using a novel eDiary-based PRO tool to assess the validity of evaluating neuropathic pain as a primary efficacy endpoint in MODIFY. Results from the PRO validation study are included. To date, MODIFY is the largest Phase 3 clinical study conducted in patients with FD. Enrollment to MODIFY is now complete, with 118 patients randomized. Results will be presented in a separate publication. Long-term effects of lucerastat are being assessed in the ongoing open-label extension study (NCT03737214).

Fabry disease (FD) is a rare lysosomal disorder caused by α-galactosidase A deficiency, and it leads to the systemic deposition of globotriasylceramide. Demonstrations of the storage material in biopsies support this diagnosis. We report a histological and ultrastructural study of biopsies that were performed on 11 individuals from a family with the variant p.Gln279Arg in GLA, which is associated with the classical phenotype of Fabry disease. Intralysosomal deposits were found in all biopsies, corresponding to the skin, kidney, and endomyocardium in both sexes and at different ages. In nine of the skin biopsies, deposits were analysed by immunofluorescence and quantified at the ultrastructural level. Then, the findings were compared according to sex, genotype, and treatment. The quantification of the deposits in the skin biopsies revealed a broader involvement in men than in women. A significant clearance of the deposits was observed in one case after treatment. Tissue involvement was remarkable at diagnosis in all individuals. The findings from the skin biopsies were demonstrative of classic FD, thus supporting the diagnosis; repeated biopsy analyses suggested the benefit of early treatment. [ABSTRACT FROM AUTHOR]

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson–Fabry disease and an elevated level of deacylated Gb3 is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1, 4βGal1, and 4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of galactokinase, UTP–glucose-1-phosphate uridylyltransferase, and α-1,4-galactosyltransferase LgtC, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes galactokinase/UTP–glucose-1-phosphate uridylyltransferase generate UDP-6-azido-6-deoxy- d -galactose, which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the 3 enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy- d -galactose-1-phosphate, UDP-6-azido-6-deoxy- d -galactose, and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V, and LgtC-Q187S, respectively, is 3–6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6-azido-6-deoxy- d -galactose with ~90% conversion yields, and products azido-globotriose and lyso-AzGb3 with substrate conversion of up to 70%. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

Globotriaosylceramide (Gb3 or CD77) is a tumor-associated carbohydrate antigen implicated in several types of cancer that serves as a potential cancer marker for developing target-specific diagnosis and therapy. However, the development of Gb3-targeted therapeutics has been challenging due to its carbohydrate nature. In the present work, taking advantage of its natural pentamer architecture and Gb3-specific targeting of shiga toxin B subunit (StxB), we constructed a pentameric antibody recruiting chimera by site-specifically conjugating StxB with the rhamnose hapten for immunotherapy of colorectal cancer. The Sortase A-catalyzed enzymatic tethering of rhamnose moieties to the C terminus of Stx1B and Stx2B had very moderate effect on their pentamer architectures and thus the resultant conjugates maintained the potent ability to bind to Gb3 antigen both immobilized on an assay plate and expressed on colorectal cancer cells. All StxB-rhamnose constructs were capable of efficiently mediating the binding of rhamnose antibodies onto HT29 colorectal cancer cells, which was further shown to be able to induce cancer cell lysis by eliciting potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro. Finally, the best StxB-rhamnose conjugate, i.e. 1B-3R, was confirmed to be able to inhibit the colorectal tumor growth using a HT29-derived xenograft murine model. Taken together, our data demonstrated the potential of repurposing StxB as an excellent multivalent scaffold for developing Gb3-targeted biotherapeutics and StxB-rhamnose conjugates might be promising candidates for targeted immunotherapy of Gb3-related colorectal cancer. [Display omitted] • StxB-rhamnose conjugates were constructed as multivalent antibody-recruiting chimera. • StxB-rhamnose conjugates directed the binding of rhamnose antibodies to tumor cells. • StxB-rhamnose conjugates mediated tumor cells killing through ADCC and CDC. • StxB-rhamnose conjugates showed potential for Gb3-targeted cancer immunotherapy. [ABSTRACT FROM AUTHOR]

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fabry Disease (FD) manifestations include organ damage, systemic inflammation, and fibrosis. • Lipidome changes possibly reflect systemic and cellular responses to FD. • Plasma ceramide ratios used in cardiovascular risk assessment were increased in FD. • Platelets accumulated lyso-Gb3, but not Gb3 in FD patients. • Identified lipids may serve as potential new screening and prognostic markers for FD. Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by the progressive accumulation of globotriaosylceramide (Gb3) leading to systemic manifestations such as chronic kidney disease, cardiomyopathy, and stroke. There is still a need for novel markers for improved FD screening and prognosis. Moreover, the pathological mechanisms in FD, which also include systemic inflammation and fibrosis, are not yet fully understood. Plasma and platelets were obtained from 11 ERT (enzyme–replacement therapy)-treated symptomatic, 4 asymptomatic FD patients, and 13 healthy participants. A comprehensive targeted lipidomics analysis was conducted quantitating more than 550 lipid species. Sphingadiene (18:2;O2)-containing sphingolipid species, including Gb3 and galabiosylceramide (Ga2), were significantly increased in FD patients. Plasma levels of lyso-dihexosylceramides, sphingoid base 1-phosphates (S1P), and GM3 ganglioside were also altered in FD patients, as well as specific plasma ceramide ratios used in cardiovascular disease risk prediction. Gb3 did not increase in patients' platelets but displayed a high inter-individual variability in patients and healthy participants. Platelets accumulated, however, lyso-Gb3, acylcarnitines, C16:0-sphingolipids, and S1P. This study identified lipidome changes in plasma and platelets from FD patients, a possible involvement of platelets in FD, and potential new markers for screening and monitoring of this disease. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is a rare lysosomal disorder caused by α-galactosidase A deficiency, and it leads to the systemic deposition of globotriasylceramide. Demonstrations of the storage material in biopsies support this diagnosis. We report a histological and ultrastructural study of biopsies that were performed on 11 individuals from a family with the variant p.Gln279Arg in GLA, which is associated with the classical phenotype of Fabry disease. Intralysosomal deposits were found in all biopsies, corresponding to the skin, kidney, and endomyocardium in both sexes and at different ages. In nine of the skin biopsies, deposits were analysed by immunofluorescence and quantified at the ultrastructural level. Then, the findings were compared according to sex, genotype, and treatment. The quantification of the deposits in the skin biopsies revealed a broader involvement in men than in women. A significant clearance of the deposits was observed in one case after treatment. Tissue involvement was remarkable at diagnosis in all individuals. The findings from the skin biopsies were demonstrative of classic FD, thus supporting the diagnosis; repeated biopsy analyses suggested the benefit of early treatment.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor. [ABSTRACT FROM AUTHOR]

Bacillus cereus is an opportunistic pathogen that can cause emetic or diarrheal foodborne illness. Previous studies have identified multiple pathogenic B. cereus strains and characterized a variety of virulence factors. Here, we demonstrate that the virulence and lethality of B. cereus for mammalian cells and host animals involve the interaction of B. cereus flagellin proteins and the host-cell-surface-localized glycosphingolipid Gb3 (CD77, Galα1-4Galβ1-4Glcβ1-Cer). We initially found that B. cereus infection was less lethal for Gb3-deficiencient A4galt−/- mice than for wild-type mice. Subsequent experiments established that some factor other than secreted toxins must account of the observed differential lethality: Gb3-deficiencient A4galt−/- mice were equally susceptible to secreted-virulence-factor-mediated death as WT mice, and we observed no differences in the bacterial loads of spleens or livers of mice treated with B. cereus strain vs. mice infected with a mutant variant of incapable of producing many secreted toxins. A screen for host-interacting B. cereus cell wall components identified the well-known flagellin protein, and both flagellin knockout strain assays and Gb3 inhibitor studies confirmed that flagellin does interact with Gb3 in a manner that affects B. cereus infection of host cells. Finally, we show that treatment with polyclonal antibody against flagellin can protect mice against B. cereus infection. Thus, beyond demonstrating a previously unappreciated interaction between a bacterial motor protein and a mammalian cell wall glycosphingolipid, our study will provide useful information for the development of therapies to treat infection of B. cereus.

At first glance, the biological function of globoside (Gb) clusters appears to be that of glycosphingolipid (GSL) receptors for bacterial toxins that mediate host-pathogen interaction. Indeed, certain bacterial toxin families have been evolutionarily arranged so that they can enter eukaryotic cells through GSL receptors. A closer look reveals this molecular arrangement allocated on a variety of eukaryotic cell membranes, with its role revolving around physiological regulation and pathological processes. What makes Gb such a ubiquitous functional arrangement? Perhaps its peculiarity is underpinned by the molecular structure itself, the nature of Gb-bound ligands, or the intracellular trafficking unleashed by those ligands. Moreover, Gb biological conspicuousness may not lie on intrinsic properties or on its enzymatic synthesis/degradation pathways. The present review traverses these biological aspects, focusing mainly on globotriaosylceramide (Gb3), a GSL molecule present in cell membranes of distinct cell types, and proposes a wrap-up discussion with a phylogenetic view and the physiological and pathological functional alternatives.

Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb 3 ). Accumulation of Gb 3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb 3 and creatinine. Samples are diluted with an internal standard solution containing Gb 3 (C17:0) and creatinine-D 3 , centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb 3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb 3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb 3 . Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb 3 /creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb 3 ) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb 3 calibration curve Support Protocol 3: Preparation of the Gb 3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT–transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT–transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin. [ABSTRACT FROM AUTHOR]

Background: Sporadic Parkinson's disease (PD) patients have lower α-galactosidase A (α-GAL A) enzymatic activity and Fabry disease (FD) patients potentially carry an increased risk of PD. Objective: Determination of PD prevalence in FD and clinical, biochemical and vascular neuroimaging description of FD pedigrees with concomitant PD. Methods: Clinical screening for PD in 229 FD patients belonging to 31 families, harbouring GLA gene mutation p.F113L, and subsequent pedigree analysis. Gender-stratified comparison of FD+/PD+ patients with their family members with FD but without PD (FD+/PD–) regarding Mainz scores, plasma & leukocytes α-GAL A enzymatic activity, urinary Gb3 and plasma Lyso-Gb3, vascular brain neuroimaging. Results: Prevalence of PD in FD was 1.3% (3/229) (3% in patients aged ≥50 years). Three FD patients, one female (73 years old) (P1) and two males (60 and 65 years old) (P2 and P3), three different pedigrees, presented akinetic-rigid PD, with weak response to levodopa (16% – 36%), and dopaminergic deficiency on 18F-DOPA PET. No pathogenic mutations were found in a PD gene panel. FD+/PD+ patients had worse clinical severity of FD (above upper 75% IQR in Mainz scores), and cortico-subcortical white matter/small vessel lesions. P3 patient was under enzyme therapy, started 1 year before PD diagnosis. P2-P3 patients had higher leucocyte α-GAL A activity (2,2-3 vs.1,0 (median)(nmol/h/mg)). Conclusion: We have shown a high prevalence of PD in a late-onset phenotype of FD, presenting high cerebrovascular burden and weak response to levodopa. Further studies will untangle how much of this PD phenotype is due to Gb3 deposition versus cerebrovascular lesions in the nigro-striatal network. [ABSTRACT FROM AUTHOR]

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs. Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3. Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects. Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients.

Shiga toxin (Stx) is associated with foodborne infections of some Shigella spp. and Shiga toxin-producing Escherichia coli (STEC), leading to life-threatening hemolytic uremic syndrome (HUS). Target-specific therapeutics against HUS are currently unavailable in clinical practice. Herein, we reported the construction and in vitro characterization of Gb3-coated bovine milk exosomes (Gb3-mExo) as a multivalent Shiga toxin neutralizer, utilizing the natural advantages of milk exosomes (mExo) in drug delivery and multivalent interactions between Stx and its receptor Gb3. Gb3-mExo constructs were achieved by conjugating mExo with the Gb3 derivatives containing stearic acid-derived lipid tail, which was prepared through an efficient chemoenzymatic approach. The constructs were able to potently neutralize the binding of the B subunit of Stx2 (Stx2B) to receptor Gb3 immobilized on the plate or expressed on model cells. General safety of the constructs was evidenced by the cytotoxicity analysis and hemolysis assay. In addition to the excellent stability under conventional storage and handling conditions, the construct can also retain most of its neutralization potency under gastrointestinal pH extremes, showing the potential for oral administration. Considering the natural availability and excellent biocompatibility of mExo, Gb3-mExo conjugates should prove to be a practical prophylactic and therapeutic for the Shiga toxin-related infections.

Pain behaviors in a Fabry mouse model are associated with the accumulation of a fat molecule that disrupts sodium ion channels in small fiber neurons.

Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla -deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

We investigated correlated µs-ms time scale motions of neighboring 13C′-15N and 13Cα-13Cβ nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100-400 s−1 range) for the 13C′-15N pairs and into the slow to intermediate regime for the 13Cα-13Cβ pairs (about 150 s−1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13Cα-13Cβ pairs. [ABSTRACT FROM AUTHOR]

A force balanced generalized molecular fractionation with conjugate caps (FB-GMFCC) method is proposed for ab initio molecular dynamic simulation of proteins. In this approach, the energy of the protein is computed by a linear combination of the QM energies of individual residues and molecular fragments that account for the two-body interaction of hydrogen bond between backbone peptides. The atomic forces on the caped H atoms were corrected to conserve the total force of the protein. Using this approach, ab initio molecular dynamic simulation of an Ace-(ALA)9-NME linear peptide showed the conservation of the total energy of the system throughout the simulation. Further a more robust 110 ps ab initio molecular dynamic simulation was performed for a protein with 56 residues and 862 atoms in explicit water. Compared with the classical force field, the ab initio molecular dynamic simulations gave better description of the geometry of peptide bonds. Although further development is still needed, the current approach is highly efficient, trivially parallel, and can be applied to ab initio molecular dynamic simulation study of large proteins.

Resonance assignment is the first stage towards solving the structure of a protein. This is normally achieved by the employment of separate inter and intra residue experiments. By utilising the mixed rotation and rotary recoupling (MIRROR) condition it is possible to double the information content through the efficient bidirectional transfer of magnetization from the CO to its adjacent Cα and the Cα of the subsequent amino acid. We have incorporated this into a 3D experiment, a 3D-MIRROR-NCOCA, where correlations present in the 3D spectrum permit the sequential assignment of the protein backbone from a single experiment as we have demonstrated on a microcrystalline preparation of GB3. Furthermore, the low-power requirements of the MIRROR recoupling sequence facilitate the development of a low-power 3D-NCOCA experiment. This has enabled us to realise significant reductions in acquisition times, allowing the acquisition of a single 3D-NCOCA spectrum suitable for a full backbone resonance assignment of GB3 in less than 24 h. [ABSTRACT FROM AUTHOR]

The cellular entry of the bacterial Shiga toxin and the related verotoxins has been scrutinized in quite some detail. This is due to their importance as a threat to human health. At the same time, the study of Shiga toxin has allowed the discovery of novel molecular mechanisms that also apply to the intracellular trafficking of endogenous proteins at the plasma membrane and in the endosomal system. In this review, the individual steps that lead to Shiga toxin uptake into cells will first be presented from a purely mechanistic perspective. Membrane-biological concepts will be highlighted that are often still poorly explored, such as fluctuation force-driven clustering, clathrin-independent membrane curvature generation, friction-driven scission, and retrograde sorting on early endosomes. It will then be explored whether and how these also apply to other pathogens, pathogenic factors, and cellular proteins. The molecular nature of Shiga toxin as a carbohydrate-binding protein and that of its cellular receptor as a glycosylated raft lipid will be an underlying theme in this discussion. It will thereby be illustrated how the study of Shiga toxin has led to the proposal of the GlycoLipid-Lectin (GL-Lect) hypothesis on the generation of endocytic pits in processes of clathrin-independent endocytosis. [ABSTRACT FROM AUTHOR]

Streptococcus suis is an important emerging zoonotic agent that causes acute bacterial meningitis in humans with high mortality and morbidity. Our previous work showed that factor H-binding protein (Fhb) contributed to virulence of S. suis, but the role of Fhb in the development of S. suis meningitis remained unclear. In this study, we demonstrated for the first time that Fhb contributed to the traversal of S. suis across the human blood-brain barrier by allelic-exchange mutagenesis, complementation and specific antibody blocking studies. We also showed that globotriaosylceramide (Gb3), the receptor of Fhb, was involved in this process and affected S. suis infection-induced activation of myosin light chain 2 through Rho/ROCK signaling in hCMEC/D3 cells. Using a murine model of S. suis meningitis, we further demonstrated that Gb3-deficiency prevented the mice from developing severe brain inflammation or injury. Our results demonstrate that the Fhb-Gb3 interaction plays an important role in the development of S. suis meningitis and might be a potential therapeutic target against S. suis infection. [ABSTRACT FROM AUTHOR]

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The relaxation interference between dipole-dipole interactions of two separate spin pairs carries structural and dynamics information. In particular, when compared to individual dynamic behavior of those spin pairs, such cross-correlated relaxation (CCR) rates report on the correlation between the spin pairs. We have recently mapped out correlated motion along the backbone of the protein GB3, using CCR rates among and between consecutive H-N and H-C dipole-dipole interactions. Here, we provide a detailed account of the measurement of the four types of CCR rates. All rates were obtained from at least two different pulse sequences, of which the yet unpublished ones are presented. Detailed comparisons between the different methods and corrections for unwanted pathways demonstrate that the averaged CCR rates are highly accurate and precise with errors of 1.5-3% of the entire value ranges. [ABSTRACT FROM AUTHOR]

Both N chemical shift anisotropy (CSA) and sufficiently rapid exchange linebroadening transitions exhibit relaxation contributions that are proportional to the square of the magnetic field. Deconvoluting these contributions is further complicated by residue-dependent variations in protein amide N CSA values which have proven difficult to accurately measure. Exploiting recently reported improvements for the implementation of T and T experiments, field strength-dependent studies have been carried out on the B3 domain of protein G (GB3) as well as on the immunophilin FKBP12 and a H87V variant of that protein in which the major conformational exchange linebroadening transition is suppressed. By applying a zero frequency spectral density rescaling analysis to the relaxation data collected at magnetic fields from 500 to 900 MHz H, differential residue-specific N CSA values have been obtained for GB3 which correlate with those derived from solid state and liquid crystalline NMR measurements to a level similar to the correlation among those previously reported studies. Application of this analysis protocol to FKBP12 demonstrated an efficient quantitation of both weak exchange linebroadening contributions and differential residue-specific N CSA values. Experimental access to such differential residue-specific N CSA values should significantly facilitate more accurate comparisons with molecular dynamics simulations of protein motion that occurs within the timeframe of global molecular tumbling. [ABSTRACT FROM AUTHOR]

Streptococcus suis is a Gram-positive bacterium, which causes sepsis and meningitis in pigs and humans. This review examines the role of known S. suis virulence factors in adhesion and S. suis carbohydrate-based adhesion mechanisms, as well as the inhibition of S. suis adhesion by anti-adhesion compounds in in vitro assays. Carbohydrate-binding specificities of S. suis have been identified, and these studies have shown that many strains recognize Galα1-4Gal-containing oligosaccharides present in host glycolipids. In the era of increasing antibiotic resistance, new means to treat infections are needed. Since microbial adhesion to carbohydrates is important to establish disease, compounds blocking adhesion could be an alternative to antibiotics. The use of oligosaccharides as drugs is generally hampered by their relatively low affinity (micromolar) to compete with multivalent binding to host receptors. However, screening of a library of chemically modified Galα1-4Gal derivatives has identified compounds that inhibit S. suis adhesion in nanomolar range. Also, design of multivalent Galα1-4Gal-containing dendrimers has resulted in a significant increase of the inhibitory potency of the disaccharide. The S. suis adhesin binding to Galα1-4Gal-oligosaccharides, Streptococcal adhesin P (SadP), was recently identified. It has a Galα1-4Gal-binding N-terminal domain and a C-terminal LPNTG-motif for cell wall anchoring. The carbohydrate-binding domain has no homology to E. coli P fimbrial adhesin, which suggests that these Gram-positive and Gram-negative bacterial adhesins recognizing the same receptor have evolved by convergent evolution. SadP adhesin may represent a promising target for the design of anti-adhesion ligands for the prevention and treatment of S. suis infections.

Inhibition of AB5-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin’s pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB5 complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor’s activity.

Shiga toxin-producing Escherichia coli is a contaminant of food and water that in humans causes a diarrheal prodrome followed by more severe disease of the kidneys and an array of symptoms of the central nervous system. The systemic disease is a complex referred to as diarrhea-associated hemolytic uremic syndrome (D+HUS). D+HUS is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. This review focuses on the renal aspects of D+HUS. Current knowledge of this renal disease is derived from a combination of human samples, animal models of D+HUS, and interaction of Shiga toxin with isolated renal cell types. Shiga toxin is a multi-subunit protein complex that binds to a glycosphingolipid receptor, Gb3, on select eukaryotic cell types. Location of Gb3 in the kidney is predictive of the sites of action of Shiga toxin. However, the toxin is cytotoxic to some, but not all cell types that express Gb3. It also can cause apoptosis or generate an inflammatory response in some cells. Together, this myriad of results is responsible for D+HUS disease.

A major problem with anti-cancer drug treatment is the development of acquired multidrug resistance (MDR) of the tumor cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the globotriaosylceramide membrane receptor (Gb3), a glycolipid associated with multidrug resistance. Gb3 is overexpressed in many human tumors and tumor cell lines with inherent or acquired MDR. Gb3 is co-expressed and interplays with the membrane efflux transporter P-gp encoded by the MDR1 gene. P-gp could act as a lipid flippase and stimulate Gb3 induction when tumor cells are exposed to cancer chemotherapy. Recent work has shown that apoptosis and inherent or acquired multidrug resistance in Gb3-expressing tumors could be affected by VT-1 holotoxin, a sub-toxic concentration of the holotoxin concomitant with chemotherapy or its Gb3-binding B-subunit coupled to cytotoxic or immunomodulatory drug, as well as chemical manipulation of Gb3 expression. The interplay between Gb3 and P-gp thus gives a possible physiological approach to augment the chemotherapeutic effect in multidrug resistant tumors.

Fabry disease is a rare lysosomal storage disorder resulting from the lack of α-Gal A gene activity. Globotriaosylceramide (GB3, ceramide trihexoside) is a novel endogenous biomarker which predicts the incidence of Fabry disease. At the early stage efficacy/biomarker study, a rapid method to determine this biomarker in plasma and in all relevant tissues related to this disease simultaneously is required. However, the limited sample volume, as well as the various levels of GB3 in different matrices makes the GB3 quantitation very challenging. Hereby we developed a rapid method to identify GB3 in mouse plasma and various tissues. Preliminary stability tests were also performed in three different conditions: short-term, freeze-thaw, long-term. The calibration curve was well fitted over the concentration range of 0.042–10 μg/mL for GB3 in plasma and 0.082–20 μg/g for GB3 in various tissues. This method was successfully applied for the comparison of GB3 levels in Fabry model mice (B6;129-Glatm1Kul/J), which has not been performed previously to the best of our knowledge.

The cellular entry of the bacterial Shiga toxin and the related verotoxins has been scrutinized in quite some detail. This is due to their importance as a threat to human health. At the same time, the study of Shiga toxin has allowed the discovery of novel molecular mechanisms that also apply to the intracellular trafficking of endogenous proteins at the plasma membrane and in the endosomal system. In this review, the individual steps that lead to Shiga toxin uptake into cells will first be presented from a purely mechanistic perspective. Membrane-biological concepts will be highlighted that are often still poorly explored, such as fluctuation force-driven clustering, clathrin-independent membrane curvature generation, friction-driven scission, and retrograde sorting on early endosomes. It will then be explored whether and how these also apply to other pathogens, pathogenic factors, and cellular proteins. The molecular nature of Shiga toxin as a carbohydrate-binding protein and that of its cellular receptor as a glycosylated raft lipid will be an underlying theme in this discussion. It will thereby be illustrated how the study of Shiga toxin has led to the proposal of the GlycoLipid-Lectin (GL-Lect) hypothesis on the generation of endocytic pits in processes of clathrin-independent endocytosis.

A new and convenient method, named ARTSY-J, is introduced that permits extraction of the 3 J HNHα couplings in proteins from the relative intensities in a pair of 15 N– 1 H TROSY-HSQC spectra. The pulse scheme includes 3 J HNHα dephasing of the narrower TROSY 1 H N –{ 15 N} doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where 3 J HNHα dephasing is suppressed. The effect of passive 1 H α spin flips downscales the apparent 3 J HNHα coupling by a uniform factor that depends approximately linearly on both the duration of the 3 J HNHα dephasing delay and the 1 H– 1 H cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and 1 H α spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3 Hz. Measurement for the HIV-1 protease homodimer (22 kDa) yields 3 J HNHα values that agree to better than 0.7 Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual 3 J HNHα couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable ϕ angle information. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls ( p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females ( p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Shiga-toxin-producing Escherichia coli (STEC) infections usually cause haemolytic uraemic syndrome (HUS) equally in male and female children. This study investigated the localization of globotriaosylceramide (Gb3) in human brain and kidney tissues removed from forensic autopsy cases in Japan. A fatal case was used as a positive control in an outbreak of diarrhoeal disease caused by STEC O157:H7 in a kindergarten in Urawa in 1990. Positive immunodetection of Gb3 was significantly more frequent in female than in male distal and collecting renal tubules. To correlate this finding with a clinical outcome, a retrospective analysis of the predictors of renal failure in the 162 patients of two outbreaks in Japan was performed: one in Tochigi in 2002 and the other in Kagawa Prefecture in 2005. This study concludes renal failure, including HUS, was significantly associated with female sex, and the odds ratio was 4·06 compared to male patients in the two outbreaks. From 2006 to 2009 in Japan, the risk factor of HUS associated with STEC infection was analysed. The number of males and females and the proportion of females who developed HUS were calculated by age and year from 2006 to 2009. In 2006, 2007 and 2009 in adults aged >20 years, adult women were significantly more at risk of developing HUS in Japan. [ABSTRACT FROM PUBLISHER]

To avoid any irreversible organ damage in patients with Fabry disease (FD), therapeutic interventions should be undertaken at an early stage. Unfortunately, early detection of the disease based on clinical symptoms alone is complicated by the fact that all complications of FD are nonspecific and clinically indistinguishable from similar abnormalities that occur in common disorders. Although the diagnosis of FD based on the determination of α-GAL A activity and GLA gene analysis can be relatively easy in affected males with classical manifestations, in male individuals with attenuated phenotypes and in heterozygous female patients the residual enzyme activity can be significant. In these cases genetic analysis may also be inconclusive due to the difficulty in establishing whether some of the encountered abnormalities in the GLA gene are truly disease-causing mutations. Because of the limitations of the current detection methods for FD, attention is now paid to the identification of biomarkers that allow solid confirmation of the diagnosis as well as monitoring of disease manifestations and therapeutic management. [ABSTRACT FROM AUTHOR]

The study of the spatial sampling of biomolecules is essential to understanding the structure–dynamics–function relationship. We have established a protocol for the determination of multiple-state ensembles based on exact measurements of the nuclear Overhauser effect (eNOE). The protocol is practical since it does not require any additional data, while all other NMR data sets must be supplemented by NOE restraints. The question arises as to how much structural and dynamics information is shared between the eNOEs and other NMR probes. We compile one of the largest and most diverse NMR data sets of a protein to date consisting of eNOEs, RDCs and J couplings for GB3. We show that the eNOEs improve the back-prediction of RDCs and J couplings, either upon use of more than one state, or in comparison to conventional NOEs. Our findings indicate that the eNOE data is self-consistent, consistent with other data, and that the structural representation with multiple states is warranted. [ABSTRACT FROM AUTHOR]

Background The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. Methods Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model. Results Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p ≤ 0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p ≤ 0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p ≤ 0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor. Conclusion The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials. [ABSTRACT FROM AUTHOR]

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