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Background: Fabry nephropathy is a consequence of the deposition of globotriaosylceramide, caused by deficient GLA enzyme activity in all types of kidney cells. These deposits are perceived as damage signals leading to activation of inflammation resulting in renal fibrosis. There are few studies related to immunophenotype characterization of the renal infiltrate in kidneys in patients with Fabry disease and its relationship to mechanisms of fibrosis. This work aims to quantify TGF-β1 and active caspase 3 expression and to analyze the profile of cells in inflammatory infiltration in kidney biopsies from Fabry naïve-patients, and to investigate correlations with clinical parameters., Methods: Renal biopsies from 15 treatment-naïve Fabry patients were included in this study. Immunostaining was performed to analyze active caspase 3, TGF-β1, TNF-α, CD3, CD20, CD68 and CD163. Clinical data were retrospectively gathered at time of kidney biopsy., Results: Our results suggest the production of TNFα and TGFβ1 by tubular cells, in Fabry patients. Active caspase 3 staining revealed that tubular cells are in apoptosis, and apoptotic levels correlated with clinical signs of chronic kidney disease, proteinuria, and inversely with glomerular filtration rate. The cell infiltrates consisted of macrophages, T and B cells. CD163 macrophages were found in biopsy specimens and their number correlates with TGFβ1 and active caspase 3 tubular expression., Conclusions: These results suggest that CD163 + cells could be relevant mediators of fibrosis in Fabry nephropathy, playing a role in the induction of TGFβ1 and apoptotic cell death by tubular cells. These cells may represent a new player in the pathogenic mechanisms of Fabry nephropathy., (© 2024. The Author(s) under exclusive licence to Italian Society of Nephrology.)

Background: The pathophysiology of renal damage in Fabry nephropathy involves a complex biological mechanism. The intracellular deposition globotriaosylceramide (Gb3) is just the first step of the mechanism. The glycolipid deposition occurs in all renal cells (endothelial, epithelial and mesangial cells). It stimulates many biological processes, including cytokine release, epithelial-mesenchymal transdifferentiation, oxidative stress and the remodelling of vascular walls, resulting in subtle initial inflammation and eventually tissue fibrosis. It has been hypothesized that the processes activated by Gb3 deposition can subsequently progress independently of cellular deposition and that even Gb3 clearance by specific therapy cannot retard or stop these pathways., Aim: This review aims to gather the reported evidence of these cellular alterations and the resulting histological changes. Our approach is similar to a routine study of kidney biopsy., Results: In the first part of the review, "histology" section, we describe the structures involved (glomeruli, vessels, tubules and interstitium) from a histological point of view. While in the second part, "pathogenesis" section, we present some interpretations about the implicated pathways based on the up-to-date available evidence., (© 2021. Japanese Society of Nephrology.)

The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP 3 and flotillin thereby facilitating efficient uptake of PAO1.

Heat shock protein Hsp70 (Hsp70) is found on the cell surface of a large variety of human and mouse tumor cell types including U87, GL261 glioblastoma, and 4T1 mammary carcinoma cells. We studied the role of membrane-bound Hsp70 (mHsp70) in the formation of cell-to-cell connections via tunneling nanotubes (TNTs) using live-cell STED nanoscopy. This technique allows the visualization of microstructures in the 100-nm range in the living cells. We could show that the presence of tumor-derived mHsp70 in TNTs with a diameter ranging from 120 to 140 nm predominantly originates from cholesterol-rich-microdomains containing the lipid compound globoyltriaosylceramide (Gb3). Under non-stress conditions, Hsp70 and Gb3 are structurally clustered in the membrane of TNTs of tumor cells that showed tumor type specific variations in the amount of cell-to-cell connection networks. Furthermore depletion of cholesterol and ionizing radiation as a stress factor results in a complete loss of Hsp70-containing TNTs.

Cell density is one of the extrinsic factors to which cells adapt their physiology when grown in culture. However, little is known about the molecular changes which occur during cell growth and how cellular responses are then modulated. In many cases, inhibitors, drugs or growth factors used for in vitro studies change the rate of cell proliferation, resulting in different cell densities in control and treated samples. Therefore, for a comprehensive data analysis, it is essential to understand the implications of cell density on the molecular level. In this study, we have investigated how lipid composition changes during cell growth, and the consequences it has for transport of Shiga toxin. By quantifying 308 individual lipid species from 17 different lipid classes, we have found that the levels and species distribution of several lipids change during cell growth, with the major changes observed for diacylglycerols, phosphatidic acids, cholesterol esters, and lysophosphatidylethanolamines. In addition, there is a reduced binding and retrograde transport of Shiga toxin in high density cells which lead to reduced intoxication by the toxin. In conclusion, our data provide novel information on how lipid composition changes during cell growth in culture, and how these changes can modulate intracellular trafficking.

The two major Shiga toxin (Stx) types, Stx1 and Stx2, produced by enterohemorrhagic Escherichia coli (EHEC) in particular injure renal and cerebral microvascular endothelial cells after transfer from the human intestine into the circulation. Stxs are AB(5) toxins composed of an enzymatically active A subunit and the pentameric B subunit, which preferentially binds to the glycosphingolipid globotriaosylceramide (Gb3Cer/CD77). This review summarizes the current knowledge on Stx-caused cellular injury and the structural diversity of Stx receptors as well as the initial molecular interaction of Stxs with the human endothelium of different vascular beds. The varying lipoforms of Stx receptors and their spatial organization in lipid rafts suggest a central role in different modes of receptor-mediated endocytosis and intracellular destiny of the toxins. The design and development of tailored Stx neutralizers targeting the oligosaccharide-toxin recognition event has become a very real prospect to ameliorate or prevent life-threatening renal and neurological complications.

Glycosphingolipids are amphipathic molecules composed of hydrophilic oligosaccharide chain and a hydrophobic ceramide part, located primarily in the membrane microdomains of animal cells. Their oligosaccharide chains make them excellent candidates for the cell surface recognition molecules. Natural glycosphingolipid, globotriaosylceramide (Gal α1-4, Gal β1-4, Glc β1-1, ceramide), is also called CD77 and its expression was previously associated with proliferating centroblasts undergoing somatic hypermutation, but it has been demonstrate that globotriaosylceramide is not a reliable marker to discriminate human centroblasts from centrocytes. Globotriaosylceramide constitutes rare P k blood group antigen on erythrocytes, and it is also known as Burkitt's lymphoma antigen. On endothelial cells, globotriaosylceramide plays as the receptor for bacterial toxins of the Shiga family, also called verotoxins. Precise biological function and significance of globotriaosylceramide expression on endothelial cells remains to be the subject of many studies and it is believed globotriaosylceramide represents an example of a glycolipid antigen able to transduce a signal leading to apoptosis. In past decade, cancer researches put a great afford in determining new therapeutic agents such as bacterial toxins against tumor malignancies. Reports have demonstrated that verotoxin-1 induces apoptosis in solid tumor cell lines expressing globotriaosylceramide such as astrocytoma, renal cell carcinoma, colon cancer and breast cancer due to verotoxin-1 high specificity and apoptosis-inducing properties, and therefore, it is suggested to be an anticancer agent. Verotoxins have been investigated weather they could reduce treatment side-effects and toxicity to normal tissues and become a new oncological tool in cancer labeling.

Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt's lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.

Shiga toxin-producing Escherichia coli causes hemolytic uremic syndrome, a constellation of disorders that includes kidney failure and central nervous system dysfunction. Shiga toxin binds the amphipathic, membrane-bound glycolipid globotriaosylceramide (Gb(3)) and uses it to enter host cells and ultimately cause cell death. Thus, cell types that express Gb(3) in target tissues should be recognized. The objective of this study was to determine whether immunohistologic detection of Gb(3) was affected by the method of tissue preparation. Tissue preparation included variations in fixation (immersion or perfusion) and processing (paraffin or frozen) steps; paraffin processing employed different dehydration solvents (acetone or ethanol). Perfusion-fixation in combination with frozen sections or acetone-dehydrated tissue for paraffin sections resulted in specific recognition of Gb(3) using immunohistochemical or immunofluorescent methods. In the mouse tissues studied, Gb(3) was associated with tubules in the kidney and neurons in the nervous system. On the other hand, Gb(3) localization to endothelial cells was determined to be an artifact generated due to immersion-fixation or tissue dehydration with ethanol. This finding was corroborated by glycolipid profiles from tissue subjected to dehydration; namely Gb(3) was subject to extraction by ethanol more than acetone during tissue dehydration. The results of this study show that tissue preparation is crucial to the persistence and preservation of the glycolipid Gb(3) in mouse tissue. These methods may serve as a basis for determining the localization of other amphipathic glycolipids in tissue.

Shiga toxin (Stx) 1 binds to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer/CD77) and injures human endothelial cells. In order to gain insight into Stx1-induced cellular impairment, we analysed in detail the molecular heterogeneity of Stx1 receptors in two endothelial cell lines differing in their Stx1-sensitivity. We observed a moderate sensitivity to Stx1 of human brain microvascular endothelial cells (HBMECs, CD(50) > 200 ng/ml), but a considerably higher mortality rate in cultures of EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (CD(50) of 0.2 ng/ml). Immunofluorescence microscopy demonstrated the presence of Gb3Cer in both cell lines, but showed an enhanced content of Gb3Cer in EA.hy 926 cells. Solid phase overlay binding assays of isolated GSLs combined with nanoelectrospray ionization quadrupole time-of-flight mass spectrometry demonstrated a balanced proportion of Gb3Cer and globotetraosylceramide (Gb4Cer) in HBMECs, but an increase of Gb3Cer and absence of Gb4Cer in EA.hy 926 cells. Gb3Cer species with C24:1/C24:0 fatty acids were found to dominate over those with C16:0 fatty acids in EA.hy 926 cells, but were similarly distributed in HBMECs. Reverse transcriptase polymerase chain reaction indicated the concomitant presence of Gb3Cer and Gb4Cer synthases in HBMECs, whereas EA.hy 926 cells expressed Gb3Cer synthase, but completely lacked Gb4Cer synthase. This deficiency, resulting in the accumulation of Gb3Cer in EA.hy 926 cells, represents the most prominent molecular reason that underlies the different Stx1 sensitivities of HBMECs and EA.hy 926 endothelial cells.

Fabry disease is an X-linked recessive inborn metabolic disorder in which a deficiency in lysosomal enzyme alpha-galactosidase A (Gal A) causes the systemic accumulation of globotriaosylceramide (Gb3). Although many investigators have attempted to treat alpha-Gal A knock-out mice (Fabry mice) with gene therapy, no report has demonstrated therapeutic effects by the retrograde renal vein injection of naked DNA. We recently developed a naked plasmid vector-mediated kidney-targeted gene transfer technique. A solution containing naked plasmid DNA encoding human alpha-Gal A (pKSCX-alpha-Gal A) was rapidly injected into the left kidney of Fabry mice (pKSCX-alpha-Gal A mice). pKSCX was used for mock transfections (pKSCX mice). We confirmed that vector-derived human alpha-Gal A mRNA was present in the left kidney but not in other tissues, by reverse transcriptase polymerase chain reaction. Compared with the pKSCX mice, the pKSCX-alpha-Gal A mice showed partial therapeutic effects: increased alpha-Gal A activity in the injected kidney and in the liver, heart, and plasma, and decreased Gb3 in the injected kidney, contralateral kidney, liver, heart, and spleen. Our results demonstrated that, although further studies are needed to improve the outcome, this method has promise as a potential treatment option for Fabry disease.

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG2c to IgG1 antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.

Purified renal globotriaosyl ceramide (Gb3)/cholesterol mixtures sonicated heated in a Triton-containing buffer placed below a discontinuous sucrose gradient form glycosphingolipid (GSL)-containing dense lipid structures at the 30/5% sucrose interface after centrifugation. Inclusion of fluorescein-labeled verotoxin 1 B subunit (FITC-VT1 B) within the most dense sucrose layer results in the fluorescent labeling of this Gb3-containing raft structure. Alternatively inclusion of I-labeled VT1 fractionation allows quantitation of binding. FITC-VT1 B effectively competes for I-VT1/Gb3 raft binding. This assay will allow the definition of the optimal raft composition for VT1 (or any other ligand) binding. The effect of several potential cellular raft components are reported. Increased cholesterol content increased VT1 binding. Addition of phosphatidylethanolamine had minimal effect while phosphatidylserine was inhibitory. Although inclusion of sphingomyelin increased the Gb3 content of the "raft" reduced VT1 binding was seen. Inclusion of other glycolipids can also be inhibitory. The addition of globotetraosyl ceramide had no effect; however addition of sulfogalactosyl ceramide but not sulfogalactoglycerolipid inhibited VT1/Gb3 raft binding. These results suggest that certain GSLs can disfavor the formation of the appropriate 'raft' structure for ligand binding that this is dependent on both their carbohydrate lipid structure. Such "deceptor" GSLs may provide an as yet unappreciated mechanism for the regulation of cellular GSL receptor activity. This model is an effective tool to approach the dynamics ligand-binding specificity of GSL/cholesterol-containing lipid microdomains.

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in alpha-interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon-alpha receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon-alpha were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.