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Role of dendritic cells and NK cells in immune hypersensitivity reactions in patients with Fabry disease
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Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
durable immune tolerance against rhGAA, leading to improved clinical outcomes.
doi:10.1016/j.ymgme.2016.11.202
194 Modulation of mTOR signaling as a therapeutic approach for Pompe disease Jeong-A Lim, Nina Raben, National Institutes of Health, Bethesda, MD, United States Mammalian target of rapamycin (mTOR) has long been known as a critical regulator of muscle mass. The recent discovery of the lysosome as the site of its activation/inactivation indisputably brings mTOR into the field of lysosomal storage diseases. The evaluation of the mTORC1 status is particularly relevant to Pompe disease, a severe muscle wasting disorder due to a deficiency of lysosomal acid alpha-glucosidase (GAA) leading to excessive accumulation of lysosomal glycogen and autophagic debris. Profound muscle wasting persists and remains a major therapeutic challenge despite the currently available enzyme replacement therapy (ERT). We have done a systematic analysis of mTOR pathway in Pompe muscle cells by evaluating mTOR activity, localization, regulation in response to nutrients, and its role in the control of protein synthesis and autophagy. We have shown that activation of the AMPK-TSC2 pathway is largely responsible for the aberrant mTOR signaling. AAV-mediated intramuscular injection of shRNA-TSC2 in GAA-KO mice led to mTOR activation, reversal of atrophy, and a striking removal of the autophagic buildup - one of the key obstacles in ERT. Of particular interest, we took advantage of the recent data on the role of arginine in the regulation of mTOR signaling and demonstrated that this amino acid reinstated mTOR activity in the diseased muscle cells and in the whole muscle of Pompe mice. This finding can be easily translated into the clinic and may have broad relevance for a large group of neuromuscular and lysosomal disorders.
doi:10.1016/j.ymgme.2016.11.203
195 Alterations in alpha-synuclein and parkin expression within PBMCs in patients and carriers of Gaucher disease with Parkinsonism Renuka P. Limgala, Chandni Jani, Ariel Badger, Ozlem Goker-Alpan, Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA, United States Disease causing mutations in both alleles of GBA gene cause Gaucher disease (GD) while mutations in one allele lead to Gaucher carrier status. GD patients display a wide range of clinical symptoms and present with serious comorbidities. It has been shown that patients with GD, even carriers with one mutated GBA gene are at a higher risk for developing Parkinson’s disease (PD), and at an earlier age. Mutations within SNCA (alpha-synuclein), LRRK2, PRKN (Parkin) are implicated in other genetic forms of Parkinsonism. Alphasynuclein is known to form aggregates in cases of PD, and shown to decrease solubility of Parkin. While the role of LRRK2 is largely unclear, the neuropathology may share similarities to GBA-associated
PD cases. As an attempt to assess whether GBA alterations impact alpha-synuclein, LRRK2 and parkin metabolism in easily accessible cell types outside the brain, we investigated the expression at both molecular and protein level in the peripheral blood mononuclear cells (PBMCs). We compared GD patients manifesting PD symptoms (GD-PD) to GD patients without PD symptoms (GD-nonPD) and nonGD controls. At the mRNA level, there was increased expression of LRRK2 and SNCA, in both GD-PD and GD-nonPD compared to controls. However, there was a significant increase in expression of LRRK2 and alpha-synuclein in subjects with GD-PD only, at protein level. Since alpha-synuclein aggregation is known to affect solubility and subsequent accumulation of Parkin, we studied Parkin in PBMCs and found a correlation with alpha-synuclein levels. Flow cytometry also revealed a fraction of PBMCs from GD-PD with overexpression of both Parkin and alpha-synuclein indicating possible aggregation of these proteins. As this finding was unique to GD-PD, the mechanisms underlying this observation may shed light to development of future biomarkers.
doi:10.1016/j.ymgme.2016.11.204
196 Role of dendritic cells and NK cells in immune hypersensitivity reactions in patients with Fabry disease Renuka P. Limgala, Tabitha Jennelle, Ozlem Goker-Alpan, Lysosomal and rare disorders research and treatment center, Fairfax, VA, United States Fabry disease (FD) is an X-linked lysosomal disorder caused by mutations in the GLA gene resulting in lack of or faulty alphagalactosidase A (α-GalA) enzyme. This leads to accumulation of globotriaosylceramide in lysosomes of various tissues and organs. Enzyme replacement therapy (ERT) with recombinant α -GalA is the standard therapy for FD. Immune hypersensitivity reactions (IHRs) are often seen in FD patients as a result of immunogenicity of the infused exogenous enzyme. Subsequent generation of antibodies in patients with no or little residual α -GalA activity can cause significant morbidity leading to interruptions and occasional discontinuation of therapy. We studied 9 subjects with FD receiving ERT who develop IHRs with symptoms ranging from rigors, fever, pain, vomiting and angioedema. Six subjects presented with NCI-CTC (National cancer institute common terminology criteria) grade 3 and three with grade 2 criteria for hypersensitivity and acute infusion reactions. All the subjects were male and had normal tryptase levels (except 1 subject). No obvious correlation was found between IHRs, GLA pathogenic variants, and the titers of anti-drug antibodies. Six subjects had neutralizing antibodies with titers ranging from 1:20 to 1:500, while three did not have any. In an attempt to better understand the role of immune system in recombinant α -GalA mediated IHRs, we performed flow cytometry based immunephenotyping of 5 subjects experiencing IHRs from peripheral blood, drawn pre- and post-infusion during IHR episode. FD subjects who do not show any IHRs to ERT were used as controls (n= 8). In all the subjects with IHRs, there was a dramatic reduction in dendritic cells post-ERT which was not seen in controls. A corresponding increase of NK cells and T cells was also seen in this cohort highlighting possible crosstalk between these immune subtypes resulting in IHRs.
doi:10.1016/j.ymgme.2016.11.205
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
durable immune tolerance against rhGAA, leading to improved clinical outcomes.
doi:10.1016/j.ymgme.2016.11.202
194 Modulation of mTOR signaling as a therapeutic approach for Pompe disease Jeong-A Lim, Nina Raben, National Institutes of Health, Bethesda, MD, United States Mammalian target of rapamycin (mTOR) has long been known as a critical regulator of muscle mass. The recent discovery of the lysosome as the site of its activation/inactivation indisputably brings mTOR into the field of lysosomal storage diseases. The evaluation of the mTORC1 status is particularly relevant to Pompe disease, a severe muscle wasting disorder due to a deficiency of lysosomal acid alpha-glucosidase (GAA) leading to excessive accumulation of lysosomal glycogen and autophagic debris. Profound muscle wasting persists and remains a major therapeutic challenge despite the currently available enzyme replacement therapy (ERT). We have done a systematic analysis of mTOR pathway in Pompe muscle cells by evaluating mTOR activity, localization, regulation in response to nutrients, and its role in the control of protein synthesis and autophagy. We have shown that activation of the AMPK-TSC2 pathway is largely responsible for the aberrant mTOR signaling. AAV-mediated intramuscular injection of shRNA-TSC2 in GAA-KO mice led to mTOR activation, reversal of atrophy, and a striking removal of the autophagic buildup - one of the key obstacles in ERT. Of particular interest, we took advantage of the recent data on the role of arginine in the regulation of mTOR signaling and demonstrated that this amino acid reinstated mTOR activity in the diseased muscle cells and in the whole muscle of Pompe mice. This finding can be easily translated into the clinic and may have broad relevance for a large group of neuromuscular and lysosomal disorders.
doi:10.1016/j.ymgme.2016.11.203
195 Alterations in alpha-synuclein and parkin expression within PBMCs in patients and carriers of Gaucher disease with Parkinsonism Renuka P. Limgala, Chandni Jani, Ariel Badger, Ozlem Goker-Alpan, Lysosomal and Rare Disorders Research and Treatment Center, Fairfax, VA, United States Disease causing mutations in both alleles of GBA gene cause Gaucher disease (GD) while mutations in one allele lead to Gaucher carrier status. GD patients display a wide range of clinical symptoms and present with serious comorbidities. It has been shown that patients with GD, even carriers with one mutated GBA gene are at a higher risk for developing Parkinson’s disease (PD), and at an earlier age. Mutations within SNCA (alpha-synuclein), LRRK2, PRKN (Parkin) are implicated in other genetic forms of Parkinsonism. Alphasynuclein is known to form aggregates in cases of PD, and shown to decrease solubility of Parkin. While the role of LRRK2 is largely unclear, the neuropathology may share similarities to GBA-associated
PD cases. As an attempt to assess whether GBA alterations impact alpha-synuclein, LRRK2 and parkin metabolism in easily accessible cell types outside the brain, we investigated the expression at both molecular and protein level in the peripheral blood mononuclear cells (PBMCs). We compared GD patients manifesting PD symptoms (GD-PD) to GD patients without PD symptoms (GD-nonPD) and nonGD controls. At the mRNA level, there was increased expression of LRRK2 and SNCA, in both GD-PD and GD-nonPD compared to controls. However, there was a significant increase in expression of LRRK2 and alpha-synuclein in subjects with GD-PD only, at protein level. Since alpha-synuclein aggregation is known to affect solubility and subsequent accumulation of Parkin, we studied Parkin in PBMCs and found a correlation with alpha-synuclein levels. Flow cytometry also revealed a fraction of PBMCs from GD-PD with overexpression of both Parkin and alpha-synuclein indicating possible aggregation of these proteins. As this finding was unique to GD-PD, the mechanisms underlying this observation may shed light to development of future biomarkers.
doi:10.1016/j.ymgme.2016.11.204
196 Role of dendritic cells and NK cells in immune hypersensitivity reactions in patients with Fabry disease Renuka P. Limgala, Tabitha Jennelle, Ozlem Goker-Alpan, Lysosomal and rare disorders research and treatment center, Fairfax, VA, United States Fabry disease (FD) is an X-linked lysosomal disorder caused by mutations in the GLA gene resulting in lack of or faulty alphagalactosidase A (α-GalA) enzyme. This leads to accumulation of globotriaosylceramide in lysosomes of various tissues and organs. Enzyme replacement therapy (ERT) with recombinant α -GalA is the standard therapy for FD. Immune hypersensitivity reactions (IHRs) are often seen in FD patients as a result of immunogenicity of the infused exogenous enzyme. Subsequent generation of antibodies in patients with no or little residual α -GalA activity can cause significant morbidity leading to interruptions and occasional discontinuation of therapy. We studied 9 subjects with FD receiving ERT who develop IHRs with symptoms ranging from rigors, fever, pain, vomiting and angioedema. Six subjects presented with NCI-CTC (National cancer institute common terminology criteria) grade 3 and three with grade 2 criteria for hypersensitivity and acute infusion reactions. All the subjects were male and had normal tryptase levels (except 1 subject). No obvious correlation was found between IHRs, GLA pathogenic variants, and the titers of anti-drug antibodies. Six subjects had neutralizing antibodies with titers ranging from 1:20 to 1:500, while three did not have any. In an attempt to better understand the role of immune system in recombinant α -GalA mediated IHRs, we performed flow cytometry based immunephenotyping of 5 subjects experiencing IHRs from peripheral blood, drawn pre- and post-infusion during IHR episode. FD subjects who do not show any IHRs to ERT were used as controls (n= 8). In all the subjects with IHRs, there was a dramatic reduction in dendritic cells post-ERT which was not seen in controls. A corresponding increase of NK cells and T cells was also seen in this cohort highlighting possible crosstalk between these immune subtypes resulting in IHRs.
doi:10.1016/j.ymgme.2016.11.205