Gaucher disease-associated alterations in mesenchymal stem cells reduce osteogenesis and favour adipogenesis processes with concomitant increased osteoclastogenesis.

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme ß-glucocerebrosidase. Until now, treatments for GD cannot completely reverse bone problems. The aim of this work was to evaluate the potential of MSCs from GD patients (GD MSCs) to differentiate towards the osteoblast (GD Ob) and adipocyte (GD Ad) lineages, and their role in osteoclastogenesis. We observed that GD Ob exhibited reduced mineralization, collagen deposition and alkaline phosphatase activity (ALP), as well as decreased gene expression of RUNX2, COLA1 and ALP. We also evaluated the process of osteoclastogenesis and observed that conditioned media from GD MSCs supernatants induced an increase in the number of osteoclasts. In this model, osteoclastogenesis was induced by RANKL and IL-1ß. Furthermore, results showed that in GD MSCs there was a promotion in NLRP3 and PPAR-γ gene expression. Adipogenic differentiation revealed that GD Ad had an increase in PPAR-γ and a reduced RUNX2 gene expression, promoting adipocyte differentiation. In conclusion, our results show that GD MSCs exhibited deficient GD Ob differentiation and increased adipogenesis. In addition, we show that GD MSCs promoted increased osteoclastogenesis through RANKL and IL-1ß. These changes in GD MSCs are likely to contribute to skeletal imbalance observed in GD patients.

Finding pathogenic commonalities between Niemann-Pick type C and other lysosomal storage disorders: Opportunities for shared therapeutic interventions.

Lysosomal storage disorders (LSDs) are diseases characterized by the accumulation of macromolecules in the late endocytic system and are caused by inherited defects in genes that encode mainly lysosomal enzymes or transmembrane lysosomal proteins. Niemann-Pick type C disease (NPCD), a LSD characterized by liver damage and progressive neurodegeneration that leads to early death, is caused by mutations in the genes encoding the NPC1 or NPC2 proteins. Both proteins are involved in the transport of cholesterol from the late endosomal compartment to the rest of the cell. Loss of function of these proteins causes primary cholesterol accumulation, and secondary accumulation of other lipids, such as sphingolipids, in lysosomes. Despite years of studying the genetic and molecular bases of NPCD and related-lysosomal disorders, the pathogenic mechanisms involved in these diseases are not fully understood. In this review we will summarize the pathogenic mechanisms described for NPCD and we will discuss their relevance for other LSDs with neurological components such as Niemann- Pick type A and Gaucher diseases. We will particularly focus on the activation of signaling pathways that may be common to these three pathologies with emphasis on how the intra-lysosomal accumulation of lipids leads to pathology, specifically to neurological impairments. We will show that although the primary lipid storage defect is different in these three LSDs, there is a similar secondary accumulation of metabolites and activation of signaling pathways that can lead to common pathogenic mechanisms. This analysis might help to delineate common pathological mechanisms and therapeutic targets for lysosomal storage diseases.

Assessment of cellular cobalamin metabolism in Gaucher disease.

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Efficacy of pentosan polysulfate in in vitro models of lysosomal storage disorders: Fabry and Gaucher Disease.

Gaucher and Fabry diseases are the most prevalent sphingolipidoses. Chronic inflammation is activated in those disorders, which could play a role in pathogenesis. Significant degrees of amelioration occur in patients upon introduction of specific therapies; however, restoration to complete health status is not always achieved. The idea of an adjunctive therapy that targets inflammation may be a suitable option for patients. PPS is a mixture of semisynthetic sulfated polyanions that have been shown to have anti-inflammatory effects in mucopolysaccharidosis type I and II patients and animal models of type I, IIIA and VI. We hypothesized PPS could be a useful adjunctive therapy to inflammation for Gaucher and Fabry diseases. The objective of this work is to analyze the in vitro effect of PPS on inflammatory cytokines in cellular models of Gaucher and Fabry diseases, and to study its effect in Gaucher disease associated in vitro bone alterations. Cultures of peripheral blood mononuclear cells from Fabry and Gaucher patients were exposed to PPS. The secretion of proinflammatory cytokines was significantly reduced. Peripheral blood cells exposed to PPS from Gaucher patients revealed a reduced tendency to differentiate to osteoclasts. Osteoblasts and osteocytes cell lines were incubated with an inhibitor of glucocerebrosidase, and conditioned media was harvested in order to analyze if those cells secrete factors that induce osteoclastogenesis. Conditioned media from this cell cultures exposed to PPS produced lower numbers of osteoclasts. We could demonstrate PPS is an effective molecule to reduce the production of proinflammatory cytokines in in vitro models of Fabry and Gaucher diseases. Moreover, it was effective at ameliorating bone alterations of in vitro models of Gaucher disease. These results serve as preclinical supportive data to start clinical trials in human patients to analyze the effect of PPS as a potential adjunctive therapy for Fabry and Gaucher diseases.

Gaucher's disease in a patient presenting with hip and abdominal pain

Summary Gaucher's disease is characterized by glucocerebroside accumulation in the cells of the reticuloendothelial system. There are three subtypes. The most common is type 1, known as the non-neuropathic form. Pancytopenia, hepatosplenomegaly and bone lesions occur as a result of glucocerebroside accumulation in the liver, lung, spleen and bone marrow in these patients. Findings associated with liver, spleen or bone involvement may be seen at radiological analysis. Improvement in extraskeletal system findings is seen with enzyme replacement therapy. Support therapy is added in patients developing infection, anemia or pain. We describe a case of hepatosplenomegaly, splenic infarction, splenic nodules and femur fracture determined at radiological imaging in a patient under monitoring due to Gaucher's disease.

In vitro osteoclastogenesis from Gaucher patients' cells correlates with bone mineral density but not with Chitotriosidase.

Gaucher disease (GD) is caused by mutations on the gene encoding for the lysosomal enzyme glucocerebrosidase. Type I GD (GD1) patients present anemia, hepatosplenomegaly and bone alterations. In spite of treatment, bone alterations in GD patients persist, including poor bone mineral density (BMD). Mechanisms leading to bone damage are not completely understood, but previous reports suggest that osteoclasts are involved. Chitotriosidase (CHIT) is the most reliable biomarker used in the follow up of patients, although its correlation with bone status is unknown. The aim of this work was to study the pro-osteoclastogenic potential in patients and to evaluate its correlation with CHIT activity levels and clinical parameters. PBMCs from treated patients and healthy controls were cultured in the presence of M-CSF, and mature osteoclasts were counted. BMD, blood CHIT activity and serum levels of CTX, BAP, and cytokines were evaluated in patients. We found that blood CHIT activity and osteoclast differentiation were significantly increased in patients, but no correlation between them was observed. Interestingly, osteoclast numbers but not CHIT, presented a negative correlation with BMD expressed as Z-score. CTX, BAP and serum cytokines involved in bone remodeling were found altered in GD1 patients. These results show for the first time a correlation between osteoclast differentiation and BMD in GD1 patients, supporting the involvement of osteoclasts in the bone pathology of GD1. Our results also suggest that an altered immune response may play an important role in bone damage.

Osteocyte Alterations Induce Osteoclastogenesis in an In Vitro Model of Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the glucosylceramidase ß (GBA 1) gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells, mainly in the monocyte/macrophage lineage. Its mildest form is Type I GD, characterized by non-neuronopathic involvement. Bone compromise is the most disabling aspect of the Gaucher disease. However, the pathophysiological aspects of skeletal alterations are not yet fully understood. The bone tissue homeostasis is maintained by a balance between resorption of old bone by osteoclasts and new bone formation by osteoblasts. A central player in this balance is the osteocyte as it controls both processes. We studied the involvement of osteocytes in an in vitro chemical model of Gaucher disease. The osteocyte cell line MLO-Y4 was exposed to conduritol-ß-epoxide (CBE), an inhibitor of GCase, for a period of 7, 14 and 21 days. Conditioned media from CBE-treated osteocytes was found to induce osteoclast differentiation. GCase inhibition caused alterations in Cx43 expression and distribution pattern and an increase in osteocyte apoptosis. Osteoclast differentiation involved osteocyte apoptotic bodies, receptor activator of nuclear factor κ-B ligand (RANKL) and soluble factors. Thus, our results indicate that osteocytes may have a role to play in the bone pathophysiology of GD.

Gaucher's disease in a patient presenting with hip and abdominal pain.

Gaucher's disease is characterized by glucocerebroside accumulation in the cells of the reticuloendothelial system. There are three subtypes. The most common is type 1, known as the non-neuropathic form. Pancytopenia, hepatosplenomegaly and bone lesions occur as a result of glucocerebroside accumulation in the liver, lung, spleen and bone marrow in these patients. Findings associated with liver, spleen or bone involvement may be seen at radiological analysis. Improvement in extraskeletal system findings is seen with enzyme replacement therapy. Support therapy is added in patients developing infection, anemia or pain. We describe a case of hepatosplenomegaly, splenic infarction, splenic nodules and femur fracture determined at radiological imaging in a patient under monitoring due to Gaucher's disease.

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Proinflammatory and proosteoclastogenic potential of peripheral blood mononuclear cells from Gaucher patients: Implication for bone pathology.

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 the accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. On the other hand it is well known that inflammation is a key player in GD pathology. In this work, we revealed increased levels of the proinflammatory CD14(+)CD16(+) monocyte subset and increased inflammatory cytokine production by monocytes and T cells in the circulation of GD patients. We showed increased levels of osteoclast precursors in PBMC from patients and a higher expression of RANKL in the surface of T cells. PBMC from patients presented higher osteoclast differentiation compared to healthy controls when cultured in the presence of M-CSF alone or in combination with RANKL. In vitro treatment with Velaglucerase reduced osteoclast levels to control levels. On the other hand THP-1 derived osteoclast precursors cultured in the presence of conditioned media from PBMC of GD patients presented higher differentiation to active osteoclasts. This induction involved TNF-α and RANKL.

Pathogenesis of Bone Alterations in Gaucher Disease: The Role of Immune System.

Gaucher, the most prevalent lysosomal disorder, is an autosomal recessive inherited disorder due to a deficiency of glucocerebrosidase. Glucocerebrosidase deficiency leads to the accumulation of glucosylceramide primarily in cells of mononuclear-macrophage lineage. Clinical alterations are visceral, hematological, and skeletal. Bone disorder in Gaucher disease produces defects on bone metabolism and structure and patients suffer from bone pain and crisis. Skeletal problems include osteopenia, osteoporosis, osteolytic lesions, and osteonecrosis. On the other hand a chronic stimulation of the immune system is a well-accepted hallmark in this disease. In this review we summarize the latest findings in the mechanisms leading to the bone pathology in Gaucher disease in relationship with the proinflammatory state.

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.

Cognitive outcome in treated patients with chronic neuronopathic Gaucher disease.

OBJECTIVE: To investigate the spectrum and prevalence of cognitive deficits among children with type 3 (chronic neuronopathic) Gaucher disease (GD). STUDY DESIGN: A case review study identified 32 children (male/female; 17:15) with type 3 GD who had received enzyme replacement therapy (ERT) or a bone marrow transplant. The diagnosis of GD was established by enzymatic assay and DNA testing. Subjects were assessed with standard neuropsychological testing, and data from the most recent evaluation were included. RESULTS: Neuropsychometric assessments demonstrated a wide spectrum of full-scale IQ scores ranging from 39 to 124 (mean 75). About 60% of subjects had intellectual skills below average. There were significant discrepancies between verbal and performance IQ, with a range between -6 and 38 points (P = .02). This gap was more prominent in older subjects, with better performance in the verbal areas. No correlation was observed between intelligence measures and genotype or the extent of systemic involvement. The dosage, age at initiation, and the length of ERT had no significant effect on IQ scores. CONCLUSIONS: In type 3 GD, cognitive deficits, characterized by visual-spatial dysfunction, are common but underappreciated and appear resistant to ERT.

Use of plain radiography to optimize skeletal outcomes in children with type 1 Gaucher disease in Brazil.

BACKGROUND: Gaucher disease is the most common lysosomal storage disease and is caused by deficient production and activity of the lysosomal enzyme beta-glucosidase (glucocerebrosidase), resulting in progressive accumulation of glucosylceramide (glucocerebroside) in lysosomes of cells of the reticuloendothelial system in the spleen, liver, and marrow. Clinical manifestations include anemia, thrombocytopenia, hepatosplenomegaly, and bone complications, including bone pain, bone marrow infiltration, lytic lesions, osteopenia, pathological fractures, and avascular necrosis. Early, adequate, and sustained treatment with enzyme replacement therapy (ERT) available since 1991 can change the natural history of the disease, particularly in children. Skeletal complications are usually the major source of disease morbidity and disability and although magnetic resonance imaging and dual-energy x-ray absorptiometry densitometry are recommended for monitoring, these are not readily available in all countries. METHODS: We describe 18 Brazilian children with type 1 Gaucher disease with bone involvement who were followed with plain radiography for at least 8 months after beginning imiglucerase ERT (initial dose, 15-60 U/kg body weight/15 days). Bone involvement noted by plain radiograph included marrow infiltration, osteopenia, pathological fractures, osteonecrosis, lytic lesions, and Erlenmeyer flask deformity. Patients were questioned about bone crises. RESULTS: Patients were followed for up to 10 years (mean follow-up, 4 years and 4 months +/- 3 years and 3 months). Bone changes were visible by plain radiographs in all patients. Clinical and radiological improvement was noted in 13 (72%) of 18 patients; bone lesions worsened in 5 (28%) of 18 patients. The final ERT dose for the 13 patients who improved was 55 +/- 10 U/kg (range, 30-60 U/kg), and the final ERT dose for the 5 who worsened was 29 +/- 2 U/kg (range, 26-30 U/kg); this difference was statistically significant (P < 0.03). CONCLUSIONS: When other imaging technologies are not available, skeletal response to ERT in children with type 1 Gaucher disease can be monitored effectively by plain radiography. Higher doses of ERT (50-60 U/kg /15 days) may be required for improvement of skeletal manifestations. CLINICAL EVIDENCE: Case series; level 4.

Biochemical properties of beta-glucosidase in leukocytes from patients and obligated heterozygotes for Gaucher disease carriers.

BACKGROUND: Gaucher's disease (GD) is a disorder caused by the deficiency of lysosomal beta-glucosidase, an enzyme that participates in the degradation of glycosphingolipids. Deficiency of this enzyme results in the storage of glucocerebrosides in lysosomes of macrophage. No studies are available in the literature comparing biochemical and kinetic behavior of this enzyme in leukocytes and fibroblasts from normal individuals, obligate heterozygotes and patients with GD. METHODS: The behavior of beta-glu in terms of optimum pH, heat stability, Km and Vmax in leukocytes from patients with GD and obligated heterozygotes with different genotypes and normal individuals were characterized. RESULTS: Optimum pH was similar in all groups analyzed. In terms of Km and Vmax, several differences among heterozygotes and homozygotes groups and among these groups and normal enzyme were observed. Enzyme from all groups were inactivated when preincubated at 60 degrees C, but some enzymes were more stable than other. Results showed a different behavior of the enzyme in the 3 groups under analysis. Such behavior varied according to individual mutation. CONCLUSIONS: The catalytic gradient presented by beta-glu allowed the correlation of N370S mutation-which presented more stable biochemical properties-with the non-neurological clinical condition of the disease and the catalytically less stable mutation (D409H), with the neurological clinical condition of GD. This study contributes to a better understanding of the repercussion of the different mutations on the protein function, thus allowing to predict the severity of such complex metabolic disorder and to anticipate the most appropriate intervention for each case specifically.

Enfermedad de Gaucher: presentación de un caso / Gaucher's disease: presentation of a case

Informe de una patología quirúrgica infrecuente. Descripción del caso clínico y revisión de la literatura. Servicio de cirugía 3. Hospital Universitario de Caracas. Venezuela. Mujer de 28 años, con antecedente familiar de enfermedad de Gaucher. Portadora de hepatomegalia, esplenomegalia e hiperesplenismo. El bazo pesó 1 300 gramos con histiocitosis xeroidea. La enfermedad de Gaucher es extraordinariamente infrecuente en nuestro medio

Doença de Gaucher / Gaucher disease

A Doença de Gaucher é caracterizada por uma falta hereditária da enzima glicocrebrosidade que leva ao acúmulo de glicosilceramida nas células monocítico-fagocitárias do fígado e baço, principalmente. Devido aos constantes avanços na terapia desta doença, é feita uma abordagem clínica inicial e subsequente atualizaçãoquanto ao diagnóstico e tratamento. Os dados foram coletados através de análise de artigos e da rede internacional de computadores. Foi feita uma seleção da bibliografia, dando ênfaseaos diagnósticos, prognósticos e terapêuticos...

Enfermedad de Gaucher tipo 1. Reporte de un caso / Gaucher disease type 1. Report of a case

Se presenta el caso de un paciente de 17 años de edad, con enfermedad de Gaucher tipo 1, cuya evolución data desde los cinco años de edad, manifestada por aumento de volumen abdominal a expensas de hepatomegalia y esplenomegalia masiva, con hipodesarrollo corporal y sexual, así como hiperesplenismo, anemia, trombocitopenia y lesiones óseas. El diagnóstico se integró con datos clínicos, radiológicos e histopatológicos

Enfermedad de Gaucher / Gaucher disease

Se presenta un caso de enfermedad de Gaucher en una paciente de 19 años quien consultó por dolor abdominal. Al examen físico presentaba palidez y hepato-esplenomegalia importante. Había pancitopenia y ß-gluco cerebrosidos disminuidos con un nivel de 1,14 mmol (V-N=6-17). La biopsia de médula ósea mostró histiocitos con corpusculos de grasa. Con esplenectomía se obtuvo brazo de 2.100 gr en el cual se encontraron células de Gaucher. Se presenta una revisión breve de la enfermedad