Romina Ceci

Fabry disease peripheral blood immune cells release inflammatory cytokines: Role of globotriaosylceramide

Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal), which leads to the accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3). Several mechanisms contribute to the diverse physiopathological alterations observed in this disease, and it has been suggested that an underlying proinflammatory state could play a significant role. The aim of this study is to investigate the presence of a proinflammatory state in the different subsets of peripheral blood mononuclear cells (PBMC) and to understand the mechanisms that contribute to its onset and perpetuation. We have shown that cultured PBMC from Fabry patients present a higher proinflammatory cytokine expression and production. Moreover, we determined that among PBMC, dendritic cells and monocytes present a basal proinflammatory cytokine production profile, which is further exacerbated with an inflammatory stimulus. Finally we established that normal, monocyte-derived dendritic cells and macrophages display the same proinflammatory profile when cultured in the presence of Gb3 and an inhibitor of αGal. Furthermore, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the process. In summary, our results demonstrate the presence of a proinflammatory state involving two key subsets of innate immunity, and provide direct evidence of Gb3 having a proinflammatory role, likely mediated by TLR4, a finding that could help in the understanding of the underlying causes of the inflammatory pathogenesis of Fabry disease.

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.

Uncoupling of osteoblast-osteoclast regulation in a chemical murine model of Gaucher disease

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. The homeostasis of bone tissue is maintained by the balanced processes of bone resorption by osteoclasts and formation by osteoblasts. We decided to test whether bone resorption and/or bone formation could be altered by the use of a chemical in vitro murine model of Gaucher disease. We used two sources of cells from monocyte/macrophages lineage isolated from normal mice, splenocytes (S) and peritoneal macrophages (PM), and were exposed to CBE, the inhibitor of GCase (S-CBE and PM-CBE, respectively). Addition of both conditioned media (CM) from S-CBE and PM-CBE induced the differentiation of osteoclasts precursors from bone marrow to mature and functional osteoclasts. TNF-α could be one of the factors responsible for this effect. On the other hand, addition of CM to an osteoblast cell culture resulted in a reduction in expression of alkaline phosphatase and mineralization process. In conclusion, these results suggest implication of changes in both bone formation and bone resorption and are consistent with the idea that both sides of the homeostatic balance are affected in GD.

An easy and sensitive method for determination of globotriaosylceramide (Gb3) from urinary sediment: Utility for Fabry disease diagnosis and treatment monitoring

BACKGROUND Fabry disease is an X-linked disorder that results from the deficiency of the lysosomal enzyme alpha-galactosidase A. The defect leads to the accumulation of globotriaosylceramide (Gb3). The detection of Gb3 accumulated in different tissues may help in the diagnosis and enzyme replacement therapy monitoring. For this reason, we developed a simple method available to clinical laboratories to measure this analyte. METHODS Gb3 excretion was determined by the incubation of urine sediment glycolipids from Fabry patients with agalsidase alpha and subsequent determination of galactose produced. RESULTS The amount of urinary Gb3 in Fabry hemizygotes was significantly higher (p = 0.00001) than the amount in normal controls. Patients undergoing enzyme replacement therapy with agalsidase alpha showed a significantly lower content of Gb3 in urine sediment. This method showed a good recovery and comparability with a previously validated method. CONCLUSIONS We developed an easy method for quantification of Gb3 in urine samples from Fabry patients, by the use of the specific recombinant enzyme for this glycolipid, that does not require complex infrastructure. Urinary Gb3 as measured by this enzymatic method could be useful for the diagnosis and monitoring of treatment in Fabry patients.

The Continuous Challenge of Diagnosing patients with Fabry disease in Argentina Genotype, Experiences, Anecdotes, and New Learnings

The lysosomal storage disorder Fabry disease (FD) is caused by pathogenic mutations in the α-galactosidase A gene, localized in X chromosome. Deficient enzymatic activity of the product of this gen...