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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.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A (AGAL/GLA) gene. The lysosomal accumulation of the substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) results in progressive renal failure, cardiomyopathy associated with cardiac arrhythmia, and recurrent strokes, significantly limiting life expectancy in affected patients. Current treatment options for FD include recombinant enzyme-replacement therapies (ERTs) with intravenous agalsidase-α (0.2 mg/kg body weight) or agalsidase-β (1 mg/kg body weight) every 2 weeks, facilitating cellular Gb3 clearance and an overall improvement of disease burden. However, ERT can lead to infusion-associated reactions, as well as the formation of neutralizing anti-drug antibodies (ADAs) in ERT-treated males, leading to an attenuation of therapy efficacy and thus disease progression. In this narrative review, we provide a brief overview of the clinical picture of FD and diagnostic confirmation. The focus is on the biochemical and clinical significance of neutralizing ADAs as a humoral response to ERT. In addition, we provide an overview of different methods for ADA measurement and characterization, as well as potential therapeutic approaches to prevent or eliminate ADAs in affected patients, which is representative for other ERT-treated lysosomal storage diseases.

Fabry disease is a rare, progressive, X-linked inherited storage disorder due to absent or deficient of lysosomal alfa galactosidase A activity. Deficient activity of alfa-galactosidase A results in progressive accumulation of globotriaosylceramide in a variety of tissues and organs including myocardium, kidney and nerve system. This disorder predominantly affects males; however, female heterozygotes may also be affected with a less severe clinical picture. Classic Fabry disease is usually diagnosed in early age of childhood because of multiorgan involvement whereas cardiac and renal variants of Fabry are manifested in 30-50 years of age because of late onset of clinical picture in which other organs involvement are uncommon. Although Fabry is known as a very rare disease, its prevalence is reported to be higher in patients with ventricular hypertrophy, chronic kidney disease and cryptogenic stroke. From the cardiology point of view, the most important key finding of the disease is unexplained ventricular hypertrophy. However, in clinical practice, ventricular hypertrophy is usually thought to be due to hypertrophic cardiomyopathy in the absence of hypertension or aortic stenosis and Fabry disease is often undiagnosed or overlooked. Early diagnosis and enzyme replacement therapy have been shown to significantly improve prognosis. The aim of this paper is to provide a comprehensive review including epidemiology, prognosis, clinical presentation, diagnosis and therapeutic approaches of cardiac variant of Fabry based on the available data in the literature.

Accumulation of amyloid-β peptide (Aβ) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aβ accumulation offers a promising approach for therapeutic strategies against AD. Aβ is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aβ is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aβ. mStx2a-treatment also inhibited the extracellular release of Aβ. Therefore, mStx2a may provide a new strategy to inhibit the production of Aβ by modulating the intracellular transport of APP.

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. We hypothesized that the orientation of the carbohydrate head group of Gb3 embedded in the lipid bilayer differentially influences LecA and StxB binding. To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and were indeed able to rebuild LecA and StxB segregation. Both, the Gb3 fatty acyl chain structure and the local membrane environment, modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes compared to LecA. Based on our findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify this approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB recognized Gb3 at the primary cilium and the periciliary membrane, whereas LecA only bound periciliary Gb3. This suggests that the ciliary membrane is of higher order than the surrounding periciliary membrane.

Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.

While females can suffer serious complications of Fabry disease, most studies are limited to males to avoid confounding by mosaicism. Here, we developed a novel unbiased method for quantifying globotriaosylceramide (GL3) inclusion volume in affected podocytes (F+) in females with Fabry disease independent of mosaicism leading to important new observations. All podocytes in male patients with Fabry are F+. The probability of observing random profiles from F+ podocytes without GL3 inclusions (estimation error) was modeled from electron microscopic studies of 99 glomeruli from 40 treatment-naïve males and this model was applied to 28 treatment-naïve females. Also, podocyte structural parameters were compared in 16 age-matched treatment-naïve males and females with classic Fabry disease and 11 normal individuals. A 4(th) degree polynomial equation best described the relationship between podocyte GL3 volume density and the estimation error (R(2) =0.94) and was confirmed by k-fold cross-validation. In females, this model showed that age related directly to F+ podocyte GL3 volume (correlation coefficient (r = 0.54) and podocyte volume (r = 0.48) and inversely to podocyte number density (r = −0.56), (all significant). F+ podocyte GL3 volume was significantly inversely related to podocyte number density (r = −0.79) and directly to proteinuria. There was no difference in F+ podocyte GL3 volume or volume fraction between age-matched males and females. Thus, in females with Fabry disease GL3 accumulation in F+ podocytes progresses with age in association with podocyte loss and proteinuria, and F+ podocyte GL3 accumulation in females with Fabry is similar to males, consistent with insignificant cross-correction between affected and non-affected podocytes. Hence, these findings have important pathophysiological and clinical implications.

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Fabry disease is an X-linked lysosomal multisystem storage disorder induced by a mutation in the alpha-galactosidase A (GLA) gene. Reduced activity or deficiency of alpha-galactosidase A (AGAL) leads to escalating storage of intracellular globotriaosylceramide (GL-3) in numerous organs, including the kidneys, heart and nerve system. The established treatment for 20 years is intravenous enzyme replacement therapy. Lately, oral chaperone therapy was introduced and is a therapeutic alternative in patients with amenable mutations. Early starting of therapy is essential for long-term improvement. This review describes chaperone therapy in Fabry disease.

Anderson–Fabry disease (AFD) is a rare disease with an incidenceof approximately 1:117,000 male births. Lysosomal accumulation of globotriaosylceramide (Gb3) is the element characterizing Fabry disease due to a hereditary deficiency α-galactosidase A (GLA) enzyme. The accumulation of Gb3 causes lysosomal dysfunction that compromises cell signaling pathways. Deposition of sphingolipids occurs in the autonomic nervous system, dorsal root ganglia, kidney epithelial cells, vascular system cells, and myocardial cells, resulting in organ failure. This manuscript will review the molecular pathogenetic pathways involved in Anderson–Fabry disease and in its organ damage. Some studies reported that inhibition of mitochondrial function and energy metabolism plays a significant role in AFD cardiomyopathy and in kidney disease of AFD patients. Furthermore, mitochondrial dysfunction has been reported as linked to the dysregulation of the autophagy–lysosomal pathway which inhibits the mechanistic target of rapamycin kinase (mTOR) mediated control of mitochondrial metabolism in AFD cells. Cerebrovascular complications due to AFD are caused by cerebral micro vessel stenosis. These are caused by wall thickening resulting from the intramural accumulation of glycolipids, luminal occlusion or thrombosis. Other pathogenetic mechanisms involved in organ damage linked to Gb3 accumulation are endocytosis and lysosomal degradation of endothelial calcium-activated intermediate-conductance potassium ion channel 3.1 (KCa3.1) via a clathrin-dependent process. This process represents a crucial event in endothelial dysfunction. Several studies have identified the deacylated form of Gb3, globotriaosylsphingosine (Lyso-Gb3), as the main catabolite that increases in plasma and urine in patients with AFD. The mean concentrations of Gb3 in all organs and plasma of Galactosidase A knockout mice were significantly higher than those of wild-type mice. The distributions of Gb3 isoforms vary from organ to organ. Various Gb3 isoforms were observed mainly in the kidneys, and kidney-specific Gb3 isoforms were hydroxylated. Furthermore, the action of Gb3 on the KCa3.1 channel suggests a possible contribution of this interaction to the Fabry disease process, as this channel is expressed in various cells, including endothelial cells, fibroblasts, smooth muscle cells in proliferation, microglia, and lymphocytes. These molecular pathways could be considered a potential therapeutic target to correct the enzyme in addition to the traditional enzyme replacement therapies (ERT) or drug chaperone therapy.

International audience; Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a., Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster).

Background Fabry disease is an X-linked lysosomal storage disorder with a highly heterogeneous clinical presentation. This complex disease is caused by a deficient activity of the enzyme α-galactosidase A, which is involved in the catabolism of glycosphingolipids. The prevalence of Fabry disease is underestimated, due to the presence of atypical variants. High-risk screening protocols are particularly relevant for this disease due to the availability of treatments, such as enzyme replacement and chaperone therapies. As kidney manifestations are present in the majority of male and many female patients with Fabry disease, a high-risk screening protocol was performed for patients with chronic kidney disease of unknown etiology. Methods Recruitment of 397 participants took place in four centers across Canada from 2011 to 2017. Globotriaosylceramide (Gb3) was analyzed in dried urine spots by liquid chromatography/tandem mass spectrometry followed by globotriaosylsphingosine (lyso-Gb3) on the repeat analysis. Results The collection and shipment of urine specimens on filter paper resulted in easier handling/shipment and significant cost-saving. No Fabry patients were detected in this study. Conclusions Increased concentrations of urinary Gb3 were observed in 13.6% of patients with chronic kidney disease suggesting that chronic kidney disease or other comorbidities might be associated with increased urinary Gb3 concentrations.

Although Gaucher disease can be accompanied by Lewy pathology (LP) and extrapyramidal symptoms, it is unknown if LP exists in Fabry disease (FD), another progressive multisystem lysosomal storage disorder. We aimed to elucidate the distribution patterns of FD-related inclusions and LP in the brain of a 58-year-old cognitively unimpaired male FD patient suffering from predominant hypokinesia. Immunohistochemistry (CD77, α-synuclein, collagen IV) and neuropathological staging were performed on 100-µm sections. Tissue from the enteric or peripheral nervous system was unavailable. As controls, a second cognitively unimpaired 50-year-old male FD patient without LP or motor symptoms and 3 age-matched individuals were examined. Inclusion body pathology was semiquantitatively evaluated. Although Lewy neurites/bodies were not present in the 50-year-old individual or in controls, severe neuronal loss in the substantia nigra pars compacta and LP corresponding to neuropathological stage 4 of Parkinson disease was seen in the 58-year-old FD patient. Major cerebrovascular lesions and/or additional pathologies were absent in this individual. We conclude that Lewy body disease with parkinsonism can occur within the context of FD. Further studies determining the frequencies of both inclusion pathologies in large autopsy-controlled FD cohorts could help clarify the implications of both lesions for disease pathogenesis, potential spreading mechanisms, and therapeutic interventions.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase-A, which results in accumulation of the glycosphingolipid (GSL) globotriaosylceramide (Gb3). Gb3 and globotriaosylsphingosine (lyso-Gb3) levels in plasma and urine are used routinely for diagnosis and treatment monitoring. FD female patients are problematic to diagnose and to predict when to begin treatment. Further biomarkers are needed to detect pre-symptomatic females that will develop the chronic symptoms associated with FD. A LC-MS/MS glycosphingolipidomic assay was developed to measure lyso-Gb3 and GSLs from the lysosomal GSL degradation pathway, including globoside (Gb4), Gb3, ceramide dihexosides (CDH) and ceramide monohexosides (CMH). We analysed plasma and urine from a cohort of Fabry patients, grouped according to clinical symptoms and independent of treatment status (asymptomatic females n = 18, symptomatic females n = 18, males n = 27 and control urines n = 16 and control plasmas n = 58). Multivariate and subsequent univariate analysis showed urine GSLs which had highest significance in identifying asymptomatic females were total levels of CDH, in particular the long chain isoforms C22:1,C22:0,C22:1-OH,C22:0-OH,C24:2,C24:0,C24:2-OH,C24:1-OH,C24:0-OH,C26:0 which likely represent Galabiosylceramide (Ga2) and not lactosylceramide. These long chain Ga2 isoforms were found to be 5-fold elevated and more statistically significant (p

Fabry disease (FD) is an X-linked inherited glycosphingolipid metabolism disorder, therefore, heterozygous female FD patients display highly variable clinical symptoms, disease severity, and pathological findings. This makes it very challenging to diagnosing female patients with FD. A 69-year-old Japanese female was introduced to the nephrologist for the evaluation of proteinuria. A renal biopsy was performed. Although the light microscopic examinations revealed that most of the glomeruli showed minor glomerular abnormalities, however, vacuolation was apparently found in the tubular epithelial cells. Immunofluorescence staining for globotriaosylceramide was positively detected in some podocytes and distal tubular epithelial cells. In addition, myelin-like structure (zebra body) was detected by electron microscopy. Pathological findings were most consistent with FD. Consequently, biochemical and genetic analysis confirmed the diagnosis of female FD. Enzyme replacement therapy was performed in conjunction with renin-angiotensin aldosterone system inhibitors and beta-blockers. The patient's family members received the analysis, and the same DNA missense mutation was detected in the patient's grandson. The enzyme replacement therapy was introduced to the grandson. The present case showed that renal biopsy can contribute towards a correct diagnosis for FD. Particularly, in female FD patients, careful examination of pathological changes is essential, for example, vacuolation of any type of renal cells may be a clue for the diagnosis.

Fabry disease (FD) is an X-linked lysosomal storage disorder associated with pain triggered by heat or febrile infections. We modelled this condition by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) from FD patients in vitro upon stimulation with heat and lipopolysaccharide (LPS). We enrolled 67 FD patients and 37 healthy controls. We isolated PBMC, assessed their gene expression of selected pro- and anti-inflammatory cytokines, incubated them with heat, LPS, globotriaosylceramide (Gb3), and tumor necrosis factor-α (TNF), and measured TNF secretion in the supernatant and intracellular Gb3 accumulation, respectively. We found increased TNF, interleukin (IL-)1β, and toll-like receptor 4 (TLR4) gene expression in FD men (p .05 to p .01). TNF and IL-10 were higher, and IL-4 was lower in the subgroup of FD men with pain compared to controls (p .05 to p .01). Hereby, TNF was only increased in FD men with pain and classical mutations (p .05) compared to those without pain. PBMC from FD patients secreted more TNF upon stimulation with LPS (p .01) than control PBMC. Incubation with Gb3 and an additional α-galactosidase A inhibitor did not further increase TNF secretion, but incubation with TNF greatly increased the Gb3 load in FD PBMC compared to controls (p .01). Also, LPS incubation and heat challenge (40 °C) increased Gb3 accumulation in PBMC of patients compared to baseline (p .05 each), while no alterations were observed in control PBMC. Our data show that TNF holds a crucial role in the pathophysiology of FD associated pain, which may open a novel perspective for analgesic treatment in FD pain.

BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.MethodsSince current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.ResultsUnder treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (pA cells.ConclusionWe conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Background/aims Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. While virtually all tissues are affected, renal damage is particularly critical for the patients' outcome. Currently, powerful diagnostic tools and in vivo research models to study FD in the kidney are lacking, which is a major obstacle for further improvements in diagnosis and therapy. The present study focuses on the effects of enzyme replacement therapy on a previously established podocyte cell culture model of Fabry disease. Methods We investigated the effect of in vitro application of α-Gal A on Fabry podocytes for 3 days, mimicking enzyme replacement therapy. We studied reduction of Gb3 levels and dysregulated molecular pathways such as autophagy, mTOR/AKT signaling and pro-fibrotic signaling by employing immunofluorescence, electron microscopy, tandem mass spectrometry and western blot. Results We detected complete resolution of Gb3 accumulation in Fabry podocytes upon α-Gal A treatment. Despite robust Gb3 clearance, dysregulation of the signaling pathways investigated was not reversed. Conclusion This study presents first evidence for Gb3-independent effects regarding dysregulation of signal transduction mechanisms in FD not recovering upon α-Gal A treatment. We assume that intracellular alterations observed in FD may have a point of no return after which a reversal of dysregulated cellular signal transduction by α-Gal A treatment is not effective, despite Gb3 clearance. Our observations suggest further research on signal transduction mechanisms altered in Fabry podocytes and on determining the appropriate time for initiation of Fabry therapy.

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.

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.

N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Gal (Gb3 and the P1 antigen) and Galα1→4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins.

Protein toxins secreted by bacteria and found in plants can be threats to human health. However, their extreme toxicity can also be exploited in different ways, e.g., to produce hybrid toxins directed against cancer cells and to study transport mechanisms in cells. Investigations during the last decades have shown how powerful these molecules are as tools in cell biological research. Here, we first present a partly historical overview, with emphasis on Shiga toxin and ricin, of how such toxins have been used to characterize processes and proteins of importance for their trafficking. In the second half of the article, we describe how one can now use toxins to investigate the role of lipid classes for intracellular transport. In recent years, it has become possible to quantify hundreds of lipid species using mass spectrometry analysis. Thus, it is also now possible to explore the importance of lipid species in intracellular transport. The detailed analyses of changes in lipids seen under conditions of inhibited toxin transport reveal previously unknown connections between syntheses of lipid classes and demonstrate the ability of cells to compensate under given conditions.

The recognition of carbohydrate receptors on host cell membranes by pathogenic lectins is a crucial step in the microbial invasion. Two bacterial lectins, the B-subunit of Shiga toxin from Shigella dysenteria (StxB) and lectin I from Pseudomonas aeruginosa (LecA), are specific to the same galactolipid-globotriaosylceramide (Gb3). In this study we present a coarse-grained (cg) model of Gb3, which we further apply to unravel the molecular details of glycolipid binding by two lectins on the surface of a DOPC/cholesterol/Gb3 bilayer. In cg molecular dynamics simulations with time scales of dozens of microseconds, Gb3 was randomly distributed. The binding of both StxB or LecA is accompanied by Gb3 clustering in a cholesterol environment and with exclusion of DOPC in protein vicinity. StxB being bound by all 15 binding sites induced membrane bending, while LecA interacted with two out of four binding sites for most of the time causing a smaller inward curvature of the model membrane. Stable interactions occurred preferably when LecA was normal to the membrane surface. Furthermore, all-atom simulations revealed that LecA bound Gb3's headgroup at only one out of two possible conformations of the carbohydrate moiety observed at protein-free conditions. The results shed light on the mechanism of interactions between two lectins and Gb3 on the membrane surface and offer a coarse-grained model to study more complex systems at large spatiotemporal scales.

In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha‐galactosidase A (GLA) or N‐sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV‐GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR‐labelled EVs were localized in brain parenchyma 1 h after intra‐arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV‐GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies., This study has been supported by ISCIII (PI18_00871 co‐founded by Fondo Europeo de Desarrollo Regional (FEDER)), and CIBER‐BBN (EXPLORE) granted to IA. Different CIBER‐BBN units of ICTS ‘NANBIOSIS’ have participated in this work (https://www.nanbiosis.es/platform-units/), more specifically the U1/Protein Production Platform for protein purification, Unit 6 for NTA analysis and TFF purification and U20/FVPR for in vivo assays.

Fabry disease (FD) is an X‐linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha‐galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life‐threatening long‐term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear. We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was up‐regulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD. Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue‐specific level points to new therapeutic targets which might enhance treatment efficacy.

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx., Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster).

6 figures, 2 tables.-- Supplementary information available.-- © 2021. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/, Identification of 19 molecular species of globotriaosylceramides (Gb3) in extracts from a Fabry's plasma patient and a healthy control was performed by High-Performance Thin-Layer Chromatography (HPTLC)-densitometry and online coupling to Mass Spectrometry (MS). Separation was carried out on LiChrospher plates using Automated Multiple Development (AMD). Densitometry was performed on twin plates by combining detection in the visible at 550 nm, through previous on-plate orcinol derivatization, and by Ultraviolet 190 nm, using a non-impregnated plate. The latter was directly coupled to an ion-trap mass spectrometer through an automated elution-based interface. Gb3 molecular species, which were identified by HPTLC- Electrospray Mass Spectrometry (+)-MS and confirmed by MS/MS or HPTLC-Atmospheric Pressure Chemical Ionization Mass Spectrometry (+)-MS, are: five isoforms of saturated Gb3; seven isoforms of methylated Gb3; and seven species with two additional double bonds. Twelve of these species were previously reported as biomarkers of Fabry's lysosomal disorder using a Liquid Chromatography-MS-based method, and the other seven are structurally similar, closely related to them. Saturated Gb3 isoforms migrated on LiChrospher plate in one of the separated peaks corresponding to the migration zone of ceramide trihexosides standard. Instead, methylated and unsaturated Gb3 species co-migrated with sphingomyelin species. Ion intensity ESI-MS profiles show that saturated Gb3 species in Fabry's plasma were in higher concentration than in control sample. Before applying the Thin-Layer Chromatography (TLC)-MS interface on HPTLC separated peaks, its positioning precision was first studied using ceramide tri-hexosides as model compound. This provided information on Gb3 peak broadening and splitting during its migration., This work was supported by DGA-FEDER (E25_17R, N&SB). One of us (J.G.) thanks to Spanish Plan Nacional I+D+i (CTQ2016-76846-R project).

Enzyme replacement therapy in Fabry disease has been available in Japan since 2004. Two post-authorization safety studies were conducted to evaluate agalsidase beta in Japanese patients with Fabry disease in real-world practice. The Special Drug Use Investigation monitored the long-term safety and efficacy of agalsidase beta, and the Drug Use Investigation monitored safety in patients not participating in the Special Drug Use Investigation. Safety and efficacy evaluations included adverse drug reactions (ADRs), infusion-associated reactions and hypersensitivity reactions, and change in blood GL-3 level over time. Of 396 patients in the aggregated data set, safety and efficacy analysis sets comprised 307 and 196 patients, respectively. ADRs occurred in 93 (30.3%) patients and serious ADRs occurred in 25 (8.1%) patients, with general disorders and administration site conditions (n=55, 17.9%), nervous system disorders (n=30, 9.8%) and skin and subcutaneous tissue disorders (n=23, 7.5%) the most common. Reductions in blood GL-3 levels occurred over the study, irrespective of age or disease phenotype. Agalsidase beta demonstrated acceptable safety and tolerability, with sustained reductions in blood GL-3 levelsin Japanese patients with Fabry disease in real-world clinical practice NCT00233870/AGAL03004 (Special Drug Use Investigation of Agalsidase beta)

Background Fabry disease (FD) is a rare X-linked disease caused by mutations in GLA gene with consequent lysosomal accumulation of globotriaosylceramide (Gb3). Women with FD often show highly heterogeneous symptoms that can manifest from mild to severe phenotype. Main body The phenotypic variability of the clinical manifestations in heterozygous women with FD mainly depends on the degree and direction of inactivation of the X chromosome. Classical approaches to measure XCI skewness might be not sufficient to explain disease manifestation in women. In addition to unbalanced XCI, allele-specific DNA methylation at promoter of GLA gene may influence the expression levels of the mutated allele, thus impacting the onset and the outcome of FD. In this regard, analyses of DNA methylation at GLA promoter, performed by approaches allowing distinction between mutated and non-mutated allele, may be much more informative. The aim of this review is to critically evaluate recent literature articles addressing the potential role of DNA methylation in the context of FD. Although up to date relatively few works have addressed this point, reviewing all pertinent studies may help to evaluate the importance of DNA methylation analysis in FD and to develop new research and technologies aimed to predict whether the carrier females will develop symptoms. Conclusions Relatively few studies have addressed the complexity of DNA methylation landscape in FD that remains poorly investigated. The hope for the future is that ad hoc and ultradeep methylation analyses of GLA gene will provide epigenetic signatures able to predict whether pre-symptomatic female carriers will develop symptoms thus helping timely interventions.

Fabry disease (FD) is a lysosomal storage disorder, depending on defects in alpha-galactosidase A (GAL) activity. At the clinical level, FD shows a high phenotype variability. Among them, cardiovascular dysfunction is often recurrent or, in some cases, is the sole symptom (cardiac variant) representing the leading cause of death in Fabry patients. The existing therapies, besides specific symptomatic treatments, are mainly based on the restoration of GAL activity. Indeed, mutations of the galactosidase alpha gene (GLA) cause a reduction or lack of GAL activity leading to globotriaosylceramide (Gb3) accumulation in several organs. However, several other mechanisms are involved in FD’s development and progression that could become useful targets for therapeutics. This review discusses FD’s cardiovascular phenotype and the last findings on molecular mechanisms that accelerate cardiac cell damage.

International audience; Background: Recent years have witnessed a considerable increase in clinical trials of new investigational agents for Fabry disease (FD). Several trials investigating different agents are currently in progress; however, lack of standardisation results in challenges to interpretation and comparison. To facilitate the standardisation of investigational programs, we have developed a common framework for future clinical trials in FD.Methods and findings: A broad consensus regarding clinical outcomes and ways to measure them was obtained via the Delphi methodology. 35 FD clinical experts from 4 continents, representing 3389 FD patients, participated in 3 rounds of Delphi procedure. The aim was to reach a consensus regarding clinical trial design, best treatment comparator, clinical outcomes, measurement of those clinical outcomes and inclusion and exclusion criteria. Consensus results of this initiative included: the selection of the adaptative clinical trial as the ideal study design and agalsidase beta as ideal comparator treatment due to its longstanding use in FD. Renal and cardiac outcomes, such as glomerular filtration rate, proteinuria and left ventricular mass index, were prioritised, whereas neurological outcomes including cerebrovascular and white matter lesions were dismissed as a primary or secondary outcome measure. Besides, there was a consensus regarding the importance of patient-related outcomes such as general quality of life, pain, and gastrointestinal symptoms. Also, unity about lysoGb3 and Gb3 tissue deposits as useful surrogate markers of the disease was obtained. The group recognised that cardiac T1 mapping still has potential but requires further development before its widespread introduction in clinical trials. Finally, patients with end-stage renal disease or renal transplant should be excluded unless a particular group for them is created inside the clinical trial.Conclusion: This consensus will help to shape the future of clinical trials in FD. We note that the FDA has, coincidentally, recently published draft guidelines on clinical trials in FD and welcome this contribution.

Fabry disease is caused by a decrease in or loss of the activity of alpha-galactosidase, which causes its substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) to accumulate in cells throughout the body. This accumulation results in progressive kidney injury due to glomerulosclerosis and in heart failure due to hypertrophy. Enzyme replacement therapy (ERT) has been used as the standard therapy for Fabry disease, but it causes a significant financial burden, and regular administration is inconvenient for patients. Because of the short half-life of alpha-galactosidase in vivo, therapeutic methods that can supplement or replace ERT are expected to involve continuous release of alpha-galactosidase, even at low doses. Cell transplantation therapy is one of these methods; however, its use has been hindered by the short-term survival of transplanted cells. CellSaic technology, which utilizes cell spheroids that form after cells are seeded simultaneously with a recombinant collagen peptide scaffold called a μ-piece, has been used to improve cell survival upon implantation. In this study, syngeneic murine embryonic fibroblasts were used to generate CellSaic that were transplanted into Fabry mice. These spheroids survived for 28 days in the renal subcapsular space with forming blood vessels. These results indicate CellSaic technology could be a platform to promote cellular graft survival and may facilitate the development of cell transplantation methods for lysosomal diseases.

Fabry disease (FD) is an X-linked disorder caused by mutations in GLA gene responsible for coding of the lysosomal enzyme alpha-galactosidase A(α-GAL). The resultant accumulation of globotriaosylceramide (Gb-3) leads to multisystemic disease including progressive chronic kidney disease, hypertrophic cardiomyopathy, stroke, angiokeratomas and corneal whorls. Current treatments include enzyme replacement therapy (ERT), along with recent advent of chaperone therapy. ERT has not shown to have dramatic improvement in outcomes for all organ systems, with benefit mostly seen in kidney disease and reduction in left ventricular hypertrophy. ERT, however, is associated with formation of anti-drug antibodies and requirement of long-term venous access, while chaperone therapy can only be used in amenable mutations. A multitude of therapies are now under investigation in various phases of clinical trials. These include pegylated form of α-GAL (pegunigalsidase alpha), gene therapy (both in-vivo and ex-vivo methods), mRNA therapy (inducing production of α-GAL) and substrate reduction therapy (inhibitors of glucosylceramide synthase leading to reduction of Gb-3). This review encapsulates literature pertaining to current and investigational therapies for FD.

Shiga toxins (Stxs) produced by Stx-producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx-mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte-derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage-like differentiated THP-1 cells treated with Stxs secreted Stx-associated exosomes (Stx-Exo) of 90-130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb3 )-dependent manner. Stx2-Exo engulfed by Gb3 -positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK-2. Stx2-Exo contained pro-inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2-Exo-mediated human renal cell death. Stx2-Exo isolated from human primary monocyte-derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx-containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin-sensitive cells. Therapeutic interventions targeting Stx-containing exosomes may prevent or ameliorate Stx-mediated acute vascular dysfunction.

Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the &alpha, galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (&minus, C2H4, &minus, C2H4+O, &minus, H2, &minus, H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-na&iuml, ve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

Fabry disease (FD) is an X-linked metabolic storage disorder arising from the deficiency of lysosomal α-galactosidase A, which leads to the gradual accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), throughout the body. Pain in the extremities is an early symptom of FD; however, the underlying pathophysiological mechanisms remain unknown. α-Galactosidase A knockout animals exhibit nociceptive behaviors, with enhanced expression levels of several ion channels. These characteristics are observed in animals treated with nerve growth factor (NGF). Here, we aimed to elucidate the potential of NGF signaling as a cause of FD-associated pain, using intraplantar Gb3-treated mice displaying mechanical allodynia. Treatment with a neutralizing antibody against a precursor of NGF (proNGF) or its receptor, p75 neurotrophin receptor (p75

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 (PIP3) 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, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.

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. 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. 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.