Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Standardising clinical outcomes measures for adult clinical trials in Fabry disease: A global Delphi consensus.

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.

Shiga toxin receptor Gb3Cer/CD77: tumor-association and promising therapeutic target in pancreas and colon cancer.

BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Recommendations and guidelines for the diagnosis and treatment of Fabry nephropathy in adults.

Progressive loss of kidney function complicates Fabry disease, an X-linked lysosomal storage disorder that arises from deficiency of alpha-galactosidase activity. Heterozygous females with Fabry disease can be as severely affected as hemizygous males, who have the classic form of the disease. Enzyme-replacement therapy with recombinant human alpha-galactosidase clears the glycosphingolipid globotriaosylceramide from kidney cells, and can stabilize renal function in adults with mild to moderate Fabry nephropathy. However, adults with more advanced nephropathy and overt proteinuria do not respond as well. For these patients, antiproteinuric therapy given in conjunction with enzyme-replacement therapy might prevent further decline in kidney function. In this Review, we propose guidelines and recommendations for the diagnosis and management of Fabry nephropathy in adults, based on published data and on the consensus of opinion of participants in the 7(th) International Fabry Nephropathy Roundtable in 2007. These organ-specific guidelines could be easier to implement than general guidelines, provided they are used in the context of an overall multisystem care approach.

A novel soluble mimic of the glycolipid, globotriaosyl ceramide inhibits HIV infection.

OBJECTIVE: To determine the effect of a gp120 binding, non-cytotoxic soluble analogue of the glycosphingolipid (GSL), globotriaosyl ceramide (Gb3) on HIV infection in vitro. DESIGN: HIV-1(IIIB) (X4 virus) infection in Jurkat and phytohaemagglutinin (PHA)/interleukin-2 (IL2) activated, peripheral blood mononuclear cells (PBMC), and HIV-1(Ba-L) (R5 virus) infection of PHA activated PBMC in vitro were assessed. We monitored cell surface markers, cell viability, and viral/host cell morphology to eliminate pleiotropic effects. Viral-host cell fusion was measured to further address any inhibitory mechanism. METHODS: HIV infection was monitored by p24(gag) ELISA. CD4, CCR5, CXCR4 and apoptosis were determined by fluorescent antibody cell sorting. A model fusion system comprising a cell line transfected with either CD4 and CXCR4 or CCR5, cocultured with a cell line expressing gp120 from either X4-, R5-tropic HIV-1 or HIV-2 virions, was used. PHA/IL2 activated PBMC GSL synthesis was monitored by metabolic radiolabelling. RESULTS: AdamantylGb3 blocked X4 and R5 virus infection with a 50% inhibitory concentration of approximately 150 microM. A reverse transcriptase and a protease-resistant X4 HIV-1 strain retained adamantylGb3 sensitivity. AdamantylGb3 had minimal effect on cell viability. Treated Jurkat cells showed a small increase in CCR5/CXCR4 expression and a slight, transient CD4 down-regulation, which was probably not related to the mechanism of inhibition. Electron microscopy showed normal viral and host cell morphology following adamantylGb3 treatment, and viral entry was blocked. AdamantylGb3 was able to prevent virus-host cell fusion irrespective of HIV strain or chemokine receptor preference. CONCLUSIONS: These results suggest that adamantylGb3 may provide a new basis for blocking HIV infections, irrespective of HIV envelope/chemokine co-receptor preference or resistance to other therapeutics.

Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections.

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

[New drugs that prevent cytotoxicity of Shiga toxins].

Shiga toxin(Stx) produced by enterohemorrhagic E. coli is the virulence factor that causes not only enterohemorrhagic colitis but also fatal complications, such as hemolytic uremic syndrome. To prevent the complications, new strategies targeted to Stx have been tested, mostly using mimics of the trisaccharide structure of neutral lipid Gb3, the receptor for Stx. One group of such new drugs are agents that can bind to Stx in gastrointestinal tract and prevent its spread to extraintestinal sites, and the other group are water-soluble neutralizers that suppress Stx cytotoxicity in the circulation. Although most of these are now under the laboratory investigations, one of these drugs may hopefully be utilized clinically to prevent hemolytic uremic syndrome in future.

Oral administration of formaldehyde-killed recombinant bacteria expressing a mimic of the Shiga toxin receptor protects mice from fatal challenge with Shiga-toxigenic Escherichia coli.

Gastrointestinal disease caused by Shiga toxin-producing Escherichia coli (STEC) is frequently complicated by life-threatening toxin-induced systemic sequelae, including the hemolytic uremic syndrome. We previously constructed a recombinant bacterium displaying a Shiga toxin receptor mimic on its surface which neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the live bacterium completely protected mice from challenge with virulent STEC. In this study, we investigated the protective capacity of formaldehyde-killed receptor mimic bacteria, as these are likely to be safer for administration to humans. The killed bacteria completely protected STEC-challenged mice when administered three times daily; incomplete protection was achieved using two doses per day. Commencement of therapy could be delayed for up to 48 h after challenge without diminishing protection, depending on the virulence of the challenge strain. Thus, administration of this agent early in the course of human STEC disease may prevent progression to life-threatening complications.

[Advances in the treatment of hemolytic uremic syndrome (HUS)].

The hemolytic uremic syndrome (HUS) is the end result of a variety of etiologic agents that can induce endothelial cell injury and thrombotic microangiopathy (TMA) mostly within the kidney. The typical, post-diarrheal verocytotoxin associated HUS (D + HUS) is the major cause of acute renal failure in children worldwide. In the course of HUS treatment, fluid overload is usually the result of overhydration in the context of oliguria or anuria which cause edema, hypertension, worsening of neurologic signs and cardiac failure. Appropriate and timely use of dialysis has dramatically reduced complications of renal failure and extra-renal complications are now the main causes of mortality and morbidity in D + HUS. The reasons for treatment by infusion of fresh frozen plasma and/or plasmapheresis for D + HUS are theoretical and their therapeutic effects are inconclusive. We believe that plasma administration for regular D + HUS has no value and is potentially harmful. Until new strategies become available in clinical practice, the general consensus for the moment is that careful supportive management with patience is still the most appropriate form of D + HUS therapy.